Synthesis of bivalent glycoclusters containing GlcNAc as hexasaccharide mimetics. Bactericidal activity against Helicobacter pylori

Article abstract of DOI:10.1016/j.carres.2012.07.011

The Cu(I) catalysed cycloaddition reaction of azides and alkynes has been used to generate a series of divalent GlcNAc clusters with both α and β configurations. These glycoclusters can be considered as potential mimetics of an anti Helicobacter pylori he

Full text of DOI:10.1016/j.carres.2012.07.011

Carbohydrate Research 360 (2012) 1–7
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of bivalent glycoclusters containing GlcNAc as hexasaccharide
mimetics. Bactericidal activity against Helicobacter pylori
Dandan Yan a,b, Julie Naughton c, Marguerite Clyne c, Paul V. Murphy a,
⇑
a School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland
b School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
c UCD School of Medicine and Medical Science and UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 June 2012
Received in revised form 16 July 2012
Accepted 16 July 2012
Available online 27 July 2012
The Cu(I) catalysed cycloaddition reaction of azides and alkynes has been used to generate a series of
divalent GlcNAc clusters with both
potential mimetics of an anti Helicobacter pylori hexasaccharide as they present two GlcNAc residues
grafted onto a core scaffold. Two bivalent compounds based on -O-GlcNAc were identified that selec-
a and b configurations. These glycoclusters can be considered as
a
tively reduced the viability of H. pylori. These compounds showed activity towards different strains of
H. pylori (Pu4 vs P12). The activity of the oligosaccharide mimetics is speculated to be due to the GlcNAc
residues being able to adopt spatial arrangements accessible to the anti H. pylori hexasaccharide which
may be important for activity.
Keywords:
Glycoclusters
GlcNAc
Glycomimetics
Helicobacter pylori
Bactericidal
Antibiotic
Ó 2012 Elsevier Ltd. All rights reserved.
Helicobacter pylori is one of the commonest infections of man-
kind. It colonizes the gastric mucosa of more than 50% of the
world’s population and is a leading cause of duodenal ulceration,
gastric carcinoma and gastric malignant lymphoma of mucosa-
associated lymphoid tissue.1 However, most infected individuals
are asymptomatic which suggests that the host defence mounted
against the organism is capable of limiting the pathology caused
by the infection. H. pylori is rarely found in deeper portions of
the gastric mucosa, where O-glycans are expressed which contain
designed and synthesized a series of bivalent GlcNAc derivatives
(1–8, Fig. 1) where two GlcNAc residues are grafted to non carbo-
hydrate scaffolds as glycocluster6 mimetics of the oligosaccharide
and report on their bactericidal activity against H. pylori. Although
the native hexasaccharide contains the
a-glycosidic linkage we
included compounds where the GlcNAc residues also contain
b-linkages.
The synthesis of 1–3 began from b-azide 9, which was prepared
from N-acetyl-D
-glucosamine as previously described.7 The bis-
terminal
that these O-glycans have antimicrobial activity against H. pylori.2
They inhibit bacterial cholesterol -glucosyltransferase, thereby
preventing the biosynthesis of -glucosyl cholesterol, a major con-
stituent of the cell wall of H. pylori.2,3 Thus, unique
-GlcNAc deriv-
atives could have potential in protecting hosts from H. pylori
a-1,4-linked N-acetylglucosamine. Lee et al. have shown
propargyl derivative 10 was prepared8 from 1,4-dihydroxybenzene
and similar conditions were used to give 11 and 12 from the appro-
priate dihydroxylated aromatic precursors. Then the azide 9 was
coupled with the bisacetylenes 10–12 using the copper catalysed
azide–alkyne cycloaddition reaction (CuAAC). Hence reaction with
Cu(II)SO4 in the presence of sodium ascorbate in 1:1 MeOH–H2O
and subsequent de-O-acetylation gave 1–3 (Scheme 1).
a
a
a
infection. An anti H. pylori hexasaccharide with
a-1,4-GlcNAc
capped glycans has been synthesized.4 The nitrophenol glycoside
of GlcNAc is known to also inhibit H. pylori growth. Promise has
been identified for 1-deoxynojirimycin and castanospermine as
The alkyne precursor 139 was prepared (26%) by adding acetyl
chloride to propargyl alcohol at 0 °C, with N-acetyl-D-glucosamine
(14) being subsequently added to the mixture at room temp.10,11
Then alkyne 13 was reacted with the azide 9 using the CuAAC reac-
tion giving the divalent compound 4 after de-O-acetylation of the
intermediate (60%, two steps, Scheme 2).
inhibitors of
lent glycoclusters based on
a
-glucosyl cholesterol.5 We hypothesized that biva-
a-GlcNAc could have activity against
H. pylori. This is based on the structure of the anti H. pylori hexasac-
charide4 (Fig. 1) which could be viewed as being comprised of two
The azide 15 (76%) was prepared from N-acetyl-D-glucosamine
a
-GlcNAc residues grafted onto a core tetrasaccharide scaffold. We
in two steps (Scheme 3). Firstly 2-bromoethanol was heated in
the presence of 14 and acetyl chloride at 70 °C for 3 h.12 The inter-
mediate obtained was subsequently treated with sodium azide and
tetrabutylammonium iodide at 60 °C overnight to give 15 (>97%).
⇑
Corresponding author.
E-mail address: paul.v.murphy@nuigalway.ie (P.V. Murphy).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.07.011

2
D. Yan et al. / Carbohydrate Research 360 (2012) 1–7
OH
OH
O
HO
HO
O
HO
HO
AcHN
AcHN
OH
O
O
OH
O
HO
O
HO
OH
O
Non-
AcHN
HO
carbohydrate
scaffold
O
OH
HO
O
O
AcHN
OR
OH
O
AcHN
AcHN
HO
HO
HO
HO
O
O
OH
OH
Oligosaccharide
mimetic
Anti H pylori
oligosaccharide
HO
HO
HO
N
N
O
HO
HO
HO
O
OH
HO
N
N
Ac
HN
N
O
N
OH
O
O
O
O
N
AcHN
OH
HO
Ac
AcHN
HN
N
N
N
OH
N
O
O
1
2
N
HO
HO
HO
HO
N
N
O
HO
O
N
HO
OH
Ac
AcHN
AcHN
HN
N
O
OH
OH
Ac
O
O
O
HN
3
OH
O
HO
N
N
N
N
4
HO
N
HO
OH
HO
HO
O
HO
HO
HO
AcHN
OH
HO
O
O
AcHN
O
N
N
O
N
N
AcHN
N
OH
HO
O
N
O
AcHN
OH
O
HO
8
N
5
O
HO
HO
O
N
N
OH
AcHN
AcHN
OH
O
N
O
N
N
N
N
N
HO
O
O
O
6
HO
HO
HO
OH
HO
O
AcHN
OH
O
AcHN
O
O
N
N
O
N
N
N
N
O
7
Figure 1. Rationale for selection of bivalent structures based on different scaffolds and structures of 1–8.
This azide was then subjected to the CuAAC reaction with dialky-
nes 10–12 and gave the desired bivalent clusters 5–7 in acceptable
yields. The direct reaction of 13 with 15 was carried out and gave 8
but it could not be purified sufficiently. Therefore 15 was acety-
lated to give 16 and the CuAAC reaction with 13 followed by
deacetylation gave 8 in higher purity (NMR) than from the direct
reaction of 15.
All bivalent clusters were tested against H. pylori. The direct
bactericidal effect of compounds on H. pylori strain Pu4 (Fig. 2)
was tested by measuring the number of viable organisms present
a concentration of 0.75 mM. The compounds were also evaluated
for their ability to inhibit growth of a second strain of H. pylori,
strain P12 (Fig. 3). In this case the bivalent a-O-GlcNAc derivative
6 was the most potent inhibitor (p < 0.0001) at 1 mM. Compound 6
was also inactive at 0.75 mM. While the concentrations at which 5
and 6 show activity are high, it is the case that high concentrations
of core O-glycan structures are also required. Monosaccharides and
oligosaccharides were evaluated at concentrations of 0.125 mM to
1 mM previously by Lee et al.2 This assay measured the ability of
compounds to inhibit H. pylori growth over a period of 5 days
after exposure to the compounds. The bivalent
a
-O-GlcNAc deriv-
and showed that a pentasaccharide, GlcNAca1?4Galb1?4Glc-
ative 5 was found to be the most toxic compound (P = 0.0001) from
the collection of compounds tested, being significantly more po-
tent at a concentration of 1 mM than the other GlcNAc derivatives
tested at the same concentration. The compound was not active at
NAcb1?6(Galb1?3)GalNAca1?octyl, which lacks a terminal Glc-
NAc residue of the hexasaccharide (Fig. 1)4 was the most potent of
the compounds tested, inhibiting growth significantly at concen-
trations of 0.5 mM or higher.

D. Yan et al. / Carbohydrate Research 360 (2012) 1–7
3
10-12
1. Dialkyne
sodium ascorbate
AcO
AcO
AcO
CuSO4, 1:1 MeOH-H2O
O
1
2
3
(83%)
(59%)
(59%)
N3
AcHN
2. NaOMe, MeOH
9
Dialkynes:
O
O
O
O
11
10
O
O
12
Scheme 1. Synthesis of 1–3.
AcO
AcO
AcO
O
N3
AcHN
1. Sodium ascorbate
Figure 2. The effect of GlcNAc containing glycoclusters 1–8 on viability of H. pylori
strain Pu4. Bacteria were exposed to the different compounds at a concentration of
1 mM. or to control diluent solutions for 1 h. Bacterial viability was assessed by
enumerating the number of CFU present in the solutions. Results are presented as
the mean result of three separate experiments the standard deviation of the
mean. ⁄ = Statistically significant (p < 0.05).
CuSO4, 1:1 MeOH-H2O
9
+
4 (60%)
HO
HO
HO
2. NaOMe, MeOH
O
AcHN
O
13
the hexasaccharide, which could more specifically direct glycomi-
metic design.
Scheme 2. Synthesis of 4.
In summary, the Cu(I) catalysed variation of the cycloaddition
reaction of azides and alkynes13,14 has simply and successfully
been used to generate a series of readily synthesized divalent Glc-
NAc-containing glycoclusters, which can be considered mimics of
an anti H. pylori hexasaccharide. Two bivalent compounds based
The distances between the GlcNAc residues in extended confor-
mations of 1–8 would be expected to vary with longer distances
between GlcNAc compounds being accessible to compounds 5–7
when compared to other more constrained structures. The antibac-
tericidal activity is speculated to be related to the distance be-
tween the GlcNAc residues, which may be optimum in 5/6.
Molecular modelling was used to investigate if this was in princi-
ple possible. Hence a model of the anti H. pylori oligosaccharide
in an extended conformation was generated. This was then over-
lapped with a model of compound 5 (Fig. 4) which was generated
to have the spacing between the GlcNAc residues (Fig. 4). In these
models the distance between the anomeric carbon atoms of the
GlcNAc residues is ꢀ20 Å. This indicates that the core scaffold in
5 can in principle mimic the core tetrasaccharide scaffold in the
natural hexasaccharide. Similarly a model of compound 6 was gen-
erated (Fig. 4) which showed it could mimic the hexasaccharide. To
date there is no information about the bioactive conformation of
on a-O-GlcNAc were identified from the series that selectively re-
duced the viability of H. pylori. It is interesting that there is activity
towards different strains of H. pylori (Pu4 vs P12) for different com-
pounds. The activity of the oligosaccharide mimetics could be due
to the ability of the GlcNAc residues to adopt spatial arrangement
which is accessible to the anti H. pylori hexasaccharide. One referee
indicated that oxidative dealkylation of hydroquinone as found in
5, might occur in vivo and increase cytotoxicity. In such a scenario
5 or a related compound would be bi-functional. This could be the
basis of a strategy to target more effectively H. pylori infection as
part of future work. Aside from the application described herein
the glycoclusters based on GlcNAc would be interesting to evaluate
against other lectins.15
1. AcCl, 2-bromoethanol
HO
HO
HO
AcO
I2, Ac2O
O
HO
70 oC, 76%
AcO
O
97%
HO
O
AcO
OH
HO
AcHN
AcHN
O
2. NaN3, Bu4NI, acetone-
water, 60 oC, 97%
O
AcHN
14
15
16
N3
N3
13
1. Alkyne
10-12
Dialkyne
sodium ascorbate
CuSO4
sodium ascorbate
CuSO4, 1:1 MeOH-H2O
5
6
7
1:1 MeOH-H2O
2. NaOMe, MeOH
(74%)
(69%)
(74%)
8
(56%)
Scheme 3. Synthesis of 5–8.

4
D. Yan et al. / Carbohydrate Research 360 (2012) 1–7
GlcNAc
GlcNAc
A
GlcNAc
B
C
GlcNAc
GlcNAc
Figure 3. The effect of GlcNAc containing glycoclusters (1–8) on viability of H.
pylori strain P12. Bacteria were exposed to the different compounds at
a
concentration of 1 mM. or to control diluent solutions for 1 h. Bacterial viability
was assessed by enumerating the number of CFU present in the solutions. Results
are presented as the mean result of three separate experiments the standard
deviation of the mean. ⁄ = Statistically significant (p < 0.05).
GlcNAc
GlcNAc
GlcNAc
GlcNAc
D
E
1. Experimental
1.1. General
Optical rotations were determined at the sodium D line at 20 °C.
NMR spectra were recorded with 400 & 500 MHz spectrometers.
Chemical shifts are reported relative to internal Me4Si in CDCl3 (d
0.0), HOD for D2O (d 4.80) for 1H and CDCl3 (d 77.0) for 13C. 1H
NMR signals were assigned with the aid of COSY and TOCSY. 13C
NMR signals were assigned with the aid of DEPT, HSQC and HMBC.
Coupling constants are reported in hertz. The IR spectra were re-
corded using a thin film between NaCl plates. Mass spectral data
were in positive and/or negative mode as indicated in each case.
Thin layer chromatography (TLC) was performed on aluminium
sheets pre-coated with silica gel and spots visualized by UV and
charring with H2SO4–EtOH (1:20), vanillin, iodine, or cerium
molybdate. Preparative TLC was carried out using analtech silica
gel HLF 20 ꢁ 20 cm. Flash chromatography was carried out with
silica gel 60 (0.040–0.630 mm) and using a stepwise solvent polar-
ity gradient correlated with TLC mobility. Chromatography sol-
vents were used as obtained from suppliers. Dichloromethane,
MeOH and THF reaction solvents were used as obtained from a
Pure Solv™ Solvent Purification System. Anhydrous acetonitrile,
DMF, pyridine and toluene were used as obtained from Sigma–
Aldrich.
GlcNAc
Figure 4. Models of: (A) anti H. pylori oligosaccharide; (B) compound 5; (C)
overlapped hexasaccharide and compound 5; (D) compound 6; (E) overlapped
hexasaccharide and compound 6.
2H, CH@C), 6.92 (4H, aromatic H), 6.12 (d, 2H, J = 10.0 Hz, H-1),
5.43 (t, 2H, J = 10.0 Hz, H-3), 5.17 (t, 2H, J = 10.0 Hz, H-4), 5.09 (s,
4H, CH@CCH2), 4.65 (apparent q, 2H, J = 9.5 Hz, H-2), 4.24 (dd,
2H, J = 5.5 Hz, J = 13.0 Hz, H-6a), 4.10 (m, 4H, overlapping signals
of H-6b and H-5), 2.04, 2.00 (s, CH3); 13C NMR (125 MHz, DMSO):
d 170.1, 170.0, 169.8, 169.3 (C@O), 152.7 (aromatic C), 143.7
(CH@C), 122.6 (CH@C), 115.9 (aromatic CH), 85.5 (C1), 74.3 (C5),
72.7 (C3), 68.3 (C4), 62.2 (CH@C–CH2), 61.9 (C6), 52.5 (C2), 22.7,
20.7, 20.6, 20.5 (each CH3). This intermediate (36 mg, 0.04 mmol)
was dissolved in dry MeOH (5 mL) and freshly prepared NaOMe
in MeOH (0.5 mL of 1 M) was added and the mixture was stirred
at room temp overnight. The reaction was neutralized to pH 7
using 10% HCl at 0 °C. The MeOH was removed under diminished
pressure. Chromatography of the residue (CH2Cl2–MeOH, 10:1,
then 5:1, then MeOH) gave 1 (20 mg, 88%) as a white solid; IR
1.2. 1,4-Bis((1-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-1H-
1,2,3-triazol-4-yl)methoxy)benzene 1
To
a
mixture of p-bispropargyloxybenzene 10 (8 mg,
0.04 mmol), the azido sugar 9 (34 mg, 0.09 mmol) in MeOH–water
(1:1 v/v, 0.5 mL), sodium ascorbate (2 mg, 8 ꢁ 10ꢂ3 mmol) and
Cu(II)SO4 (7 mg, 4 ꢁ 10ꢂ3 mmol) were subsequently added. The
mixture was stirred at room temp overnight. The solvent was re-
moved under diminished pressure and chromatography of the res-
idue (CH2Cl2–MeOH, 10:1) gave the protected glycocluster as a
white solid (36 mg, 94%); 1H NMR (500 MHz, DMSO): d 8.13 (m,
m
max/cmꢂ1: 3298, 2922, 1649 (C@O), 1560, 1511, 1415, 1234,
1211, 1105, 1046, 896; [
a
]
ꢂ19.5° (c 1.06, CH3OH); 1H NMR
D

D. Yan et al. / Carbohydrate Research 360 (2012) 1–7
5
(500 MHz, D2O): d 8.17 (s, 2H, CH@C), 6.93 (s, 4H, aromatic CH),
5.78 (d, 2H, J = 9.5 Hz, H-1), 5.16 (s, 4H, CH@C–CH2), 4.19 (t, 2H,
J = 10.0 Hz, H-2), 3.87 (dd, 2H, J = 13.0, 2.0 Hz, H-6b), 3.77–
3.673(m, 8H, overlapping signals of H-6b, H-3, H-4, H-5), 1.84 (s,
6H, CH3); 13C NMR (125 MHz, D2O) d 173.8 (C@O), 151.9 (aromatic
C), 143.5 (CH@C), 123.8 (CH@C), 116.8 (aromatic CH), 86.2 (C1),
78.9, 73.5, 69.2 (C3, C4 and C5), 61.7 (CH@C–CH2), 60.3 (C6), 55.1
(C2), 21.4 (CH3). ESI-HRMS: calcd for C28H37N8O12 677.2531, found
m/z 677.2553 [MꢂH]ꢂ.
(125 MHz, CF3CD2OD) d 175.7, 175.4, 174.7, 174.1 (each C@O),
156.8 (aromatic C), 146.4 (CH@C), 132.3 (aromatic C), 130.5 (aro-
matic CH), 124.6 (CH@C), 120.9, 109.9 (aromatic CH), 87.9 (C1),
76.7 (C5), 74.2 (C3), 70.4 (C4), 63.9 (C6), 62.9 (CH@C–CH2), 55.7
(C2), 22.7, 20.9, 20.9, 20.8 (each CH3); ESI-HRMS calcd for
C
44H53N8O18 981.3478; found m/z 981.3460 [M+H]+. The title com-
pound 3 (19 mg, 97%) was obtained after deacetylation according
to the procedure used for the preparation of 1; [
a]
ꢂ50° (c 0.09,
D
DMSO); 1H NMR (500 MHz, DMSO): d 8.31 (s, 2H, CH@C), 7.87 (d,
2H, J = 9.5 Hz, NH), 7.75 (d, 2H, J = 9.0 Hz, aromatic CH), 7.44 (d,
2H, J = 2.0 Hz, aromatic CH), 7.17 (dd, 2H, J = 9.0, 2.5 Hz, aromatic
CH), 5.74 (d, 2H, J = 10.0 Hz, H-1), 5.26 (dd, 4H, J = 17.0, 5.5 Hz,
CH@C–CH2), 4.09 (apt q, 2H, J = 9.5 Hz, H-2), 3.71 (dd, 2H,
J = 10.5, 5.5 Hz, H-6a), 3.56 (m, 2H, H-3), 3.50–3.43 (m, 4H, overlap-
1.3. 1,3-Bis((1-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-1H-
1,2,3-triazol-4-yl)methoxy)benzene 2
To a mixture of m-bispropargyloxybenzene 11 (9 mg, 0.05
mmol), the azido sugar 9 (35 mg, 0.09 mmol) in MeOH–water
(2 mL, 1:1 v/v) sodium ascorbate (2 mg, 9.5 ꢁ 10ꢂ3 mmol) and
Cu(II)SO4 (1 mg, 4.7 ꢁ 10ꢂ3 mmol) were subsequently added and
the mixture was stirred at room temp overnight. The solvent was
removed under diminished pressure and chromatography of the
residue (CH2Cl2–acetone, gradient elution, 5:2 to 1:1) gave the pro-
ping signals of H-6b and H-5), 3.29 (m, 2H, H-4), 2.09 (s, CH3); 13
C
NMR (125 MHz, DMSO) d 169.0 (C@O), 154.4 (aromatic C), 142.4
(CH@C), 129.3 (aromatic CH), 128.2 (aromatic C), 123.1 (CH@C),
118.8, 107.4 (aromatic CH), 85.9 (C1), 80.0 (C5), 73.8 (C3), 69.8
(C4), 61.5 (CH@C–CH2), 61.1 (C6), 54.9 (C2), 22.6 (CH3); ESI-HRMS
calcd for C32H39N8O12 727.2687; found m/z 727.2684 [MꢂH]ꢂ.
tected intermediate as a white solid (28 mg, 60%); IR m :
max/cmꢂ1
3265, 1745 (C@O), 1668, 1530, 1375, 1228, 1039; 1H NMR
(500 MHz, CD3CN): d 8.09 (s, 2H, CH@C), 7.23 (t, 1H, J = 8.5 Hz, aro-
matic CH), 6.69 (t, 1H, J = 2.0 Hz, aromatic CH), 6.64 (dd, 2H, J = 8.5,
2.5 Hz, aromatic CH), 6.59 (d, 2H, J = 9.5 Hz, NH), 6.13 (d, 2H,
J = 10.0 Hz, H-1), 5.45 (t, 2H, J = 10.0 Hz, H-3), 5.18 (6H, overlapping
signals of H-4 and CH@CCH2), 4.59 (q, 2H, J = 9.5 Hz, H-2), 4.23–
4.12 (m, 6H, overlapping signal of H-6 and H-5), 2.04, 2.01, 2.00,
1.63 (s, CH3); 13C NMR (125 MHz, CD3CN) d 170.4, 170.2 (2s),
169.8 (C@O), 159.7 (aromatic C), 143.9 (CH@C), 130.3 (aromatic
CH), 122.9 (CH@C), 107.9, 102.0 (aromatic CH), 85.5 (C1), 74.6
(C5), 72.3 (C3), 68.3 (C4), 61.9 (C6), 61.5 (CH@C-CH2), 53.0 (C2),
21.9, 20.1, 20.0, 20.0 (each CH3); ESI-HRMS calcd for C40H49N8O18
929.3165, found m/z 929.3168 [MꢂH]ꢂ. The title compound 2
was obtained (98%) after deacetylation of this intermediate accord-
1.5. Progargyl 2-acetamido-2-deoxy-a-D-glucopyranoside 13
Acetyl chloride (1.1 mL, 15.4 mmol) was added dropwise to
propargyl alcohol (4 mL), under N2 and at 0 °C. N-Acetyl- -glucosa-
D
mine 14 (1.0 g, 1.36 mmol) was then added at room temp. The
reaction mixture was stirred at 70 °C for 3 h. Solid NaHCO3 was
added until the pH was 7 and the suspension was filtered through
celite and washed several times with MeOH. The solvent was re-
moved under diminished pressure and chromatography (CHCl3–
MeOH, 10:1 and 5:1) of the residue gave the previously known
1316 as a white solid (0.30 g, 26%); 1H NMR (CD3OD, 400 MHz): d
4.92 (d, 1H, J1,2 = 3.6 Hz, H-1), 4.26 (broad AB d, 2H, J = 12 Hz,
OCH2), 3.93 (dd, 1H, J = 3.6 Hz, J = 10.8 Hz, H-2), 3.81 (dd, 1H,
J = 2.0 Hz, J = 12.0 Hz, H-6), 3.61–3.70 (m, 2H), 3.57 (ddd, 1H,
J = 2.4 Hz, J = 5.6 Hz, J = 10.0 Hz, H-5), 3.36 (apt t, 1H, J = 9 Hz, H-
4), 2.83 (t, 1H, J = 1 Hz, CCH), 1.98 (s, CH3); 13C NMR (100 MHz,
CD3OD) d 172.4 (C@O), 95.8 (C1), 79.2 (CCH), 73.0 (C5), 71.3 (C3),
70.9 (C4), 61.2 (C6), 53.9 (CH2), 53.7 (CH), 21.2 (CH3).
ing to procedure used to give 1; IR
(C@O), 1593, 1374, 1035, 898; [
m
max/cmꢂ1: 3265, 2924, 1658
] +1° (c 0.50, CH3OH–H2O, 1:1);
D
a
1H NMR (500 MHz, D2O): d 8.10 (s, 2H, CH@C), 7.12 (apt t, 1H,
J = 8.0 Hz, aromatic CH), 6.54–6.51 (overlapping signals, 3H, aro-
matic CH), 5.69 (d, 2H, J = 10.0 Hz, H-1), 5.08 (s, 4H, CH@CCH2),
4.10 (t, 2H, J = 10.0 Hz, H-2), 3.77 (dd, 2H, J = 12.5, 1.5 Hz, H-6b),
3.50–3.58 (m, 6H, overlapping signals of H-3, H-4, H-5 and H-6a),
1.55 (s, 6H, CH3); 13C NMR (125 MHz, D2O): d 173.9 (C@O), 158.3
(aromatic C), 143.4 (CH@C), 130.5 (aromatic CH), 123.8 (CH@C),
108.6, 103.0 (aromatic CH), 86.2 (C1), 76.9, 73.5 (C3 and C4),
69.2 (C5), 60.9 (CH@CCH2), 60.4 (C6), 55.2 (C2), 21.4 (CH3); ESI-
1.6. 4-(2-Acetamido-2-deoxy-a-D-glucopyranosyloxymethyl)-1-
(2-acetamido-2-deoxy-b- -glucopyranosyl)-1H-1,2,3-triazole 4
D
To a mixture of 13 (21 mg, 0.08 mmol), the azido sugar 9 (28 mg,
0.07 mmol) in MeOH–water (1:1 v/v, 0.5 mL) sodium ascorbate
(1 mg, 5 ꢁ 10ꢂ3 mmol) and Cu(II)SO4 (0.5 mg, 3 ꢁ 10ꢂ3 mmol) were
subsequently added. The mixture was stirred at room temp over-
night. The solvent was removed under diminished pressure and
chromatography of the residue (CH2Cl2–MeOH, 10:1) gave the pro-
tected intermediate as a white solid (34 mg, 72%); 1H NMR (CD3OD,
500 MHz): d 8.26 (s, 1H, CH@C), 6.10 (d, 1H, J = 10.0 Hz, H-10), 5.47
(t, 1H, J = 10.0 Hz, H-30), 5.22 (t, 1H, J = 10.0 Hz, H-40), 4.88 (d, 1H,
J = 3.5 Hz, H-1), 4.81 (d, 1H, J = 12.0 Hz, CH@C–CH(H)), 4.60 (d, 1H,
J = 12.0 Hz, CH@C–CH(H)), 4.56 (d, 1H, J = 10.0 Hz, H-20), 4.32 (dd,
1H, J = 5.0 Hz, J = 12.5 Hz, H-60a), 4.16 (m, 2H, overlapping signals
of H-60b and H-50), 3.93 (dd, 1H, J = 3.5 Hz, J = 11.0 Hz, H-2), 3.84
(br d, 1H, J = 12.0 Hz, H-6), 3.66 (m, 3H, overlapping signals of H-
6, H-5 and H-3), 3.35 (t, 1H, J = 9.0 Hz, H-4), 2.05, 2.04, 2.01, 1.73
(each CH3); 13C NMR (125 MHz, CD3OD): d 172.3, 172.0, 170.8,
170.3, 169.8 (each C@O), 144.4 (C@CH), 122.6 (C=CH), 96.7 (C1),
85.7 (C10), 74.6 (C50), 72.8 (C3), 72.3 (C30), 71.4 (C5), 70.9 (C4),
68.2 (C40), 61.7 (C60), 61.3 (C6), 59.6 (CH2–C@CH), 53.8 (C2), 53.3
(C20), 21.2, 21.0, 19.2, 19.1, 19.0 (each CH3); ESI-HRMS calcd for
HRMS calcd for
C28H37N8O12 677.2531, found m/z 677.2531
[MꢂH]ꢂ.
1.4. 2,6-Bis((1-(2-acetamido-2-deoxy-b-D-glucopyranosyl)-1H-
1,2,3-triazol-4-yl)methoxy)naphthalene 3
To a mixture of 2,6-bispropargyloxynaphthalene 12 (11 mg,
0.05 mmol), the azido sugar 9 (34 mg, 0.09 mmol) in MeOH–water
(1:1 v/v, 2 mL) sodium ascorbate (2.0 mg, 9.1 ꢁ 10ꢂ3 mmol) and
Cu(II)SO4 (1 mg, 4.7 ꢁ 10ꢂ3 mmol) were subsequently added. The
mixture was stirred at room temp overnight. The solvent was re-
moved under diminished pressure and chromatography of the res-
idue (CH2Cl2–acetone, gradient elution of 5:2 to 1:1) gave the
protected intermediate as a white solid (26 mg, 60%); 1H NMR
(500 MHz, CF3CD2OD):
d 7.97 (s, 2H, CH=C), 7.64 (d, 2H,
J = 9.0 Hz, aromatic CH), 7.19 (s, 2H, aromatic CH), 7.11 (d, 2H,
J = 8.5 Hz, aromatic CH), 5.96 (d, 2H, J = 9.5 Hz, H-1), 5.43 (t, 2H,
J = 9.5 Hz, H-3), 5.22 (t, 2H, J = 10.0 Hz, H-4), 5.17 (s, 4H, CH@C–
CH2), 4.45 (t, 2H, J = 10.0 Hz, H-2), 4.18 (s, 4H, H-6), 3.97 (d, 2H,
J = 10.0 Hz, H-5), 1.99, 1.97, 1.96, 1.63 (each CH3); 13C NMR
C
25H36 N5O14 630.2259; found m/z 630.2284 [MꢂH]ꢂ. The title
compound 4 (15.9 mg, 83%) was obtained from the deacetylation
of the intermediate according to the procedure used to prepare 1;

6
D. Yan et al. / Carbohydrate Research 360 (2012) 1–7
IR
m
]
max/cmꢂ1: 3270, 2924, 1650, 1555, 1376, 1314, 1097, 1034, 950;
D +50.3° (c 0.94, CH3OH); 1H NMR (CD3OD, 500 MHz): d 8.16 (s,
ping signals of OCH2CH2 and H-2), 3.58–3.76 (5H, overlapping sig-
nals H6b, H-6a, H-3), 3.35 (s, 2H, H-4), 3.31 (s, 2H, H-5), 1.96 (s,
CH3); 13C NMR (125 MHz, CD3OD): d 172.2 (C@O), 152.9 (C),
143.9 (CH), 124.4 (CH), 115.6 (C), 97.3 (C1), 72.7 (C5), 71.3 (C3),
70.4 (C4), 65.8 (OCH2), 61.7 (CH@CCH2), 61.1 (C-6), 53.8 (C2),
49.8 (OCH2CH2), 21.3 (CH3); ESI-HRMS calcd for C32H45N8O14
765.3055; found m/z found: 765.3088 [MꢂH]ꢂ.
[a
1H, C@CH), 5.73 (d, 1H, J = 10.0 Hz, H-10), 4.86 (d, 1H, J = 3 Hz, H-
1), 4.74 (d, 1H, J = 12.0 Hz, CHHC=CH), 4.54 (d, 1H, J = 12.0 Hz,
CHHC=CH), 4.15 (apt t, 1H, J = 10.0 Hz, H-20), 3.83 (3H, overlapping
signals, H-2, H-60b and H-6), 3.70 (dd, 1H, J = 5.0 Hz, J = 12.5 Hz, H-
60a), 3.64 (2H, overlapping signals, H-6 and H-30), 3.53–3.29 (5H,
overlapping signals of H-3, H-5, H-50, H-40, H-4), 1.90, 1.73 (each
s, each CH3); 13C NMR (125 MHz, CD3OD): d 172.3, 172.1 (each
C@O), 143.8 (C=CH), 122.5 (C@CH), 96.5 (C1), 86.8 (C10), 79.8
(C50), 74.2 (C30), 72.8 (C5), 71.4 (C3), 71.0 (C4), 70.0 (C40), 61.4
(C60), 60.9 (C6), 59.6 (CH2-C@CH), 55.4 (C20), 53.8 (C2), 21.2, 21.1
(each CH3); ESI-HRMS calcd for C19H30N5O11 504.1942; found m/z
504.1950 [MꢂH]ꢂ.
1.9. 1,3-Bis((1-(2-(2-acetamido-2-deoxy)-a-D-
glucopyranosyloxyethyl)-1H-1,2,3-triazol-4-
yl)methoxy)benzene 6
The title compound 6 (38 mg, 69%) was obtained from 11 and
15 according to the same procedure used to give 5; IR mmax
cmꢂ1: 3306, 2926, 1732, 1636, 1603, 1461, 1380, 1229, 1145,
1085, 1031, 768; [
+71.7° (c 0.63, H2O); 1H NMR (500 MHz,
/
1.7. 2-Azidoethyl 2-acetamido-2-deoxy-
a
-D
-glucopyranoside
a]
D
D2O): d 8.03 (s, 2H, CH@C), 7.16 (t, 1H, J = 8.5 Hz, aromatic CH),
6.59 (dd, 2H, J = 8.5, 1.5 Hz, aromatic H), 6.55 (broad s, 1H, aro-
matic H), 5.08 (s, 4H, CH@CCH2), 4.60 (d, 2H, J = 3.5 Hz, H-1), 4.53
(broad s, 4H), 3.95 (m, 2H), 3.69–3.75 (m, 4H), 3.54 (m, 4H, H-6),
3.49 (t, 2H, J = 10.0 Hz, H-3), 3.29 (t, 2H, J = 9.5 Hz, H-4), 3.04 (br
s, 2H, H-5), 1.82 (s, 6H, CH3); 13C NMR (125 MHz, D2O): d 174.0
(C@O), 158.6 (aromatic C), 143.3 (CH@C), 130.6 (aromatic CH),
125.3 (CH@C), 108.3, 102.7 (aromatic CH), 96.7 (C1), 71.9 (C5),
70.8 (C3), 69.5 (C4), 65.7 (OCH2CH2), 61.0 (CH@CCH2), 60.2 (C6),
53.4 (C2), 50.0 (OCH2CH2), 21.8 (CH3). ESI-HRMS calcd for
Acetyl chloride (0.33 mL, 4.61 mmol) was added dropwise to 2-
bromoethanol (3 mL), under N2 and at 0 °C. N-Acetyl- - glucosa-
D
mine (0.3 g, 1.36 mmol) was then added at room temp. The reac-
tion mixture was stirred at 70 °C for 3 h and then solid NaHCO3
was added until the pH was 7 and the suspension was filtered
through celite, washing with MeOH. The solvent was removed un-
der diminished pressure and chromatography (CHCl3–MeOH,
8:1and 5:1) of the residue gave the bromide containing intermedi-
ate as light brown solid (0.34 g, 76%); 1H NMR (CD3OD, 500 MHz):
d 4.87 (d, 1H, J1,2 = 4.0 Hz, H-1), 3.99 (m, 1H, m OCHHCH2), 3.89 (dd,
1H, J = 4.5 Hz, J = 13.5 Hz, H-2), 3.79 (m, 2H, overlapping signals of
H-6 and OCH2CH2), 3.68 (m, 2H, overlapping signal of H-60 and H-
3), 3.58 (m, 2H, OCH2CH2), 3.36 (m, 1H, H-5), 3.30 (t, 1H, J = 2.0 Hz,
H-4), 2.00 (s, CH3); 13C NMR (125 MHz, CD3OD): d 172.3 (C@O),
97.5 (C1), 72.7 (C3), 71.3 (C5), 68.0 (OCH2CH2), 61.2 (C6), 54.0
(CH), 47.6 (CH), 30.3 (OCH2CH2), 21.3 (CH3); ESI-HRMS calcd for
C
32H45N8O14 765.3055; found m/z found: 765.3055 [MꢂH]ꢂ.
1.10. 2,6-Bis((1-(2-(2-acetamido-2-deoxy)-a-D-
glucopyranosyloxyethyl)-1H-1,2,3-triazol-4-
yl)methoxy)naphthalene 7
The title compound 7 (12 mg, 74%) was obtained from 12 and
15 according to the procedure used for the preparation of 5; IR
C
10H18 NO6Br 350.0215; found m/z found: 350.0204 [M+Na]+. To
this bromide (0.26 g, 0.78 mmol) in acetone–water (1:1, 6 mL),
NaN3 (0.30 g, 4.68 mmol) and Bu4NI (0.29 g, 0.78 mmol) were sub-
sequently added. The light brown solution was heated at reflux
overnight. The solvent was removed under diminished pressure
and chromatography (CH2Cl2–MeOH, 8:1 and 5:1) of the residue
gave the title azide 15 as light brown solid (0.24 g, 99%); 1H NMR
(500 MHz, CD3OD): d 4.86 (d, 1H, J1,2 = 2.0 Hz, H-1), 3.90 (m, 2H,
overlapping signals of OCH2CH2 and H-2), 3.83 (d, 1H, J = 12.0 Hz,
H-6), 3.70 (m, 2H, overlapping signals of H-60 and H-5), 3.61 (2H,
overlapping signals of OCH2CH2 and H-3), 3.47 (t, 2H, J = 4.5 Hz,
OCH2CH2), 3.37 (m, 1H, H-4), 2.00 (s, CH3); 13C NMR (125 MHz,
CD3OD): d 172.4 (C@O), 97.4 (C1), 72.6 (C3), 71.3 (C5), 70.8 (C4),
66.4 (OCH2CH2), 61.2 (C6), 53.9 (C2), 50.3 (OCH2CH2), 21.4 (CH3).
ESI-HRMS calcd for C10H17 N4O6 289.1148; found m/z found:
289.1157 [MꢂH]ꢂ.
m
max/cmꢂ1: 3307, 2926, 1730, 1646 (C@O), 1603 (NH), 1510,
1393, 1225, 1116, 1031, 852; [
a
]
D
+79° (c 0.66, H2O); 1H NMR
(500 MHz, D2O): d 7.79 (s, 2H, CH@C), 7.38 (d, 2H, J = 9.0 Hz, aro-
matic CH), 6.96 (s, 2H, aromatic CH), 6.90 (d, 2H, J = 8.5 Hz, aro-
matic CH), 4.89 (s, 4H, CH@CCH2), 4.51 (d, 2H, J = 3.5 Hz, H-1),
4.37 (broad s, 4H), 3.83 (m, 2H), 3.66 (m, 2H), 3.61 (m, 2H), 3.52
(m, 4H, H-6), 3.47 (t, 2H, J = 10.5 Hz, H-3), 3.28 (t, 2H, J = 9.5 Hz,
H-4), 3.02 (m, 2H, H-5), 1.79 (s, 6H, CH3); 13C NMR (125 MHz,
D2O) d 173.9 (C@O), 154.0 (aromatic C), 143.3 (CH@C), 129.5 (aro-
matic C), 128.5 (aromatic CH), 125.0 (CH@C), 118.8, 107.8 (aro-
matic CH), 96.7 (C1), 71.9 (C5), 70.8 (C3), 69.5 (C4), 65.6 (O-
CH2CH2), 60.8 (CH@CCH2), 60.2 (C6), 53.3 (C2), 49.9 (CH2), 21.8
(CH3); ESI-HRMS calcd for C36H49N8O14 817.3368; found m/z
found: 817.3355 [M+H]+.
1.11. Propargyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
a-D-
1.8. 1,4-Bis((1-(2-(2-acetamido-2-deoxy)-a-D-
glucopyranoside 16
glucopyranosyloxyethyl)-1H-1,2,3-triazol-4-
yl)methoxy)benzene 5
Iodine (1.8 mg, 6.9 lmol) was added to a stirred suspension of
15 (30 mg, 0.12 mmol) in acetic anhydride (1 mL) at room temp.
The mixture was then diluted with CH2Cl2 (2 mL) and 10% Na2S2O3
(2 mL) was added and the mixture left to stir for 40 min. It was
then filtered and the filtrate was removed. Chromatography of
the residue (CH2Cl2–MeOH, 20:1) gave 16 as a light yellow solid
(40 mg). 1H NMR (500 MHz, CDCl3): d 5.74 (d, 1H, J = 9.5 Hz, NH),
5.22 (t, 1H, J = 9.5 Hz, H-3), 5.15 (t, 1H, J = 10.0 Hz, H-4), 5.04 (d,
1H, J = 4.0 Hz, H-1), 4.39 (ddd, 1H, J = 3.5 Hz, J = 9.5 Hz,
J = 10.5 Hz, H-2), 4.28 (dd, 2H, J = 2.5 Hz, J = 6.0 Hz, OCH2CCH),
4.24 (dd, 1H, J = 4.5 Hz, J = 12.5 Hz, CH2), 4.11 (dd, 1H, J = 2.0 Hz,
J = 12.0 Hz, CH2), 4.00 (ddd, 1H, J = 2.5 Hz, J = 4.0 Hz, J = 10.0 Hz,
H-5), 2.49 (apt t., 1H, J = 2.0 Hz, CCH), 2.10, 2.03, 2.02, 1.96 (each
s, each CH3); 13C NMR (125 MHz, CDCl3): d 171.2, 170.6, 170.0,
To
a
mixture of p-bispropargyloxybenzene 10 (6 mg,
0.03 mmol), the azido sugar 15 (19 mg, 0.06 mmol) in MeOH–
water (1:1 v/v, 0.5 mL) sodium ascorbate (1 mg, 6 ꢁ 10ꢂ3 mmol)
and Cu(II)SO4 (0.5 mg, 3 ꢁ 10ꢂ3 mmol) were subsequently added.
The mixture was stirred at room temp overnight. The solvent
was removed under diminished pressure and chromatography
(CH2Cl2–MeOH, 5:1 then MeOH) of the residue gave 5 as a gel
(17 mg, 74%); IR
1207, 1084, 1027, 829; [
(500 MHz, CD3OD): d 8.12 (s, 2H, CH@C), 6.96 (broad s, 4H, aro-
matic CH), 5.12 (s, 4H, CH@CCH2), 4.78 (d, 2H, J = 2.0 Hz, H-1),
4.65 (m, OCH2CH2), 4.09 (m, 2H, OCH2CH2), 4.09 (m, 4H, overlap-
m
max/cmꢂ1: 3287, 2923, 1647, 1552, 1504, 1376,
+83° (c 0.93, CH3OH); 1H NMR
a]
D

D. Yan et al. / Carbohydrate Research 360 (2012) 1–7
7
169.2 (each C@O), 96.2 (C1), 78.1 (CH2CCH), 75.4 (CCH), 71.0 (C3),
68.3 (C5), 68.0 (C4), 61.8 (C6), 55.3 (CH2CCH), 51.6 (C2), 23.1, 20.7,
20.6, 20.5 (CH3).
was calculated. Each assay was performed in duplicate on three
separate occasions.
1.14. Molecular modelling
1.12. 1-(2-(2-Acetamido-2-deoxy-a-D-
glucopyranosyl)oxyethyl)-4-((2-acetamido-2-deoxy-a-D-
glucopyranosyl)oxymethyl)-1H-1,2,3-triazole 8
A model of the anti H. pylori hexasaccharide was built using
Maestro version 6.0 (Schrödinger Inc., LLC, New York, USA). Con-
straints were then applied during energy minimization using Mac-
romodel version 8.5 (Schrödinger Inc.) so as to generate an
extended conformation for the hexasaccharide where the GlcNAc
residues were constrained at a distance of ꢀ20 Å. Minimization
(gas phase) was carried out using the OPLSAA force field & PRCG
method. A model of 5 was then built with the GlcNAc residues con-
strained in the same spatial arrangement as the extended hexasac-
charide. Then minimization was carried out with constraints
applied to all GlcNAc atoms and allowing the remainder of the
structure, the scaffold, to minimize. This led to generation of the
structures shown in Figure 4.
The acetylated intermediate was obtained from alkyne 13 and
16 (70%) as described above. IR
max/cmꢂ1: 3346.3, 1747, 1660,
1545, 1373, 1238, 1043; [
+112° (c 0.51, CH3OH); 1H NMR
m
a
]
D
(500 MHz, CD3OD): d 5.04 (dd, 1H, J = 11.0, 9.5 Hz, CH), 4.85 (t,
1H, J = 10.5 Hz, CH), 4.80 (d, 1H, J = 3.5 Hz, H-1), 4.71 (d, 1H,
J = 12.5 Hz, CH2), 4.72 (d, 1H, J = 4.5 Hz, H-10), 4.60 (t, 2H,
J = 5.0 Hz, CH2), 4.55 (d, 1H, J = 12.5 Hz, CH2), 4.11 (dd, 1H,
J = 11.0, 3.5 Hz, CH), 4.06 (dd, 1H, J = 12.0, 4.5 Hz, CH2), 4.00 (dt,
1H, J = 11.5, 6.0 Hz, CH2), 3.92 (dd, 1H, J = 12.5, 2.0 Hz, CH2), 3.85
(dt, 1H, J = 11.0, 4.5 Hz, CH2), 3.82 (dd, 1H, J = 10.5, 4.0 Hz, CH),
3.74 (dd, 1H, J = 12.0, 2.0 Hz, CH2), 3.61–3.51 (m, 3H, overlapping
signals of 2 ꢁ CH and 1H of CH2), 3.44 (dq, 1H, J = 10.5, 2.5 Hz,
CH), 3.26 (t, 1H, J = 9.5 Hz, CH), 1.95, 1.90, 1.86, 1.86, 1.85 (s,
15H, CH3). 13C NMR (125 MHz, CD3OD): d 172.2, 172.0, 170.9,
170.6, 169.8 (C@O), 143.9 (CH@C), 124.6 (CH@C), 97.1 (C10), 96.6
(C1), 72.8, 71.4, 70.9, 70.7, 68.6, 67.7 (CH), 66.0, 61.6, 61.4, 59.7
(CH2), 53.8, 51.3 (CH), 49.6 (CH2), 21.2, 21.1, 19.2, 19.2, 19.1
(CH3); ESI-HRMS calcd for C27H40N5O15 674.2521; found m/z
found: 674.2523 [MꢂH]ꢂ. The title compound 8 (10.9 mg) was ob-
Acknowledgements
The authors are grateful for financial support from Science
Foundation Ireland (08/SRC/B1393) and IRCSET for a Ph.D. Scholar-
ship (D.Y.) and to referees for useful comments.
Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.carres.
2012.07.011.
tained (80%) after de-O-acetylation of this intermediate; IR mmax
cmꢂ1: 3297, 2922, 1650, 1550, 1376, 1323, 1229, 1118, 1034;
+136° (c 0.09, CH3OH); 1H NMR (500 MHz, CD3OD): d 4.88
/
[a]
D
(d, 1H, J = 3.5 Hz, H-1), 4.82 (d, 1H, J = 13.0 Hz, CH2), 4.78 (d, 1H,
J = 3.5 Hz, H-10), 4.65 (t, 2H, J = 5.5 Hz, CH2), 4.61 (d, 1H,
J = 12.0 Hz, CH2), 4.09 (dt, 1H, J = 11.5, 4.5 Hz, CH2), 3.92 (dd, 1H,
J = 10.5, 3.0 Hz, CH), 3.87–3.83 (m, 3H, overlapping signals of CH
and CH2), 3.76 (dd, 1H, J = 11.5, 2.0 Hz, CH2), 3.71–3.60 (m, 4H,
overlapping signals of 2 ꢁ CH and CH2), 3.54 (t, 1H, J = 9.0 Hz,
CH), 3.36–3.29 (m, 2H, overlapping signals of 2 ꢁ CH), 3.23–3.20
(m, 1H, CH), 1.98, 1.96 (each s, each CH3). 13C NMR (125 MHz,
CD3OD) d 172.3, 172.2 (C@O), 143.9 (CH@C), 124.4 (CH@C), 97.4
(C10), 96.6 (C1), 72.8, 72.7, 71.3, 71.3, 70.9, 70.7 (each CH), 65.7,
61.4, 61.2, 59.8 (each CH2), 53.8, 53.7 (each CH), 49.8 (CH2), 21.3,
21.2 (each CH3). ESI-HRMS calcd for for C21H34N5O12 548.2204;
found m/z found: 548.2204 [MꢂH]ꢂ.
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1.13. Assay to test the bactericidal activity of test compounds
H. pylori strains PU4, and P12 were used in this study. Bacteria
were grown on Columbia blood agar containing 7% (v/v) defibrin-
ated horse blood for 48 h at 37 °C under microaerophilic conditions
generated using Campygen gaspaks. Bacteria were harvested from
agar plates and suspended in brain heat infusion broth containing
10% (v/v) foetal calf serum. The OD600nm of the bacterial suspension
was adjusted to 0.5 and 100 ll aliquots were added to the wells of
a 96 well microtitre plate. Stock solutions of the test compounds
were dissolved in PBS (Dulbecco’s formula A, pH 7.3) or PBS con-
taining DMSO. The test compounds were added to the wells con-
taining the bacteria so that the final concentration of the
compound was 1 mM. PBS and PBS containing DMSO were added
to wells to act as controls. Bacteria and test solutions were incu-
bated together for one hour at 37 °C under microaerophilic condi-
tions after which time serial dilutions of the bacterial suspensions
were prepared. 100 ll volumes of the dilutions were spread on
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Columbia blood agar plates which were incubated at 37 °C under
microaerophilic conditions for up to 5 days. The number of viable
organisms (colony forming units per ml) present in each solution