The development of selective inhibitors of nagz: Increased susceptibility of gram-negative bacteria to β-lactams

Article abstract of DOI:10.1002/cbic.201300395

The increasing incidence of inducible chromosomal AmpC β-lactamases within the clinic is a growing concern because these enzymes deactivate a broad range of even the most recently developed β-lactam antibiotics. As a result, new strategies are needed to block the action of this antibiotic resistance enzyme. Presented here is a strategy to combat the action of inducible AmpC by inhibiting the β-glucosaminidase NagZ, which is an enzyme involved in regulating the induction of AmpC expression. A divergent route facilitating the rapid synthesis of a series of N-acyl analogues of 2-acetamido-2-deoxynojirimycin is reported here. Among these compounds are potent NagZ inhibitors that are selective against functionally related human enzymes. These compounds reduce minimum inhibitory concentration values for β-lactams against a clinically relevant Gram-negative bacterium bearing inducible chromosomal AmpC β-lactamase, Pseudomonas aeruginosa. The structure of a NagZ-inhibitor complex provides insight into the molecular basis for inhibition by these compounds. The development of selective and potent inhibitors of the β-glucosaminidase NagZ, which is an important enzyme in AmpC β-lactamase expression, based on the inhibitor 2-acetamido-2-deoxynojirimycin is described. In addition, the structure of a NagZ-inhibitor complex provides insight into the molecular basis for inhibition by these compounds.

Full text of DOI:10.1002/cbic.201300395

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DOI: 10.1002/cbic.201300395
The Development of Selective Inhibitors of NagZ:
Increased Susceptibility of Gram-Negative Bacteria to b-
Lactams
Keith A. Stubbs,*[a] John-Paul Bacik,[b, d] G. Evan Perley-Robertson,[c] Garrett E. Whitworth,[c, e]
Tracey M. Gloster,[c, f] David J. Vocadlo,[c] and Brian L. Mark*[b]
The increasing incidence of inducible chromosomal AmpC b-
lactamases within the clinic is a growing concern because
these enzymes deactivate a broad range of even the most re-
cently developed b-lactam antibiotics. As a result, new strat-
egies are needed to block the action of this antibiotic resist-
ance enzyme. Presented here is a strategy to combat the
action of inducible AmpC by inhibiting the b-glucosaminidase
NagZ, which is an enzyme involved in regulating the induction
of AmpC expression. A divergent route facilitating the rapid
synthesis of a series of N-acyl analogues of 2-acetamido-2-de-
oxynojirimycin is reported here. Among these compounds are
potent NagZ inhibitors that are selective against functionally
related human enzymes. These compounds reduce minimum
inhibitory concentration values for b-lactams against a clinically
relevant Gram-negative bacterium bearing inducible chromo-
somal AmpC b-lactamase, Pseudomonas aeruginosa. The struc-
ture of a NagZ–inhibitor complex provides insight into the
molecular basis for inhibition by these compounds.
Introduction
Resistance to even the most recent generation of b-lactams is
steadily developing as various resistance mechanisms dissemi-
nate through the bacterial domain. Resistance mechanisms to
b-lactams are varied, but the most important involve the b-lac-
tamases—enzymes that deactivate b-lactam antibiotics by
cleaving the cyclic amide moiety. One specific class of these
enzymes, inducible chromosomal AmpC b-lactamases,[1–3] is
increasingly problematic in many Gram-negative bacteria,
because these enzymes deactivate a broad range of even the
most recent b-lactam antibiotics,[4–6] and are resistant to clini-
cally available b-lactamase inhibitors.[7,8] As a result, new strat-
egies for blocking the action of this class of enzyme are of con-
siderable interest.[9]
AmpC b-lactamase expression depends on the activity of
a number of proteins that are engaged in peptidoglycan me-
tabolism. The peptidoglycan is an essential component of the
bacterial cell and is a highly cross-linked hetero-polymer that
forms an exoskeleton around the organism, defining its shape
and protecting it from osmotic lysis.[10] During normal cell divi-
sion, a considerable amount of the peptidoglycan is degraded
and recycled.[10,11] The resulting GlcNAc-1,6-anhydroMurNAc-
peptide degradation products have their non-reducing GlcNAc
residue removed by a cytosolic b-glucosaminidase known as
NagZ.[12,13] The resulting products are N-acetyl-d-glucosamine
and a series of 1,6-anhydroMurNAc-tri-, tetra- and pentapepti-
des (Scheme 1A). These 1,6-anhydroMurNAc catabolic frag-
ments activate the transcription of inducible ampC by binding
to the transcriptional regulator AmpR.[14] To prevent the contin-
uous expression of ampC, another molecule, UDP-MurNAc-pen-
tapeptide, which is a biosynthetic building block of the cell
wall derived from these catabolic products, is involved in
repressing ampC transcription by binding to AmpR.[14,15] The
relative concentrations of these molecules enable bacteria to
sense b-lactams and so regulate AmpC expression.[2]
[a] Dr. K. A. Stubbs
School of Chemistry and Biochemistry, University of Western Australia
35 Stirling Highway, Crawley, WA 6009 (Australia)
E-mail: keith.stubbs@uwa.edu.au
[b] Dr. J.-P. Bacik, Dr. B. L. Mark
Department of Microbiology, University of Manitoba
45 Chancellors Circle, Winnipeg, MB R3T2N2 (Canada)
E-mail: Brian.Mark@ad.umanitoba.ca
[c] G. E. Perley-Robertson, Dr. G. E. Whitworth, Dr. T. M. Gloster,
Dr. D. J. Vocadlo
Department of Chemistry, Simon Fraser University
8888 University Drive, Burnaby, BC V5A1S6 (Canada)
[d] Dr. J.-P. Bacik
Current address: Bioscience Division, Los Alamos National Laboratory
P.O. Box 1663, Los Alamos, NM 87545 (USA)
[e] Dr. G. E. Whitworth
Current address: Department of Chemistry
Massachusetts Institute of Technology
77 Massachusetts Avenue, Cambridge, MA 02139 (USA)
[f] Dr. T. M. Gloster
Current address: Biomedical Sciences Research Complex
University of St Andrews
North Haugh, St Andrews, Fife, KY16 9ST (UK)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201300395.
One strategy to combat inducible AmpC would be to inhibit
NagZ using small-molecule inhibitors. NagZ, a member of
family GH3 (for an overview of the CAZy classification system
of glycoside hydrolases see http://www.cazy.org[16]) uses a two-
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This is an open access article under the terms of the Creative Commons At-
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medium, provided the original work is properly cited.
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Scheme 1. A) NagZ catalyzed hydrolysis of peptidoglycan cell-wall fragments releases the series of 1,6-anhydroMurNAc peptide inducer molecules that acti-
vate transcription of ampC expression (tripeptide shown). B) The putative transition state of the NagZ catalyzed hydrolysis of N-acetylglucosaminides (denot-
ed by °). C) Structures of known inhibitors of NagZ enzymes.
step, double-displacement mechanism involving the formation
and breakdown of a covalent glycosyl-enzyme intermediate via
oxocarbenium-ion-like transition states (Scheme 1B).[17–20] By in-
hibiting NagZ, the formation of the inducer molecules compris-
ing 1,6-anhydroMurNAc peptides would be impeded thus lead-
ing to reduced AmpC production and increased sensitivity to
b-lactams. The approach of targeting NagZ has received recent
validation using both chemical,[21,22] and genetic studies,[23–25]
while structural studies of NagZ are now enabling the design
of improved inhibitors.[26]
lective inhibitors for NagZ enzymes. Such compounds might
be valuable tools, not only by inhibiting NagZ and thus render-
ing increased susceptibility to b-lactams of Gram-negative bac-
teria harbouring inducible ampC, but also by aiding improved
understanding of the binding of selective inhibitors to this
family of enzymes and the role played by NagZ in peptidogly-
can recycling.
Results and Discussion
The main focus of NagZ inhibitor design has centred around
the known b-glucosaminidase inhibitor, O-(2-acetamido-2-
Multiple syntheses of 1 previously described in the literature
use N-acetyl-d-glucosamine as a starting material.[36–40] To pre-
pare the series of target compounds we envisaged that a diver-
gent synthesis would facilitate their rapid preparation. Accord-
ingly, N-acetyl-d-glucosamine was not a viable starting material
since we aimed to generate a panel of compounds with vari-
ous N-acyl groups. We felt, due to the nature of the chemical
transformations necessary, that an azido group at C-2 would
be stable throughout the synthesis enabling us to prepare
a common synthetic intermediate (2, Scheme 2), which would
be amenable to rapid diversification to generate the desired
panel of N-acyl compounds. In addition, the 5,6-alkene could
be used in the preparation of the desired iminosugars, through
their intermediate ulososides. This general approach has
shown value in the preparation of 2-acetamido-1,2-dideoxyno-
jirimycin-lysine hybrids[41] and other iminosugars.[42]
deoxy-d-glucopyranosylidene)amino
N-phenylcarbamate
(PUGNAc, Scheme 1C).[27] Despite its potency for NagZ,[21]
a downside to using this molecule in a complex biological con-
text is that it lacks selectivity for NagZ over important human
enzymes. PUGNAc has been demonstrated to inhibit family
GH84 human O-GlcNAcase (OGA),[28,29] family GH20 human b-
hexosaminidases[30] and family GH89 hexosaminidases related
to NAGLU.[31] An area gaining increasing attention in carbohy-
drate enzymology is the need for inhibitors with improved se-
lectivity between functionally related enzymes.[32] Therefore, in
an effort to overcome problems associated with concomitant
inhibition of these enzymes, a series of PUGNAc derivatives
were prepared through modification of the pendant N-acyl
chain[21,33] and were found to be selective for NagZ as well as
useful at reducing AmpC b-lactamase expression.[21,23]
Starting from the readily accessible hydrochloride 3, avail-
able in three steps from d-glucosamine hydrochloride,[43] the
azido group was introduced to protect the amine moiety
using an azido-transfer reagent[44] to give the triol 4[45]
(Scheme 2). With triol 4 in hand, a one-pot activation at O-6,
using a tosylate group followed by displacement of the tosy-
late with sodium iodide and in situ acetylation gave the
iodide 5 in excellent yield. Elimination of hydroiodic acid
across C-5/6 was achieved using 1,8-diazabicyclo[5.4.0]undec-
Another molecule that suffers from the same selectivity
problem, yet has been used as an inhibitor of b-hexosamini-
dases, is 2-acetamido-2-deoxynojirimycin (NHAcDNJ, 1,
Scheme 1C). This compound is known to potently inhibit hex-
osaminidases from families GH20[34,35] and GH89[31] and has re-
cently been found to inhibit NagZ from E. coli.[22] Based on
these observations we envisaged that making modifications to
the pendant N-acyl chain of 1 might yield new potent and se-
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Scheme 2. Reagents: a) ImSO2N3·HCl, K2CO3, CuSO4, MeOH; b) i: TsCl, pyridine, CH2Cl2; ii: NaI, DMF; iii: Ac2O, C5H5N; c) DBU, THF; d) i: PBu3, THF, H2O;
ii: (RCO)2O; e) i: 3-chloroperbenzoic acid, CH2Cl2, BnOH; ii: NaOMe, MeOH; f) NH4OAc, Pd(OH)2/C, H2, MeOH/H2O (15:1).
7-ene (DBU) in THF to give the desired intermediate alkene 2.
Treatment of the alkene 2 with tributylphosphine in THF/H2O,
followed by acylation with the appropriate acyl anhydride
gave the series of amides 6–12. Oxidation of the alkenes 6–12
with 3-chloroperbenzoic acid provided the presumed inter-
mediate ulososides which were deprotected to give the triols
13–19. Finally, debenzylation by hydrogenolysis and in situ re-
ductive amination of 13–19 with ammonium acetate in the
presence of hydrogen gratifyingly gave the desired iminosu-
gars 1 and 20–25 in excellent overall yield, exclusively as the
d-gluco-configured materials.
It has been previously established that 1 is a potent compet-
itive inhibitor of lysosomal family GH20 b-hexosaminidases, the
lysosomal family GH89 a-hexosaminidase NAGLU, and E. coli
NagZ. The Ki value for the human lysosomal family GH20 b-
hexosaminidases is 540 nm[35] (determined against b-hexosami-
nidase B) and for human NAGLU it is 450 nm.[46] As previously
discussed, developing selective inhibitors of NagZ is important
to block NagZ function in bacteria but also to ensure that
there is no concomitant inhibition of these human enzymes.
With the synthesised panel of inhibitors in hand, we evaluat-
ed them against representative NagZ enzymes found in Vibrio
cholerae (VcNagZ) and Salmonella typhimurium (StNagZ) and
found them to be potent competitive inhibitors of these en-
zymes (Table 1). What was also of interest was that increasing
the N-acyl chain length leads to a greater increase in Ki value
for both b-hexosaminidase B and NAGLU as compared to NagZ
enzymes, consistent with those observed for the N-acyl
PUGNAc analogues and presumably a consequence of the
more spacious active site found in NagZ enzymes.[21] Further-
more these compounds were also poor inhibitors of OGA, with
the parent compound showing a Ki value of 23 mm, consistent
with an independent report for this enzyme.[35] We find that
the selectivity ratio for the NagZ enzymes improves as the
chain length increases; this is illustrated best for compound 21,
which shows over 50-fold selectivity for VcNagZ (>20-fold for
StNagZ) over the human enzymes, whilst retaining potency for
the NagZ enzymes. To gain a more detailed understanding of
the molecular basis for the inhibition of NagZ enzymes by 21,
which we deemed to be the most potentially promising com-
pound in this series in terms of potency as well as selectivity,
we determined the three-dimensional structure of StNagZ in
complex with 21 at 1.45 ꢁ resolution (Figure 1).
With the enzyme–inhibitor complex we find that the
pseudo-glycoside ring structure of 21 adopts a relaxed 4C1
chair conformation that closely resembles that observed for
the reaction product GlcNAc. Accordingly, similar hydrogen-
bonding interactions are observed between 21 and residues in
the active site (for a detailed description of the structure and
mechanism of NagZ, see ref. [20]). However, we also note that
the NH group within the ring of 21, which replaces the endo-
cyclic oxygen of GlcNAc, forms a hydrogen bond with the
side-chain of Asp249, which likely confers the increased bind-
ing affinity relative to GlcNAc. The butyl moiety of the N-acyl
group of 21 appears to form hydrophobic interactions with
a small surface created by the side chains of Ala129 and Ile130
Table 1. Inhibition constants and selectivity ratio (S.R.) of inhibitors for O-GlcNAcase, b-hexosaminidase B, NAGLU, VcNagZ and StNagZ.
Compound
O-GlcNAcase Ki
[mm]
b-Hexosaminidase B
Ki [mm]
NAGLU Ki
[mm]
VcNagZ Ki
[mm]
StNagZ Ki
[mm]
S.R.
S.R.
(Ki OGA/Ki VcNagZ)
(Ki OGA/Ki StNagZ)
1
20
21
22
23
24
25
23
130
0.54[35]
61
0.45[46]
8.5
4.2
9.4
25.2
45.4
23.2
47.3
102
2.8
31
>53
>9
>0.9
63
>1.8
0.94
2.9
96
>1000
>1000
>1000
800
>500
>1000
>1000
>1000
>1000
1460
>22
>21
>10
>40
>3.4
>5000
>5000
670
110
1135
15.8
555
24.6
294
>5000
>1000
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atic for patients suffering from cystic fibrosis, severe burns and
pulmonary disease.[48–50] Importantly, P. aeruginosa PA01 con-
tains a functional NagZ and strains lacking the nagZ gene are
known to have increased susceptibility to b-lactams, support-
ing the validity of this strain in such b-lactam susceptibility
assays.[23,24]
A series of b-lactam antibiotics, cefoxitin, ceftazidime, ampi-
cillin, the monobactam aztreonam and the carbapenem imipe-
nem, were chosen as they are commonly used in clinical anti-
biotic susceptibility experiments. Using minimal inhibitory con-
centration (MIC) assays we found that cultures treated with the
selective inhibitor 21 are more susceptible to these b-lactam
antibiotics when compared to control cultures that were not
treated with 21 (Table 2).
Table 2. Susceptibility of P. aeruginosa PA01 against various b-lactam
antibiotics.
[a]
Antibiotic
MIC [mgmLÀ1
]
Clearing radius [mm][b]
À21
+21
À21
+21
Ceftazidime
Aztreonam
Imipenem
Ampicillin
Cefoxitin
2
1
8
0.5
0.5
4
128
512
12.2
13.8
11.9
14.3
16.0
13.3
512
2048
[a] MIC determined by standard serial dilution. [b] Susceptibility deter-
mined in an agar diffusion assay using 6 mm filter disks loaded with
30 mg of antibiotic with or without 21. The zone of clearance was mea-
sured after incubation overnight.
To use a separate assessment of antibiotic susceptibility we
took the more potent b-lactams, ceftazidime, aztreonam and
imipenem and assessed them in agar diffusion assays (Table 2).
In accord with the MIC data we found that agar-diffusion
assays gave similar results, revealing enhanced susceptibility to
b-lactams in the presence of 21. As a control we evaluated 21
as an inhibitor of bacterial growth but observed no differences
in growth rates even at a concentration of 1 mm (data not
shown), indicating that the inhibitor, on its own, is not antibac-
terial or bacteriostatic. Of note is that the concentration of 21
required to give rise to the increased susceptibility observed is
fivefold less than the concentration of the PUGNAc analogue
O-(2-deoxy-2-N-2-ethylbutyryl-d-glucopyranosylidene)amino N-
phenylcarbamate (EtBuPUG) used previously to induce a similar
effect.[23] Together, these results suggest that 21 might gain
more ready access to the cytosol, either through a transporter
protein or passive diffusion as compared to EtBuPUG.
Figure 1. Crystal structure of StNagZ bound to 21. Active-site residues and
21 are drawn as sticks with oxygen and nitrogen atoms shown in red and
blue, respectively. Carbon atoms of the enzyme are shown in grey, while the
carbon atoms of the inhibitor are yellow. The hydrophobic surface formed
by side chains of Ala129 and Ile130, against which the butyl chain sits, is
shown in grey. The catalytic nucleophile, Asp248, is shown in stick format.
Occupancy values for the dual conformations of Asp248 are 0.48/0.52, and
0.53/0.47 for Asp249 (flipped in or out towards the inhibitor, respectively).
Hydrogen bonds are shown as yellow dashed lines. Electron density around
21 is a maximum-likelihood-weighted omit map (FobsÀFcalcd) contoured to
2.5s.
adopting a similar conformation as that seen in the VcNagZ
structures bound to PUGNAc-derived inhibitors.[21,26] Finally, the
structure shows the butyl chain is exposed to solvent on the
outer side, supporting the idea that the relatively open active-
site arrangement of NagZ accounts for the much greater selec-
tivity demonstrated toward the bacterial hydrolases by the
NHAcDNJ-derived inhibitors with more bulky modified N-acyl
chains.
Conclusions
The incidence of inducible chromosomal AmpC b-lactamases is
increasing and since these enzymes deactivate a broad range
of b-lactam antibiotics they are a pressing health issue. We
have devised a divergent route that enables the rapid synthe-
sis of a series of potent and selective NHAcDNJ-based inhibi-
tors bearing different N-acyl groups that target NagZ, an im-
portant enzyme in the regulation of AmpC activity. One of
We next set out to obtain insight into whether these com-
pounds could increase the susceptibility of bacteria harbouring
inducible AmpC b-lactamase to b-lactams. We therefore evalu-
ated them against Pseudomonas aeruginosa PA01, a Gram-
negative bacterium that harbours a chromosomally inducible
AmpC b-lactamase.[47] This opportunistic pathogen is problem-
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Methyl 3,4-di-O-acetyl-2-azido-2,6-dideoxy-b-d-xylo-hex-5-eno-
side (2): DBU (4.9 mL, 33 mmol) was added to iodide 5 (4.5 g,
11 mmol) in THF (40 mL) and the mixture refluxed (2 h). The mix-
ture was then concentrated and the resultant residue diluted with
EtOAc (150 mL) and was washed with water (2ꢂ40 mL), ice-cold
aq. HCl (1m, 1ꢂ50 mL), saturated aq. NaHCO3 solution (1ꢂ50 mL),
brine (1ꢂ50 mL), dried (MgSO4), filtered and concentrated. Flash
chromatography of the resultant residue (EtOAc/hexane 3:7) yield-
ed 2 as a colourless oil (2.5 g, 80%). Rf =0.4 (EtOAc/hexane 1:4);
1H NMR (500 MHz, CDCl3): d=5.41 (ddd, J=1.6, 1.6, 10.0 Hz, 1H),
4.93 (dd, J=9.2, 9.2 Hz, 1H), 4.82 (dd, J=1.6, 1.6 Hz, 1H), 4.59 (dd,
J=1.6, 1.6 Hz, 1H), 4.37 (d, J=7.2 Hz, 1H), 3.59 (s, 3H), 2.08 (s, 3H),
2.07 ppm (s, 3H); 13C NMR (125 MHz, CDCl3): d=169.6, 169.3, 103.2,
97.5, 71.4, 69.2, 63.4, 57.3, 20.6 ppm; nmax =2108 cmÀ1 (N3); HRMS:
m/z calcd: 286.1039 [M+H]+, found: 286.1028; elemental analysis
calcd (%) for C11H15N3O6: C 46.32, H 5.30, N 14.73; found: C 46.28,
H 5.33, N 14.80.
these compounds reduces MIC values for b-lactams against
a clinically relevant Gram-negative bacterium bearing inducible
chromosomal AmpC b-lactamase, P. aeruginosa. Using a struc-
ture of a NagZ–inhibitor complex we provide insight into the
molecular basis for the selectivity and potency of this inhibitor.
It is anticipated that further structure-guided refinement will
lead to candidates with increased potency for NagZ and, using
the strategy outlined here, systematic elaboration of other
b-N-acetylglucosaminidase inhibitor scaffolds might also yield
selective and potent inhibitors for NagZ.
Experimental Section
1
General methods for chemical synthesis: H and 13C NMR spectra
1
were recorded on a Bruker ARX500 (500 MHz for H and 125 MHz
for 13C) or a Bruker AV600 (600 MHz for 1H and 150 MHz for 13C;
chemical shifts quoted relative to CHCl3 for CDCl3 and CH3OH for
D2O where appropriate) spectrometer. Mass spectra were recorded
with a VG-Autospec spectrometer using the fast atom bombard-
ment technique, with 3-nitrobenzyl alcohol as a matrix. Elemental
analyses of all synthesised compounds used in enzyme assays
were performed at the Australian National University Microanalyti-
cal Facility. Flash chromatography was performed on silica gel
(BDH) with the specified solvents. Thin-layer chromatography (TLC)
was effected on Merck silica gel 60 F254 aluminium-backed plates
that were stained by heating (>2008C) with 5% sulfuric acid in
EtOH. Percentage yields for chemical reactions as described are
quoted only for those compounds that were purified by recrystalli-
zation or by column chromatography, and purity was assessed
General procedure for formation of methyl 3,4-di-O-acetyl-2-
acylamido-2,6-dideoxy-b-d-xylo-hex-5-enosides (6–12): Tributyl
phosphine (0.2 mL, 0.8 mmol) was added to azide 2 (200 mg,
0.7 mmol) in a solution of THF (5 mL) and H2O (0.5 mL) at 08C and
the solution stirred. This was followed by the addition of the
appropriate acyl anhydride (3 equiv) and the mixture stirred (5 h).
Concentration followed by flash chromatography of the residue
(EtOAc/hexane 7:3) gave the desired compounds 6–12 in yields
ranging from 52% to 73%.
Methyl 2-acetamido-3,4-di-O-acetyl-2,6-dideoxy-b-d-xylo-hex-5-
1
enoside (6): Yield: 60%. H and 13C NMR spectra were consistent
with those found in the literature.[41]
Methyl 3,4-di-O-acetyl-2,6-dideoxy-2-propamido-b-d-xylo-hex-5-
enoside (7): Yield: 73%; Rf =0.3 (EtOAc/hexane 7:3); 1H NMR
(600 MHz, CDCl3): d=5.81 (d, J=8.8 Hz, 1H), 5.61 (ddd, J=1.3, 1.3,
7.5 Hz, 1H), 4.94 (dd, J=6.0, 7.5 Hz, 1H), 4.78 (dd, J=1.3, 1.3 Hz,
1H), 4.66 (d, J=4.0 Hz, 1H), 4.57 (dd, J=1.3, 1.3 Hz, 1H), 4.24 (ddd,
J=4.0, 6.0, 8.8 Hz, 1H), 3.48 (s, 3H), 2.24–2.19 (m, 2H), 2.11 (s, 3H),
2.06 (s, 3H), 1.14 ppm (t, J=7.5 Hz, 3H); 13C NMR (150 MHz, CDCl3):
d=173.3, 170.4, 168.9, 150.5, 102.3, 97.3, 71.4, 68.6, 56.2, 52.1, 29.6,
20.8, 20.7, 9.5 ppm; HRMS: m/z calcd: 316.1396 [M+H]+, found:
316.1371; elemental analysis calcd (%) for C14H21NO7: C 53.33, H
6.71, N 4.44; found: C 53.30, H 6.66, N 4.51.
1
using TLC or H NMR spectroscopy.
Methyl 3,4-di-O-acetyl-2-azido-2-deoxy-6-iodo-b-d-glucopyrano-
side (5): p-Toluenesulfonyl chloride (3.9 g, 20 mmol) was added to
a solution of azide 4[45] (4.0 g, 18 mmol) in pyridine (20 mL) and
CH2Cl2 (80 mL) and the solution was stirred (RT, 6 h). The reaction
was quenched by the addition of water and the resultant mixture
stirred (1 h). The organic layer was then collected and was washed
with water (1ꢂ50 mL), aq. HCl (1m, 1ꢂ50 mL), saturated aq.
NaHCO3 solution (1ꢂ50 mL), brine (1ꢂ20 mL), dried (MgSO4), fil-
tered and concentrated. The residue was then dissolved in DMF
(60 mL) and to it was added NaI (8.0 g, 53 mmol) and the resultant
mixture stirred (1008C, 4 h). The mixture was then concentrated
and the resultant residue diluted with EtOAc (150 mL) and was
washed with water (2ꢂ20 mL), brine (1ꢂ20 mL), dried (MgSO4), fil-
tered and concentrated. The residue was then dissolved in a mix-
ture of CH2Cl2 (30 mL) and pyridine (10 mL) and Ac2O (10 mL) was
added. The resultant solution was left at RT overnight. The mixture
was quenched with MeOH and concentrated. The resultant residue
was then diluted with CH2Cl2 (100 mL) and washed with water (2ꢂ
20 mL), aq. HCl (1m, 1ꢂ50 mL), saturated aq. NaHCO3 solution (1ꢂ
50 mL), brine (1ꢂ20 mL), dried (MgSO4), filtered and concentrated.
Flash chromatography of the resultant residue (EtOAc/hexane 3:7)
yielded 5 as a colourless oil (4.8 g, 64%, over three steps). Rf =0.7
Methyl 3,4-di-O-acetyl-2-butamido-2,6-dideoxy-b-d-xylo-hex-5-
enoside (8): Yield: 70%; Rf =0.4 (EtOAc/hexane 7:3); 1H NMR
(600 MHz, CDCl3): d=5.76 (d, J=9.3 Hz, 1H), 5.61 (d, J=7.5 Hz,
1H), 4.95 (dd, J=6.3, 6.4 Hz, 1H), 4.79 (s, 1H), 4.66 (d, J=4.1 Hz,
1H), 4.57 (s, 1H), 4.24 (ddd, J=4.1, 7.5, 9.3 Hz, 1H), 3.48 (s, 3H),
2.16 (t, J=8.0 Hz, 2H), 2.12 (s, 3H), 2.06 (s, 3H), 1.68–1.63 (m, 2H),
0.94 ppm (t, J=7.3 Hz, 3H); 13C NMR (150 MHz, CDCl3): d=172.5,
170.4, 169.0, 150.6, 102.4, 97.2, 71.4, 68.6, 56.2, 52.2, 38.6, 20.84,
20.8, 18.9, 13.6 ppm; HRMS: m/z calcd: 330.1553 [M+H]+, found:
330.1564; elemental analysis calcd (%) for C15H23NO7: C 54.70, H
7.04, N 4.25; found: C 54.65, H 7.07, N 4.33.
Methyl 3,4-di-O-acetyl-2,6-dideoxy-2-valeramido-b-d-xylo-hex-5-
enoside (9): Yield: 67%; Rf =0.5 (EtOAc/hexane 7:3); 1H NMR
(600 MHz, CDCl3): d=5.79 (d, J=8.6 Hz, 1H), 5.60 (d, J=7.5 Hz,
1H), 4.95 (dd, J=6.4, 7.3 Hz, 1H), 4.78 (s, 1H), 4.65 (d, J=4.1 Hz,
1H), 4.57 (s, 1H), 4.23 (ddd, J=4.1, 6.4, 8.6 Hz, 1H), 3.48 (s, 3H),
2.19–2.16 (m, 2H), 2.12 (s, 3H), 2.05 (s, 3H), 1.61–1.56 (m, 2H),
1.36–1.29 (m, 2H), 0.90 ppm (t, J=6.8 Hz, 3H); 13C NMR (150 MHz,
CDCl3): d=172.7, 170.4, 169.0, 150.6, 102.4, 97.2, 71.4, 68.6, 56.2,
52.2, 36.7, 27.5, 22.2, 20.82, 20.8, 13.7 ppm; HRMS: m/z calcd:
344.1709 [M+H]+, found: 344.1722; elemental analysis calcd (%)
1
(EtOAc/hexane 2:3); H NMR (600 MHz, CDCl3): d=4.98 (dd, J=9.3,
9.3 Hz, 1H), 4.80 (dd, J=9.5, 9.5 Hz, 1H), 4.31 (d, J=8.0 Hz, 1H),
3.63 (s, 3H), 3.49–3.44 (m, 2H), 3.29 (dd, J=2.7, 11.0 Hz, 1H), 3.14
(dd, J=8.4, 11.0 Hz, 1H), 2.07 (s, 3H), 2.04 ppm (s, 3H); 13C NMR
(150 MHz, CDCl3): d=169.9, 169.6, 102.5, 73.2, 72.3, 72.2, 63.8, 57.4,
20.64, 20.63, 2.8 ppm; nmax =2114 cmÀ1 (N3); HRMS: m/z calcd:
414.0162 [M+H]+, found: 414.0156; elemental analysis calcd (%)
for C11H16IN3O6: C 31.98, H 3.90, N 10.17; found: C 31.93, H 3.92, N
10.15.
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for C16H25NO7: C 55.97, H 7.34, N 4.08; found: C 55.89, H 7.39, N
4.09.
7.6 Hz, 3H); 13C NMR (125 MHz, CD3OD): d=177.4, 140.0, 129.2,
129.0, 128.3, 102.9, 101.9, 76.0, 73.6, 64.6, 62.3, 56.8, 56.3, 30.4,
10.3 ppm; HRMS: m/z calcd: 356.1709 [M+H]+, found: 356.1711; el-
emental analysis calcd (%) for C17H25NO7: C 57.45, H 7.09, N 3.94;
found: C 57.28, H 7.01, N 3.99.
Methyl 3,4-di-O-acetyl-2,6-dideoxy-2-hexamido-b-d-xylo-hex-5-
enoside (10): Yield: 52%; Rf =0.7 (EtOAc/hexane 7:3); 1H NMR
(500 MHz, CDCl3): d=5.76 (d, J=8.7 Hz, 1H), 5.60 (dt, J=1.1, 1.1,
7.6 Hz, 1H), 4.95 (dd, J=6.3, 7.4 Hz, 1H), 4.78 (d, J=1.1 Hz, 1H),
4.65 (d, J=4.1 Hz, 1H), 4.57 (d, J=1.1 Hz, 1H), 4.24 (ddd, J=4.1,
6.3, 8.7 Hz, 1H), 3.49 (s, 3H), 2.19–2.16 (m, 2H), 2.12 (s, 3H), 2.06 (s,
3H), 1.65–1.59 (m, 4H), 1.35–1.25 (m, 4H), 0.89 ppm (t, J=6.8 Hz,
3H); 13C NMR (125 MHz, CDCl3): d=172.7, 170.4, 169.0, 150.6,
102.4, 97.2, 71.4, 68.6, 56.2, 52.2, 36.6, 31.3, 25.2, 22.3, 20.84, 20.8,
13.9 ppm; HRMS: m/z calcd: 358.1866 [M+H]+, found: 358.1857;
elemental analysis calcd (%) for C17H27NO7: C 57.13, H 7.61, N 3.92;
found: C 57.01, H 7.53, N 3.99.
Methyl (5R/S)-5-C-benzyloxy-2-butamido-2-deoxy-b-d-xylo-hexo-
pyranoside (15): Yield: 53%. (5S) epimer—Rf =0.25 (EtOAc);
1H NMR (500 MHz, CD3OD): d=7.45–7.41 (m, 2H), 7.32–7.28 (m,
2H), 7.25–7.22 (m, 1H), 4.75 (ABq, J=9.0 Hz, 1H), 4.71–4.68 (m,
2H), 4.03 (dd, J=7.4, 9.6 Hz, 1H), 3.90–3.83 (m, 3H), 3.71 (dd, J=
8.1, 9.3 Hz, 1H), 3.44 (s, 3H), 2.20 (t, J=6.4 Hz, 2H), 1.69–1.62 (m,
2H), 0.96 ppm (t, J=7.4 Hz, 3H); 13C NMR (125 MHz, CD3OD): d=
176.5, 140.0, 129.1, 129.0, 128.3, 102.8, 101.9, 76.1, 73.5, 64.6, 62.3,
56.8, 56.3, 39.3, 20.3, 13.9 ppm; HRMS: m/z calcd: 370.1866
[M+H]+, found: 370.1889; elemental analysis calcd (%) for
C18H27NO7: C 58.52, H 7.37, N 3.79; found: C 58.49, H 7.51, N 3.71.
Methyl 3,4-di-O-acetyl-2,6-dideoxy-2-isobutamido-b-d-xylo-hex-
5-enoside (11): Yield: 54%; Rf =0.45 (EtOAc/hexane 7:3); 1H NMR
(500 MHz, CDCl3): d=5.85 (d, J=8.4 Hz, 1H), 5.60 (d, J=7.5 Hz,
1H), 4.95 (dd, J=6.3, 6.3 Hz, 1H), 4.79 (s, 1H), 4.65 (d, J=4.0 Hz,
1H), 4.57 (s, 1H), 4.22 (ddd, J=4.0, 6.3, 8.4 Hz, 1H), 3.48 (s, 3H),
2.38–2.33 (m, 1H), 2.12 (s, 3H), 2.05 (s, 3H), 1.15 (d, J=6.9 Hz, 3H),
1.14 ppm (d, J=6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3): d=176.5,
170.4, 168.9, 150.5, 102.4, 97.5, 71.3, 68.6, 56.2, 51.9, 35.5, 20.83,
20.8, 19.4, 19.3 ppm; HRMS: m/z calcd: 330.1553 [M+H]+, found:
330.1570; elemental analysis calcd (%) for C15H23NO7: C 54.70, H
7.04, N 4.25; found: C 54.63, H 7.09, N 4.28.
Methyl (5R/S)-5-C-benzyloxy-2-deoxy-2-valeramido-b-d-xylo-hex-
opyranoside (16): Yield: 68%. (5S) epimer—Rf =0.3 (EtOAc);
1H NMR (600 MHz, CD3OD): d=7.45–7.43 (m, 2H), 7.32–7.28 (m,
2H), 7.25–7.22 (m, 1H), 4.76 (ABq, J=10.9 Hz, 1H), 4.71–4.68 (m,
2H), 4.02 (dd, J=7.3, 9.6 Hz, 1H), 3.89–3.82 (m, 3H), 3.71 (dd, J=
8.1, 9.3 Hz, 1H), 3.44 (s, 3H), 2.24–2.20 (m, 2H), 1.62–1.58 (m, 2H),
1.40–1.35 (m, 2H), 0.94 ppm (t, J=7.3 Hz, 3H); 13C NMR (150 MHz,
CD3OD): d=176.7, 140.0, 129.2, 129.0, 128.3, 102.9, 101.9, 76.1,
73.6, 64.6, 62.3, 56.8, 56.3, 37.2, 29.1, 23.2, 14.1 ppm; HRMS: m/z
calcd: 384.2022 [M+H]+, found: 384.2010; elemental analysis calcd
(%) for C19H29NO7: C 59.52, H 7.62, N 3.65; found: C 59.64, H 7.59, N
3.61.
Methyl 3,4-di-O-acetyl-2,6-dideoxy-2-isovalermido-b-d-xylo-hex-
5-enoside (12): Yield: 68%; Rf =0.5 (EtOAc/hexane 7:3); 1H NMR
(600 MHz, CDCl3): d=5.75 (d, J=8.9 Hz, 1H), 5.60 (dt, J=1.3, 1.3,
7.6 Hz, 1H), 4.96 (dd, J=6.5, 7.5 Hz, 1H), 4.79 (d, J=1.3 Hz, 1H),
4.64 (d, J=4.2 Hz, 1H), 4.57 (d, J=1.3 Hz, 1H), 4.24 (ddd, J=4.2,
6.5, 8.9 Hz, 1H), 3.48 (s, 3H), 2.12 (s, 3H), 2.10–2.04 (m, 3H), 2.06 (s,
3H), 0.96–0.93 ppm (m, 6H); 13C NMR (150 MHz, CDCl3): d=172.1,
170.4, 169.0, 150.6, 102.5, 97.1, 71.4, 68.7, 56.2, 52.2, 46.0, 26.1,
22.35, 22.3, 20.82, 20.8 ppm; HRMS: m/z calcd: 344.1709 [M+H]+,
found: 344.1729; elemental analysis calcd (%) for C16H25NO7: C
55.97, H 7.34, N 4.08; found: C 56.02, H 7.41, N 4.04.
Methyl (5R/S)-5-C-benzyloxy-2-deoxy-2-hexamido-b-d-xylo-hexo-
1
pyranoside (17): Yield: 61%. (5S) epimer—Rf =0.4 (EtOAc); H NMR
(500 MHz, CD3OD): d=7.45–7.41 (m, 2H), 7.32–7.28 (m, 2H), 7.25–
7.21 (m, 1H), 4.76–4.70 (m, 3H), 4.03 (dd, J=7.2, 9.5 Hz, 1H), 3.90–
3.84 (m, 3H), 3.73 (dd, J=8.1, 9.6 Hz, 1H), 3.44 (s, 3H), 2.24–2.20
(m, 2H), 1.66–1.59 (m, 2H), 1.40–1.30 (m, 4H), 0.91 ppm (t, J=
6.8 Hz, 3H); 13C NMR (125 MHz, CD3OD): d=176.7, 140.0, 129.1,
129.0, 128.3, 102.8, 101.8, 76.0, 73.4, 64.5, 62.3, 56.8, 56.3, 37.3,
32.3, 26.6, 23.4, 14.3 ppm; HRMS: m/z calcd: 398.2179 [M+H]+,
found: 398.2171; elemental analysis calcd (%) for C20H31NO7: C
60.44, H 7.86, N 3.52; found: C 60.51, H 7.93, N 3.51.
General procedure for formation of methyl (5R/S)-2-acylamino-
5-C-benzyloxy-2-deoxy-b-d-xylo-hexopyranosides (13–19): To
a 1% solution of 6–12 (0.5 mmol) in a mixture of CH2Cl2/benzyl al-
cohol (1:1 v/v, 12 mL), 3-chloroperbenzoic acid (70%, 0.6 mmol)
was added, and the mixture was stirred (2 h). The mixture was
then diluted with CH2Cl2 (20 mL) and washed with saturated aq.
NaHCO3 solution (1ꢂ50 mL), dried (MgSO4), filtered and concen-
trated. The resulting residue was chromatographed (EtOAc) to
yield a residue that was dissolved in MeOH and treated with
sodium methoxide (10 mg) and the solution stirred (RT, 30 min).
The mixture was quenched with resin (Amberlite IR-120, H+), fil-
tered and concentrated to give 13–19 as a mixture of diastereo-
mers at C-5 from which the major (5S) epimer was purified by
flash chromatography (MeOH/CHCl3 1:9) in yields ranging from
53% to 68% over two steps.
Methyl
(5R/S)-5-C-benzyloxy-2-deoxy-2-isobutamido-b-d-xylo-
hexopyranoside (18): Yield: 55%. (5S) epimer—Rf =0.3 (EtOAc);
1H NMR (500 MHz, CD3OD): d=7.45–7.42 (m, 2H), 7.32–7.28 (m,
2H), 7.25–7.21 (m, 1H), 4.76 (ABq, J=11.0 Hz, 1H), 4.71–4.68 (m,
2H), 4.00 (dd, J=6.9, 9.3 Hz, 1H), 3.90–3.82 (m, 3H), 3.73 (dd, J=
7.8, 9.3 Hz, 1H), 3.44 (s, 3H), 2.48–2.42 (m, 1H), 1.14 (d, J=6.7 Hz,
3H), 1.13 ppm (d, J=6.7 Hz, 3H); 13C NMR (125 MHz, CD3OD): d=
180.5, 140.0, 129.2, 129.0, 128.3, 102.9, 102.0, 76.0, 73.5, 64.6, 62.2,
56.8, 56.2, 36.5, 20.0, 19.8 ppm; HRMS: m/z calcd: 370.1866
[M+H]+, found: 370.1872; elemental analysis calcd (%) for
C18H27NO7: C 58.52, H 7.37, N 3.79; found: C 58.50, H 7.31, N 3.75.
Methyl (5R/S)-5-C-benzyloxy-2-deoxy-2-isovaleramido-b-d-xylo-
hexopyranoside (19): Yield: 64%. (5S) epimer—Rf =0.4 (EtOAc);
1H NMR (600 MHz, CD3OD): d=7.45–7.42 (m, 2H), 7.33–7.29 (m,
2H), 7.25–7.21 (m, 1H), 4.75 (ABq, J=11.0 Hz, 1H), 4.72–4.69 (m,
2H), 4.04 (dd, J=7.4, 9.7 Hz, 1H), 3.90–3.83 (m, 3H), 3.72 (dd, J=
8.1, 9.5 Hz, 1H), 3.44 (s, 3H), 2.12–2.05 (m, 3H), 0.98–0.94 ppm (m,
6H); 13C NMR (150 MHz, CD3OD): d=176.0, 140.0, 129.1, 129.0,
128.3, 102.8, 101.8, 76.1, 73.5, 64.6, 62.3, 56.7, 56.2, 46.7, 27.5, 22.8,
22.6 ppm; HRMS: m/z calcd: 384.2022 [M+H]+, found: 384.2024,
elemental analysis calcd (%) for C19H29NO7: C 59.52, H 7.62, N 3.65;
found: C 59.60, H 7.65, N 3.70.
Methyl (5R/S)-2-acetamido-5-C-benzyloxy-2-deoxy-b-d-xylo-hex-
1
opyranoside (13): Yield: 68%. H and 13C NMR spectra of the (5S)
epimer were consistent with those found in the literature.[41]
Methyl (5R/S)-5-C-benzyloxy-2-deoxy-2-propamido-b-d-xylo-hex-
opyranoside (14): Yield: 64%. (5S) epimer—Rf =0.2 (EtOAc);
1H NMR (500 MHz, CD3OD): d=7.45–7.42 (m, 2H), 7.32–7.28 (m,
2H), 7.25–7.23 (m, 1H), 4.76 (ABq, J=10.9 Hz, 1H), 4.71–4.68 (m,
2H), 4.02 (dd, J=7.0, 9.7 Hz, 1H), 3.89–3.83 (m, 3H), 3.71 (dd, J=
7.9, 9.6 Hz, 1H), 3.44 (s, 3H), 2.26–2.21 (m, 2H), 1.12 ppm (t, J=
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General procedure for formation of 2-acylamido-1,5-imino-1,2,5-
trideoxy-d-glucitols (1, 20–25): To a solution of 13–19 in MeOH/
H2O (15:1 v/v, 0.03m), NH4HCO2 (1 equiv) and 20% Pd(OH)2/C
(0.1 equiv) were added, and the heterogeneous reaction mixture
was stirred under hydrogen at RT and ambient pressure (1 atm,
48 h). After filtration and evaporation, the resulting residue was
chromatographed (CHCl3/MeOH/conc. NH3 12:8:1) to yield the
desired compounds 1, 20–25 in yields ranging from 48 to 63%.
48.8, 36.4, 20.1, 19.8 ppm; HRMS: m/z calcd: 233.1501 [M+H]+,
found: 233.1511; elemental analysis calcd (%) for C10H20N2O4: C
51.71, H 8.68, N 12.06; found: C 51.83, H 8.51, N 12.16.
1,5-Imino-2-isovaleramido-1,2,5-trideoxy-d-glucitol (25): Yield:
63%; Rf =0.5 (CHCl3/MeOH/conc. NH3 12:8:1); 1H NMR (600 MHz,
CD3OD): d=3.81–3.74 (m, 2H), 3.64 (dd, J=6.1, 10.5 Hz, 1H), 3.27–
3.20 (m, 2H), 3.10 (dd, J=4.4, 12.3 Hz, 1H), 2.47–2.43 (m, 1H), 2.39
(dd, J=11.7, 11.7 Hz, 1H), 2.11–2.03 (m, 3H), 0.94 ppm (m, 6H);
13C NMR (150 MHz, CD3OD): d=175.9, 77.7, 74.1, 62.9, 62.8, 53.8,
48.8, 46.5, 27.4, 22.8, 22.6 ppm; HRMS: m/z calcd: 247.1658
[M+H]+, found: 247.1661; elemental analysis calcd (%) for
C11H22N2O4: C 53.64, H 9.00, N 11.37; found: C 53.59, H 8.98, N
11.31.
2-Acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol (1): Yield: 58%.
1H and 13C NMR spectra were consistent with those found in the
literature.[39]
1,5-Imino-2-propamido-1,2,5-trideoxy-d-glucitol (20): Yield: 52%;
Rf =0.25 (CHCl3/MeOH/conc. NH3 12:8:1); 1H NMR (600 MHz,
CD3OD): d=3.86–3.80 (m, 2H), 3.71 (dd, J=5.9, 11.4 Hz, 1H), 3.38–
3.34 (m, 2H), 3.21 (dd, J=4.7, 12.5 Hz, 1H), 2.71–2.66 (m, 1H), 2.56
(dd, J=11.9, 11.9 Hz, 1H), 2.26–2.20 (m, 2H), 1.12 ppm (t, J=
7.6 Hz, 3H); 13C NMR (150 MHz, CD3OD): d=177.5, 76.7, 72.6, 62.5,
61.4, 52.3, 47.8, 30.1, 10.3 ppm; HRMS: m/z calcd: 219.1345
[M+H]+, found: 219.1344; elemental analysis calcd (%) for
C9H18N2O4: C 49.53, H 8.31, N 12.84; found: C 49.51, H 8.26, N
12.89.
Kinetic analysis of inhibitors: Assays against O-GlcNAcase,
VcNagZ and StNagZ were performed in NaPi buffer (50 mm, NaCl
(100 mm), pH 6.5) and for b-hexosaminidase B citrate buffer
(50 mm, NaCl (100 mm), pH 4.25) using 4-methylumbelliferyl N-
acetyl-b-d-glucosaminide as substrate. For NAGLU, assays were
performed in acetate buffer (100 mm, pH 4.3), containing bovine
serum albumin (0.5 mgmLÀ1) using 4-methylumbelliferyl N-acetyl-
a-d-glucosaminide as substrate. Release of 4-methylumbelliferone
was monitored continuously using a fluorimeter plate reader for
OGA, VcNagZ and StNagZ, and a 30 min stopped-assay procedure
was used for b-hexosaminidase B and NAGLU (quenched with four-
fold excess of quenching buffer, glycine (200 mm), pH 10.75). For
OGA, the inhibitors were preincubated with the enzyme for 10 min
before the addition of the substrate. Readings were taken at exci-
tation and emission wavelengths of 368 nm and 450 nm respec-
tively. Assays contained substrate at the previously determined Km
value of the substrate for the enzyme, and the enzyme typically at
a concentration of 100–200 nm. Inhibitors were added at a range
of concentrations encompassing their Ki values. The rates at each
inhibitor concentration were plotted and the best fit line through
the points ascertained. The À1/Ki was taken as the point where
2-Butamido-1,5-imino-1,2,5-trideoxy-d-glucitol (21): Yield: 63%;
1
Rf =0.4 (CHCl3/MeOH/conc. NH3 12:8:1); H NMR (500 MHz, CD3OD):
d=3.90–3.85 (m, 2H), 3.75 (dd, J=5.3, 11.5 Hz, 1H), 3.48 (dd, J=
9.7, 9.7 Hz, 1H), 3.43 (dd, J=9.4, 9.4 Hz, 1H), 3.21 (dd, J=4.8,
12.7 Hz, 1H), 2.81–2.76 (m, 1H), 2.64 (dd, J=11.9, 11.9 Hz, 1H),
2.28–2.20 (m, 2H), 1.65–1.59 (m, 2H), 0.91 ppm (t, J=7.5 Hz, 3H);
13C NMR (125 MHz, CD3OD): d=178.4, 75.9, 71.7, 61.2, 60.9, 51.6,
47.0, 38.7, 19.9, 13.6 ppm; HRMS: m/z calcd: 233.1501 [M+H]+,
found: 233.1516; elemental analysis calcd (%) for C10H20N2O4: C
51.71, H 8.68, N 12.06; found: C 51.77, H 8.59, N 12.13.
1,5-Imino-1,2,5-trideoxy-2-valeramido-d-glucitol (22): Yield: 61%;
1
Rf =0.5 (CHCl3/MeOH/conc. NH3 12:8:1); H NMR (500 MHz, CD3OD):
the line of best fit intersected with 1/Vmax
.
d=3.82 (dd, J=2.9, 11.1 Hz, 1H), 3.77 (ddd, J=4.7, 10.6, 11.0 Hz,
1H), 3.65 (dd, J=6.0, 11.1 Hz, 1H), 3.30–3.25 (m, 2H), 3.14 (dd, J=
4.8, 12.4 Hz, 1H), 2.56–2.51 (m, 1H), 2.44 (dd, J=12.1, 12.1 Hz, 1H),
2.25–2.19 (m, 2H), 1.63–1.56 (m, 2H), 1.40–1.34 (m, 2H), 0.93 ppm
(t, J=7.3 Hz, 3H); 13C NMR (125 MHz, CD3OD): d=176.7, 77.3, 73.5,
62.7, 62.3, 53.2, 48.7, 36.9, 29.1, 23.3, 14.1 ppm; HRMS: m/z calcd:
247.1658 [M+H]+, found: 247.1651; elemental analysis calcd (%)
for C11H22N2O4: C 53.64, H 9.00, N 11.37; found: C 53.57, H 9.03, N
11.29.
NagZ crystallization, structure determination and refinement:
StNagZ plasmid construction, expression and purification have
been previously described.[20] StNagZ crystals were grown at RT
using the hanging drop vapor-diffusion method by mixing equal
volumes of reservoir buffer (25% PEG1000, MES (0.1m), pH 6.5)
and protein solution (6 mgmLÀ1) in crystallization buffer (NaCl
(150 mm), BisTris (20 mm), pH 6.5). A single StNagZ crystal was
soaked overnight in a drop containing reservoir buffer and 21 at
a concentration of 30 mm to obtain the protein–inhibitor complex.
The 25% PEG1000 present in the buffer was sufficient for cryo-pro-
tection, and crystals were harvested by flash-cooling in liquid nitro-
gen. X-ray diffraction data were collected at beamline 08ID-1 at
the Canadian Light Source (Saskatoon, Canada). Diffraction data
were integrated using XDS[51] and scaled and merged using
SCALA[52] (see Table S1). The structure was solved by molecular re-
placement using the program PHASER[53] and the crystal structure
of native unliganded StNagZ (PDB ID: 4GVG). Subsequent rounds
of refinement were performed using phenix.refine and COOT.[54,55]
A ligand restraint file was generated for 21 using PHENIX eLBOW
and the inhibitor was initially fit into electron density using PHENIX
Ligandfit.[54] Solvent molecules were added using phenix.refine and
final refinement was performed using COOT and phenix.refine. Ste-
reochemical quality of the final model was assessed by using Mol-
Probity.[56] The final refinement statistics are presented in Table S1.
2-Hexamido-1,5-imino-1,2,5-trideoxy-d-glucitol (23): Yield: 48%;
Rf =0.55 (CHCl3/MeOH/conc. NH3 12:8:1); 1H NMR (600 MHz,
CD3OD): d=3.81 (dd, J=3.1, 11.2 Hz, 1H), 3.74 (ddd, J=4.7, 11.0,
11.0 Hz, 1H), 3.64 (dd, J=4.1, 11.1 Hz, 1H), 3.28–3.23 (m, 2H), 3.11
(dd, J=4.7, 12.3 Hz, 1H), 2.50–2.47 (m, 1H), 2.40 (dd, J=11.9,
11.9 Hz, 1H), 2.25–2.19 (m, 2H), 1.64–1.59 (m, 2H), 1.37–1.29 (m,
4H), 0.90 ppm (t, J=6.8 Hz, 3H); 13C NMR (150 MHz, CD3OD): d=
176.7, 77.5, 73.8, 62.7, 62.6, 53.5, 48.9, 37.2, 32.5, 26.7, 23.4,
14.3 ppm; HRMS: m/z calcd: 261.1814 [M+H]+, found: 261.1807;
elemental analysis calcd (%) for C12H24N2O4: C 55.36, H 9.29, N
10.76; found: C 55.31, H 9.25, N 10.68.
1,5-Imino-2-isobutamido-1,2,5-trideoxy-d-glucitol (24): Yield:
54%; Rf =0.4 (CHCl3/MeOH/conc. NH3 12:8:1); 1H NMR (500 MHz,
CD3OD): d=3.81 (dd, J=3.0, 11.2 Hz, 1H), 3.71 (ddd, J=4.8, 10.9,
10.9 Hz, 1H), 3.64 (dd, J=5.0, 11.2 Hz, 1H), 3.34–3.30 (m, 1H), 3.24
(dd, J=9.6, 9.6 Hz, 1H), 3.07 (dd, J=4.9, 12.4 Hz, 1H), 2.50–2.44 (m,
2H), 2.39 (dd, J=12.3, 12.3 Hz, 1H), 1.10 ppm (t, J=7.8 Hz, 3H);
13C NMR (125 MHz, CD3OD): d=180.9, 77.5, 73.8, 62.8, 62.5, 53.6,
Determination of the Minimal Inhibitory Concentration of b-lac-
tams: Cultures were prepared by inoculating Mueller–Hinton broth
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2013, 14, 1973 – 1981 1979

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K.A.S. thanks the Australian Research Council for support and the
Centre for Microscopy, Characterization and Analysis, The Univer-
sity of Western Australia, which are supported by University, State
and Federal Government funding. D.J.V. and B.L.M. thank the
Canadian Institutes of Health Research (MOP 97818) and Cystic
Fibrosis Canada for funding. J.P.B. was supported by a postdoctor-
al fellowship from the Manitoba Health Research Council
(MHRC). B.L.M. holds a Manitoba Research Chair in Structural
Biology. D.J.V. is a Canada Research Chair in Chemical Glycobiol-
ogy and a EWR Steacie Memorial Fellow. G.E.W. was supported
by a fellowship from the Natural Sciences and Engineering Re-
search Council of Canada (NSERC) and T.M.G. was supported by
a Wellcome Trust Sir Henry Wellcome Postdoctoral fellowship
and a research trainee award from MSFHR. We also thank Veron-
ica Larmour for technical assistance and Shaun Labiuk and the
staff of the Canadian Light Source (CLS) beamline 08ID-1 for as-
sistance with data collection. The CLS is supported by the NSERC,
National Research Council, the Canadian Institutes of Health Re-
search (CIHR), and the University of Saskatchewan.
Keywords: carbohydrates · enzymes · inhibitors · selectivity ·
synthesis
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ChemBioChem 2013, 14, 1973 – 1981 1981