Synthesis of glucose derivatives modified at the 4-OH as potential chain-terminators of cellulose biosynthesis; Herbicidal activity of simple monosaccharide derivatives

Article abstract of DOI:10.1039/b820830a

A series of d-glucose derivatives that have been modified at C-4 were synthesised from D-galactose as potential chain terminators of cellulose biosynthesis. Two compounds displayed herbicidal activity in pre-emergence tests and in addition a cell expansio

Full text of DOI:10.1039/b820830a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of glucose derivatives modified at the 4-OH as potential
chain-terminators of cellulose biosynthesis; herbicidal activity of simple
monosaccharide derivatives†
Emma van Dijkum,a Ramona Danac,a David J. Hughes,b Richard Wood,b Anne Rees,b Brendan L. Wilkinsona
and Antony J. Fairbanks*a,c
Received 21st November 2008, Accepted 17th December 2008
First published as an Advance Article on the web 30th January 2009
DOI: 10.1039/b820830a
A series of D-glucose derivatives that have been modified at C-4 were synthesised from D-galactose as
potential chain terminators of cellulose biosynthesis. Two compounds displayed herbicidal activity in
pre-emergence tests and in addition a cell expansion assay at higher concentrations revealed
symptomology of a third compound that was indicative of inhibition of cellulose biosynthesis.
to display significant biological activity in vivo is fraught with
Introduction
difficulty, since such inhibitors are required to mimic a reaction
Increasing global food output becomes a key objective as the popu-
transition state that effectively involves three distinct species—the
lation of the world itself continues to rise.1 However, achieving this
glycosyl acceptor, the nucleoside di-phosphate, and the glycosyl
goal by increasing the proportion of the planet’s surface used for
donor. The fact that none of the known inhibitors of cellulose
agricultural purposes, for example by the clearing of native forests,
biosynthesis have yet been identified as competitive inhibitors of
would not only be catastrophic from an immediate ecological
cellulose synthases is therefore unsurprising.
perspective, but may have serious long term consequences for the
An alternative strategy to inhibition of cellulose biosynthesis
sustainability of the biosphere.2 In future, continued increases in
may prove useful, which would be complementary to the differ-
agricultural productivity from the currently farmed areas of the
ent modes of action of other cellulose biosynthesis inhibitors.
planet will therefore be necessary in order to feed an increased
Such an approach would be to affect chain termination of
population. One of several undertakings that will facilitate this
cellulose biosynthesis using glucose derivatives that have been
objective is the development of new herbicides to substantially
modified at the hydroxyl group at the 4-position,9 which is
increase agricultural output from currently farmed areas. However
where subsequent glucose units would be added to the growing
not all herbicides are environmentally benign, and their increased
oligosaccharide chain. Chain-termination processes have found
and indiscriminate use could in certain cases have serious environ-
widespread use as a means of DNA sequencing,10 and as the
mental and health effects. Future herbicides should therefore be
basis of several anti-viral therapies currently in clinical use.11
as safe as possible.
Moreover chain termination has previously been implicated in
Inhibition of cellulose biosynthesis3 represents a potentially safe
the biological effects of some modified monosaccharide deriva-
herbicidal mode of action, which should in principle be non-toxic
tives on mammalian glycoconjugate12 and glycosoaminoglycan
to animals, fish, and other wildlife. Several chemically diverse
biosynthesis.13 More recently attempts to develop both new anti-
classes of herbicidal cellulose biosynthesis inhibitors4,5 are known,
fungal14 and anti-mycobacterial agents15 by the use of a chain
which include compounds such as dichlobenil and isoxaben. The
termination approach have been reported, with varying degrees of
specific molecular targets have not yet been fully characterised for
success.
these herbicides, but it is unlikely that they act by competitive
Cellulose is comprised of 36 parallel b(1–4) glucan chains,
inhibition6 of cellulose synthase enzymes. Cellulose synthases,
consisting of between 8000 and 15 000 repeating monosaccha-
encoded for by a family of genes called CesA,7 contain motifs
ride units. Although the precise details of cellulose biosynthesis are
still quite poorly understood16 it is known that the b(1–4) glucan
chains, which are the constituents of cellulose fibrils, are assembled
stepwise by large complexes of cellulose synthases by transfer of
characteristic of family 28 glycosyl transferase enzymes, and use
UDP-glucose as the donor substrate to catalyse the addition of
glucose units to growing b(1–4) glucan chains. The design and
synthesis of competitive inhibitors of glycosyl transferases that are
glucose residues to growing oligomeric chains. Potential chain
potent enough and posses the correct pharmacokinetic properties
terminators of this process are therefore D-glucose residues in
which the 4-OH has been modified; if such materials are processed
aDepartment of Chemistry, Chemistry Research Laboratory, University of
Oxford, Mansfield Road, Oxford, UK OX1 3TA
by a cellulose synthase then their transfer to the terminus of a
growing b(1–4) glucan chain will result in a chain termination
step since the required 4-OH at which subsequent units would
be added will now be lacking. Alternatively glucose derivatives
modified at the 4-position could act as competitive inhibitors of
cellulose synthase enzymes, or additionally interfere with cellulose
formation in another manner.
bSyngenta Limited, Jealott’s Hill International Research Centre, Bracknell,
Berkshire, UK RG42 6EY
cDepartment of Chemistry, University of Canterbury, Private Bag 4800,
Christchurch 8140, New Zealand
† Electronic supplementary information (ESI) available: NMR data for
compounds 6a–d, 7a–d, 8a–d and 9a–d. See DOI: 10.1039/b820830a
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In order to investigate the potential for simple monosaccharide
derivatives to display herbicidal activity a variety of glucose
derivatives modified at the 4-hydroxyl were synthesised. These
materials were screened for potential herbicidal activity in pre- and
post-emergence assays, and in certain cases plant cell morphology
was also investigated to elucidate if any compounds produced
particular cellular effects that were indicative of inhibition of
cellulose biosynthesis.
protecting groups yielded 4-azido glucose 4b,9 which was also
screened for herbicidal activity.
Conversion of the peracetylated azide 4a into a selection of
triazoles was undertaken by the use of the modified Huisgen
1,3-dipolar cycloaddition in which azide 4a was reacted with a
selection of alkynes 5a–d in the presence of a Cu(I) salt.22,23 Cu(I)
was generated in situ by the addition of sodium ascorbate to a
solution of CuSO4 and the mixture of azide and alkyne was heated
to 40 ◦C in a 1 : 1 mixture of water and tert-butanol.24 Reactions
proceeded smoothly and the desired triazoles 6a–d were produced
in good to excellent yield (83–91%, Scheme 2). Finally, in order
to assess any herbicidal activity of a series of fully deprotected
derivatives, the acetates were removed by treatment of the triazoles
6a–d with a solution of sodium methoxide in methanol, yielding
the corresponding polyols 7a–d in quantitative yield.
Results and discussion
Synthesis
Replacement of the 4-hydroxyl group of glucose was envisaged
by substitution with azide, which had the attraction that this
functional group could be further elaborated into a variety of other
derivatives by click chemistry,17 or amide synthesis. In order to
probe chemical space in the search for compounds that may either
be processed by cellulose synthases or competitively inhibit them,
two alkyl derivatives with different chain lengths and two aromatic
derivatives were targeted. The decision to install hydrophobic
residues at C-4, whilst perhaps appearing counter-intuitive, would
have the advantage of reducing compound polarity and thus of
improving the ability of de-protected compounds to penetrate
intracellularly.
Introduction of nitrogen at C-4 would necessitate an inversion
of configuration and so a galacto configured starting material was
required. Selective access to the 4-OH of a galacto monosaccharide
derivative was achieved by regioselective reductive ring-opening
of the 4,6-benzylidene acetal of the methyl pyranoside 1,18 which
was achieved by treatment with sodium cyanoborohydride and
HCl in ether.11 Reaction of the secondary alcohol 2a19 with triflic
anhydride and pyridine in dichloromethane gave the triflate 2b,
which was immediately reacted with sodium azide in DMF to give
the gluco configured azide 320 (Scheme 1). Exhaustive acetolysis of
the methyl pyranoside 3 with a mixture of sulfuric acid, acetic
anhydride, and acetic acid then yielded the tetra-acetate 4a,21
as a mixture of anomers. Tetraacetate 4a acted as a divergent
intermediate for the synthesis of a variety of protected and
deprotected triazoles and amides in which structural variation was
introduced at the 4-position. Additionally removal of the acetate
Scheme 2 Reagents and conditions: (i) 5a–d, CuSO4·5H2O, sodium
ascorbate, BuOH, H2O, 40 ◦C; 6a, 91%, 6b, 86%, 6c, 83%, 6d, 84%;
t
(ii) NaOMe, MeOH, quant.
In order to avoid potential complications of acetyl migration
and the formation of acetamide side-products, conversion of
the azide 4a into a series of amides was undertaken via a
direct Staudinger ligation using the appropriate acid chloride25
(Scheme 3). An initial reaction of azide 4a with isobutyryl chloride
and triphenylphosphine produced the corresponding amide 8a in
low yield, and additionally this was difficult to separate from the
triphenylphosphine oxide by-product. However the use of tri-n-
butylphosphine as an alternative was found to be beneficial; reac-
tion of 4a with isobutyryl chloride and n-Bu3P in dichloromethane
at reflux resulted in production of the corresponding amide 8a
Scheme 1 Reagents and conditions: (i) NaBH3CN, HCl, THF, 0 ◦C to
rt, 83%; (ii) Tf2O, pyridine; (iii) NaN3, DMF, rt; 84% over two steps;
(iv) H2SO4, Ac2O, AcOH, rt, 88%; (v) NaOMe, MeOH, rt, quant.
Scheme 3 Reagents and conditions: (i) 4a, RCOCl, n-Bu3P, CH2Cl2, reflux;
8a, 37%, 8d, 61%; (ii) 4a, RCO2Cl, Ph3P, CH2Cl2, reflux; 8b, 33%, 8c, 59%;
(iii) NaOMe, MeOH, quant.
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Table 1 Pre- and post-emergence test results on four different weed species
Post-emergence test
Rate
Pre-emergence test
Compounda
(g/ha) AMARE DIGSA ALOMY CHEAL Pt typeb AMARE DIGSA ALOMY CHEAL Pt typeb
1000
0
0
0
0
—
70
90
20
70
ST
1000
0
0
0
0
—
40
30
70
50
ST
a Test compounds were sprayed on plants at 2500 L/ha after one-day cultivation (pre-emergence) and after 8 days cultivation (post-emergence). Controls
of unsprayed plants, and plants sprayed with a known herbicide were also cultivated in order to provide a comparison with which to gauge results. After
an incubation period of 13 days results were evaluated as percentage herbicide effect (100% = total damage to plant; 0% = no damage to plant). b The
symptoms recorded (Pt type) were Stunt (ST).
in an improved, but still moderate yield (37%). Similar reaction
using n-Bu3P and 4-trifluoromethylbenzoyl chloride produced the
corresponding amide 8d in a more acceptable 61% yield. Although
the use of tri-n-butylphosphine was beneficial in these two cases,
particularly with respect to product purification, it was not found
to be an absolute requirement for the success of the reaction. For
example treatment of both hexanoyl chloride and benzoyl chloride
with 4a and triphenyl phosphine in refluxing dichloromethane
gave the corresponding amides 8b and 8c in moderate yields (33%
and 59% respectively). Variation of other parameters was also
investigated. In the case of the synthesis of 8d neither changing
the reaction solvent to toluene, nor the corresponding increase in
reaction temperature achieved at reflux did little to alter either the
rate of reaction or yield of product. For the synthesis of benzamide
8c a Staudinger reaction of 4a with benzoic acid26 was also
investigated to provide direct comparison of the reactivity of an
acid and an acid chloride in the Staudinger process. Interestingly
reaction of azide 4a with triphenylphosphine and benzoic acid
in refluxing dichloromethane did not result in any appreciable
reaction. Finally with the selection of desired amides 8a–d in hand,
removal of theacetate protectinggroups was achieved bytreatment
with a solution of sodium methoxide in methanol, yielding the
corresponding tetraols 9a–d in quantitative yields (Scheme 3).
azido glucose 4b showed stunting and almost complete inhibition
of growth of DIGSA (rating: 90%), and also significant effects on
both AMARE (rating: 70%) and CHEAL (rating: 70%) (Table 1).
The protected trifluorophenyl triazole 6d also showed weaker
activity, though interestingly was most active against ALOMY
(rating: 70%) (Table 1). However neither of these compounds
displayed any activity in the post emergence tests.27 None of the
other compounds tested displayed significant herbicidal activity
in either the pre- or the post-emergence studies.
Secondly a cell expansion assay on bright yellow 2 (BY2)
tobacco cells was undertaken with a selection of the putative chain
termination compounds, which were chosen as the acetylated
and deprotected pairs and 8b/9b, 8c/9c, and 6c/7c. Several
control experiments were also carried out on BY2 cells including
monitoring the effects of DMSO alone, and the actions of a
known thiazolidinone herbicide 1028 and Oryzalin 11,29 a stan-
dard cellulose biosynthesis inhibitor and a standard microtubule
inhibitor respectively. Treatment of the BY2 cells with Oryzalin
caused round swelling typical of microtubule inhibition which
occurs as the internal scaffolding of cells is disrupted. In contrast
treatment with the thiazolidinone caused large and non-uniform
swelling and a high degree of clumping, which presumably occurs
to compensate for the lack of cellulose that provides the cells’
scaffolding, and is typical of cellulose biosynthesis inhibition.
Whilst the majority of the tested compounds did not display any
effect at concentrations up to 500 ppm, the pyridinyl acetylated
triazole 6c showed toxicity at 500 ppm, and cellulose biosynthesis
type symptomatology at 167 ppm (Fig. 1); cells showed large non-
uniform clumping as was observed in the assay with thiazolidinone
10, suggesting that 6c acted via inhibition of cellulose biosynthe-
sis, rather than by microtubule inhibition or another mode of
action.
Herbicidal screening
Potential herbicidal activity of the putative chain termination
compounds 4a,b, 6a–d, 7a–d, 8a–d, 9a–d was assessed using
two types of assay. Firstly, a series of pre-emergence and post-
emergence tests were performed on two broadleaf weeds: Amaran-
thus retroflexus (AMARE) and Chenopodium album (CHEAL)
and two grass weeds: Alopecurus myosuroides (ALOMY) and
Digitaria sanguinalis (DIGSA). Plants for the pre-emergence test
were grown in sand, and plants for the post emergence test were
grown in sandy loam soil. Damage to the plant was qualified as
stunting of emerging plant growth, chlorosis of leaves, or com-
plete inhibition of growth and recorded as percentage herbicide
effect. Pre-emergence activity was recorded in two compounds at
application rates of 1000 grams per hectare (g/ha). Deprotected 4-
Finally the cell expansion assay on BY2 tobacco cells was
repeated with the two compounds which had proved active in the
pre-emergence test, namely azide 4b and trifluoromethylphenyl
triazole 6d. Both of these compounds did display cell toxicity,
though only at the highest concentration investigated (500 ppm).
However neither displayed symptomology diagnostic for either
inhibition of cellulose biosynthesis or for de-polymerisation of
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and on a Bruker micrOTOF (ESI) spectrometer, respectively. m/z
values are reported in Daltons and are followed by their percentage
abundance in parentheses. Optical rotations were measured on a
Perkin-Elmer 241 polarimeter with a path length of 1 dm. Concen-
trations are given in g/100 mL. Microanalyses were performed by
the Inorganic Chemistry Laboratory Elemental Analysis service,
Oxford University, UK. Thin layer chromatography (t.l.c.) was
carried out on Merck Kieselgel 60F254 pre-coated glass-backed
plates. Visualisation of the plates was achieved using a u.v. lamp
(lmax = 254 or 365 nm), and/or ammonium molybdate (5% in
2 M sulfuric acid), or sulfuric acid (5% in ethanol). Flash column
chromatography was carried out using Sorbsil C60 40/60 silica.
Dichloromethane was distilled from calcium hydride, or dried on
an alumina column. Anhydrous THF, DMF, pyridine, methanol
and toluene were purchased from Fluka over molecular sieves.
‘Petrol’ refers to ◦the fraction of light petrol ether boiling in the
range of 40–60 C. Compounds 2a,19 3,20 4a21 and 4b9 were
prepared using the routes shown in Scheme 1, and exhibited
spectroscopic data in agreement with those reported previously.
Fig. 1 Cell expansion studies of effect of compounds on tobacco BY2
cells; (i) DMSO alone, and control; (ii) thiazolidinone 10; (iii) Oryzalin 11;
(iv) acetylated pyridinyl triazole 6c.
microtubules, indicating a different mode of action against BY2
cells in these cases.
4-Butyl-1-(1¢,2¢,3¢,6¢-tetra-O-acetyl-4¢-deoxy-D-glucopyranosyl)-
1,2,3-triazole (6a). Azide 4a (139 mg, 0.37 mmol) and 1-hexyne
5a (43 mL, 2.3 mmol) were suspended in a 1 : 1 mixture of
t-BuOH and distilled water (1.6 mL). Sodium L-ascorbate (30 mg,
0.15 mmol), and CuSO4·5H2O (19 mg, 0.08 mmol) were then
added. The resulting mixture was stirred at 40 ◦C for 30 min after
which t.l.c. (petrol–ethyl acetate, 3 : 2) indicated formation of
a single product (Rf 0.3) and complete consumption of starting
material (Rf 0.6). The resulting mixture was diluted with ethyl
acetate (10 mL), washed with water (10 mL) and brine (10 mL),
dried (MgSO4), filtered and concentrated in vacuo. The residue
was purified by flash column chromatography (petrol–ethyl
acetate, 1 : 1) to afford the n-butyl triazole 6a (155 mg, 91%), as
In conclusion a series of D-glucose derivatives that have been
modified at C-4 have been synthesised as potential inhibitors of
cellulose biosynthesis. Either click chemistry or Staudinger reac-
tion allowed the rapid, if un-optimised, synthesis of a small series
of compounds for herbicidal screening. The herbicidal activity of
both 4-azido glucose 4b and the trifluoromethylphenyl triazole
6d in pre-emergence tests demonstrated for the first time that
simple monosaccharide derivatives can indeed display herbicidal
activity, though cell expansion assays indicated that their mode
of action, at least on BY2 cells, was probably not by inhibition
of cellulose biosynthesis. However at higher concentrations a
similar cell expansion assay indicated that another member of the
set, pyridyl triazole 6c, not only displayed toxicity to plant cells
but did indeed give symptomology consistent with inhibition of
cellulose biosynthesis. Despite the herbicidal activity of 4-azido
glucose 4b, a caveat to these findings is that triazoles 6c and
6d do contain extended functionality which may on its own be
responsible for their observed herbicidal activity; in these cases
the possibility that the carbohydrate portion of these molecules
acts largely as a bystander cannot as yet be ruled out.30 Further
investigations into the mode of action of these compounds and
the synthesis and screening of further monosaccharide derivatives
as potential inhibitors of cellulose biosynthesis or other plant-
specific pathways are currently underway, and their results will be
reported in due course.
=
=
a clear oil; nmax (KBr) 3071 (m, C CH), 1749 (br, C O), 1552
-1
=
(s, C C) cm ; dH (400 MHz, CDCl3) (a : b, 6 : 1), 0.93 (0.5H,
t, J 7.17 Hz, CH3-b), 0.93 (3H, t, J 7.25 Hz, CH3-a), 1.24–1.28
(0.3H, m, CH2-b), 1.32–1.39 (2H, m, CH2-a), 1.60–1.68 (2.3H, m,
CH2-a, CH2-b), 1.86 (0.5H, s, COCH3-b), 1.88 (3H, s, COCH3-a),
2.03 (3H, s, COCH3-a), 2.05 (0.5H, s, COCH3-b), 2.06 (0.5H, s,
COCH3-b), 2.07 (3H, s, COCH3-a), 2.15 (0.5H, s, COCH3-b),
2.26 (3H, s, COCH3-a), 2.70–2.73 (2.3H, m, CH2-a, CH2-b), 3.78
(1H, dd, J5,6 3.5 Hz, J6,6¢ 12.6 Hz, H-6¢a), 3.81–3.84 (0.2H, m,
H-6¢b), 4.12–4.14 (0.2H, m, H-6¢¢b), 4.17 (1H, dd, J5,6¢ 2.0 Hz, J6,6¢
12.3 Hz, H-6¢¢a), 4.44 (0.2H, d at, J 3.8 Hz, J 10.2 Hz, H-5¢b),
4.68–4.73 (2.2H, m, H-4¢a, H-4¢b, H-5¢a), 5.17–5.20 (1.2H,
m, H-2¢a, H-2¢b), 5.65–5.70 (0.2H, m, H-3¢b), 5.87 (1H, at, J
9.9 Hz, H-3¢a), 5.90 (0.2H, d, J1,2 8.2 Hz, H-1¢b), 6.42 (1H, d, J1,2
=
=
3.8 Hz, H-1¢a), 7.26 (0.2H, s, C CH-b), 7.29 (1H, s, C CH-a);
dC (100.6 MHz, CDCl3) a (major) anomer only: 13.8 (CH3) 20.5,
20.6, 20.7, 20.8 (COCH3), 21.2, 23.4, 31.7, (CH2), 59.6 (C-4¢),
60.2 (C-6¢) 70.0 (C-3¢), 70.1, 70.4 (C-2¢, C-5¢), 95.5 (C-1¢), 120.7
Experimental
General
Melting points were recorded on a Kofler hot block and are
uncorrected. Proton and carbon nuclear magnetic resonance (dH,
dC) spectra were recorded on Bruker DPX 250 (250 MHz), Bruker
DPX 400 (400 MHz), Bruker DQX 400 (400 MHz), Bruker AVC
500 (500 MHz) or Bruker AMX 500 (500 MHz) spectrometers.
All chemical shifts are quoted on the d-scale in ppm using
residual solvent as an internal standard. Low-resolution and high-
resolution mass spectra were recorded on a Waters 31 LCT Premier
XE spectrometer with electrospray ionisation (ESI+ or ESI-)
=
=
(C CH), 148.8 (C CH), 168.9, 168.9, 169.8, 170.2 (COCH3);
m/z (ES+) 911 (2M + H+, 100), 478 (M + Na+, 5), 456 (M +
H+, 50%); HRMS (ES+) Calcd. for C20H29N3O9 (MH+) 456.1977.
Found 456.1972.
4-Octyl-1-(1¢,2¢,3¢,6¢-tetra-O-acetyl-4¢-deoxy-D-glucopyranosyl)-
1,2,3-triazole (6b). Azide 4a (225 mg, 0.60 mmol) and 1-decyne
5b (110 mL, 0.60 mmol), were suspended in a mixture of 1 : 1
t-BuOH and distilled water (2.4 mL). Sodium L-ascorbate (48 mg,
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0.24 mmol), and CuSO4·5H2O (30 mg, 0.12 mmol) were then
added. The resulting mixture was stirred at 40 ◦C for 1 h after
which t.l.c. (petrol–ethyl acetate, 3 : 2) indicated formation of
a single product (Rf 0.4) and complete consumption of starting
material (Rf 0.6). The resulting mixture was diluted with ethyl
acetate (10 mL), washed with water (10 mL) and brine (10 mL),
dried (MgSO4), filtered and concentrated in vacuo. The residue
was purified by flash column chromatography (petrol–ethyl
acetate, 1 : 1) to afford the octyl triazole 6b (266 mg, 86%), as
(ArCH, 2 ¥ ArC), 168.8, 169.0, 169.8, 170.1 (COCH3); m/z (ES+)
953 (2M + H+, 90), 477 (M + H+, 100%); HRMS (ES+) Calcd. for
C21H24N4O9 (MNa+) 499.1435. Found 499.1434.
4-Trifluoromethylphen-2-yl-1-(1¢,2¢,3¢,6¢-tetra-O-acetyl-4¢-de-
oxy-D-glucopyranosyl)-1,2,3-triazole (6d). Azide 4a (180 mg,
0.48 mmol) and 2-ethynyl-a,a,a-trifluorotoluene 5d (67 mL,
0.48 mmol) were suspended in a 1 : 1 mixture of t-BuOH and
distilled water (2 mL). Sodium L-ascorbate (38 mg, 0.19 mmol),
and CuSO4·5H2O (24 mg, 0.1 mmol) were then added. The
resulting mixture was stirred at 40 ◦C for 4 h after which t.l.c.
(petrol–ethyl acetate, 3 : 2) indicated formation of a single product
(Rf 0.3) and complete consumption of starting material (Rf 0.6).
The resulting mixture was diluted with ethyl acetate (10 mL),
washed with water (10 mL) and brine (10 mL), dried (MgSO4),
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography (petrol–ethyl acetate, 3 : 2) to give
the trifluoromethylphenyl triazole 6d (217 mg, 84%) as a colourless
=
a colourless waxy solid; nmax (thin film) 3065 (m, C C-H), 1744
-1
=
=
(br, C O), 1552 (m, C C) cm ; dH (400 MHz, CDCl3) (a : b,
6 : 1), 0.86–0.89 (3.5H, m, CH3-a, CH3-b), 1.19–1.32 (11.7H,
m, 5 ¥ CH2-a, 5 ¥ CH2-b), 1.62–1.66 (2.3H, m, CH2-a, CH2-b),
1.86 (0.5H, s, COCH3-b), 1.87 (3H, s, COCH3-a), 2.03 (3H, s,
COCH3-a), 2.04 (0.5H, s, COCH3-b), 2.05 (0.5H, s, COCH3-b),
2.07 (3H, s, COCH3-a), 2.14 (0.5H, s, COCH3-b), 2.25 (3H, s,
COCH3-a), 2.67–2.71 (2.3H, m, CH2-a, CH2-b), 3.77 (1H, dd,
J5,6 3.1 Hz, J6,6¢ 12.4 Hz, H-6¢a), 3.81 (0.2H, dd, J5,6¢ 4.1 Hz,
J6,6¢ 12.4 Hz, H-6¢b), 4.14 (0.2H, dd, J5,6¢ 2.7 Hz, J6,6¢ 12.3 Hz,
H-6¢¢b), 4.17 (1H, dd, J5,6¢ 1.9 Hz, J6,6¢ 12.6 Hz, H-6¢¢a), 4.44–4.48
(0.2H, m, H-5¢b), 4.66–4.68 (0.2H, m, H-4¢b), 4.70–4.72 (2H, m,
H-4¢a, H-5¢a), 5.17–5.23 (1.2H, m, H-2¢a, H-2¢b), 5.67 (0.2H,
at, J 9.7 Hz, H-3¢b), 5.84–5.91 (1.2H, m, H-3¢a, H-1¢b), 6.42
=
=
foam; nmax (thin film) 1753 (br, C O), 1643 (m, C C), 1316 (s, C-F)
cm-1; dH (400 MHz, CDCl3) (a : b, 6 : 1), 1.9 (0.5H, s, COCH3-b),
1.91 (3H, s, COCH3-a), 2.05 (3H, s, COCH3-a), 2.06 (0.5H, s,
COCH3-b), 2.07 (0.5H, s, COCH3-b), 2.08 (3H, s, COCH3-a),
2.16 (0.5H, s, COCH3-b), 2.28 (3H, s, COCH3-a), 3.82 (1H, dd,
J5,6 3.4 Hz, J6,6¢ 12.8 Hz, H-6¢a), 3.85–3.89 (0.2H, m, H-6¢b),
4.25–4.29 (1.2H, m, H-6¢¢a, H-6¢¢b), 4.63 (0.2H, d at, J 2.5 Hz,
J 10.6 Hz, H-5¢b), 4.75–4.83 (1.2H, m, H-4¢a, H-4¢b), 4.91 (1H,
d at, J 2.7 Hz, J4,5 10.6 Hz, H-5¢a), 5.23 (1H, dd, J1,2 3.8 Hz,
J2,3 10.0 Hz, H-2¢a), 5.25–5.26 (0.2H, m, H-2¢b), 5.79 (0.2H, at,
J 10.2 Hz, H-3¢b), 5.95–6.02 (1.2H, m, H-3¢a, H-1¢b), 6.45 (1H,
d, J 3.8 Hz, H-1¢a), 7.50–7.54 (1.2H, m, ArH), 7.64–7.68 (1.2H,
=
(1H, d, J1,2 3.8 Hz, H-1¢a), 7.26 (0.2H, s, C CH-b), 7.29 (1H, s,
C CH-a); dC (100.6 MHz, CDCl3) a (major) anomer: 14.1 (2 ¥
=
CH3), 20.2, 20.4, 20.6, 21.0 (COCH3), 22.6, 25.5, 29.1, 29.2, 29.3,
29.3, 31.8 (CH2), 59.6 (C-4¢), 61.6 (C-6¢), 69.1 (C-3¢), 69.6 (C-2¢),
=
=
69.8 (C-5¢), 89.1 (C-1¢), 120.6 (C CH), 148.7 (C CH), 168.8,
168.8, 169.8, 170.2 (COCH3); m/z (ES+) 534 (M+ Na+, 90), 512
(M + H+, 60%); HRMS (ES+) Calcd. for C24H37N3O9 (MNa+)
534.2422. Found 534.2421. Found C, 56.54; H, 7.35; N, 8.21.
C24H37N3O9 requires C, 56.35; H, 7.29; N, 8.21%.
=
m, ArH), 7.75–7.78 (2.3H, m, C CH, ArH), 7.97–7.99 (1.2H, m,
ArH); dC (100.6 MHz, CDCl3) a (major) anomer only: 20.0, 20.4,
20.5, 20.9 (COCH3), 59.7 (C-4¢), 61.6 (C-6¢), 69.2 (C-5¢), 69.6
(C-2¢ and C-3¢), 89.1 (C-1¢), 124.1 (d, JC–F 273.7 Hz, CF3), 126.2
(q, JC–F 5.9 Hz, ArCH), 123.5 (q, JC–F 5.9 Hz, ArCH), 127.3
4-Pyridin-2-yl-1-(1¢,2¢,3¢,6¢-tetra-O-acetyl-4¢-deoxy-D-glucopy-
ranosyl)-1,2,3-triazole (6c). Azide 4a (180 mg, 0.48 mmol) and
2-ethynyl pyridine 5c (59 mL, 0.48 mmol) were suspended in a 1 : 1
mixture of t-BuOH and distilled water (2 mL). Sodium L-ascorbate
(38 mg, 0.19 mmol), and CuSO4·5H2O (24 mg, 0.1 mmol) were
then added. The resulting mixture was stirred at 40 ◦C for 1 h
after which t.l.c. (petrol–ethyl acetate, 1 : 1) indicated formation
of a single product (Rf 0.2) and complete consumption of starting
material (Rf 0.7). The resulting mixture was diluted with ethyl
acetate (10 mL), washed with water (10 mL) and brine (10 mL),
dried (MgSO4), filtered and concentrated in vacuo. The residue was
purified by flash column chromatography (petrol–ethyl acetate,
1 : 1) to afford the pyridyl triazole 6c (191 mg, 83%), as a colourless
foam. Recrystallisation from hot absolute◦ethanol gave pure a-
anomer as colourless crystals; mp 188–189 C; [a]2D5 +43.7 (c, 0.75
=
(q, JC–F 30.5 Hz, ArC), 128.7, 128.8 (ArC, C CH), 131.8, 132.2
=
(ArCH), 144.2 (C CH), 168.7, 168.8, 169.8, 170.0 (COCH3); m/z
(ES+) 566 (M + Na+, 80), 544 (M + H+, 100%); HRMS (ES+)
Calcd. for C23H24N3O9F3 (MNa+) 566.1357. Found 566.1349;
Found: C, 50.82; H, 4.58; N, 7.74. C23H24N3O9F3 requires C,
50.83; H, 4.58; N, 7.73%.
4-Butyl-1-(4¢-deoxy-D-glucopyranose)-1,2,3-triazole (7a). Ace-
tylated triazole 6a (115 mg, 0.25 mmol) was dissolved in methanol
(5 mL). Freshly prepared NaOMe (0.025 mL of a 1 M solution)
was then added portion-wise. The reaction was monitored by t.l.c.
(DCM–methanol, 4 : 1) which upon completion indicated the
formation of a single product (Rf 0.3). Amberlight IR-120 (H+)
was then added until the solution reached pH 7; the solution
was filtered and concentrated in vacuo to give the de-protected
=
=
in CHCl3); nmax (KBr) 3071 (m, C C-H), 1749 (br, C O), 1552
◦
-1
=
(s, C N) cm ; dH (400 MHz, CDCl3) 1.89 (3H, s, COCH3), 2.03
triazole 7a (73 mg, 100%) as a white solid, mp 109–110 C; nmax
-1
=
(3H, s, COCH3), 2.06 (3H, s, COCH3), 2.27 (3H, s, COCH3), 3.79
(1H, dd, J5,6 3.4 Hz, J6,6¢ 12.7 Hz, H-6¢), 4.23 (1H, dd, J5,6¢ 2.4 Hz,
J6,6¢ 12.7 Hz, H-6¢¢), 4.77–4.87 (2H, m, H-4¢, H-5¢), 5.23 (1H, dd,
J1,2 3.6 Hz, J2,3 10.0 Hz, H-2¢), 5.96 (1H, at, J 10.0 Hz, H-3¢),
6.44 (1H, d, J 3.6 Hz, H-1¢), 7.24–7.28 (1H, m, ArH), 7.79 (1H,
(thin film) 3453 (br, OH), 2385 (w, C-H), 1642 (m, C C) cm ; dH
(400 MHz, methanol-d4) (a : b, 1.5 : 1) 0.98 (5.1H, t, J 7.6 Hz,
CH3-a, CH3-b), 1.35–1.45 (3.4H, m, CH2-a, CH2-b), 1.65–1.71
(3.4H, m, CH2-a, CH2-b), 2.71–2.75 (3.4H, m, CH2-a, CH2-b),
3.14–3.20 (3.4H, m, H-6¢a, H-6¢b), 3.30 (0.7H, ad, J 9.1 Hz,
H-2¢b), 3.46–3.48 (1.7H, m, H-6¢¢a, H-6¢¢b), 3.55 (1H, dd, J1,2
3.6 Hz, J2,3 9.5 Hz, H-2¢a), 4.02–4.05 (0.7H, m, H-5¢b), 4.09 (0.7H,
at, J 9.5 Hz, H-3¢b), 4.37 (1H, at, J 9.5 Hz, H-3¢a), 4.42–4.47
(1.7H, m, H-4¢a, H-4b), 4.50–4.52 (1H, m, H-5¢a), 4.72 (0.7H, d,
=
m, ArH), 8.17–8.18 (1H, m, ArH), 8.21 (1H, s, C CH), 8.57–
8.59 (1H, m, ArH); dC (100.6 MHz, CDCl3) 20.2, 20.4, 20.6, 20.9
(CH3), 59.9 (C-4¢), 61.5 (C-6¢), 69.5 (C-3¢), 69.6 (C-2¢), 69.8 (C-5¢),
89.1 (C-1¢), 120.4, 122.1, 123.3, 137.0 (ArCH), 148.6, 149.4, 149.5
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J1,2 7.9 Hz, H-1¢b), 5.28 (1H, d, J1–2 3.6 Hz, H-1¢a), 7.75 (0.7H, s,
C CH-b), 7.78 (1H, s, C CH-a); dC (100.6 MHz, methanol-d4)
14.4 (2 ¥ CH3), 23.3, 26.0, 26.1, 32.7 (CH2), 61.7, 63.2, 63.7, 63.8,
The reaction was monitored by t.l.c. (DCM–methanol, 4 : 1)
which upon completion indicated the formation of a single
product (Rf 0.5). Amberlight IR-120 (H+) was added until the
solution reached pH 7; the solution was filtered and concentrated
in vacuo to afford de-protected triazole 7d (66 mg, 100%) as a
=
=
71.3, 72.1, 74.5, 75.4, 76.1, 77.0 (C-2¢, C-3¢, C-4¢, C-5¢, C-6¢), 94.3,
+
=
=
98.5 (C-1¢), 124.2, 124.4 (C CH), 148.7, 148.8 (C CH); m/z (ES )
575 (2M + H+, 50), 288 (M + H+, 100%); HRMS (ES+) Calcd. for
C12H21N3O5 (MNa+) 310.1373. Found 310.1363.
yellow solid; mp 69–70 ◦C; nmax (thin film) 3233 (br, OH), 1652
-1
=
(m, C C), 1320 (s, C-F) cm ; dH (400 MHz, methanol-d4) (a : b,
1 : 1), 3.21–3.25 (2H, m, H-6¢a, H-6¢b), 3.26–3.35 (1H, m, H-2¢b),
3.52–3.54 (2H, m, H-6¢¢a, H-6¢¢b), 3.56 (1H, dd, J1,2 3.5 Hz,
J2,3 9.1 Hz, H-2¢a), 3.71–3.74 (1H, m, H-5¢b), 4.18 (1H, at, J
9.5 Hz, H-3¢b), 4.45 (1H, at, J 9.1 Hz, H-3¢b), 4.56–4.6 (3H,
m, H-4¢a, H-4¢b, H-3¢a), 4.76 (1H, d, J1,2 7.9 Hz, H-1¢b), 5.3
(1H, d, J 3.6 Hz, H-1¢a), 7.64 (2H, m, ArH), 7.73–7.79 (4H, m,
4-Octyl-1-(4¢-deoxy-D-glucopyranose)-1,2,3-triazole
(7b).
Octyl triazole 6b (90 mg, 0.18 mmol) was dissolved in methanol
(5 mL). Freshly prepared NaOMe (0.02 mL of a 1 M solution)
was then added portion-wise. The reaction was monitored by
t.l.c. (DCM–methanol, 4 : 1) which upon completion indicated
the formation of a single product (Rf 0.5). Amberlight IR-120
(H+) was added until the solution reached pH 7; the solution was
filtered and concentrated in vacuo to give de-protected triazole 7b
=
ArH), 7.86 (2H, m, ArH), 8.14, 8.17 (C CH); dC (100.6 MHz,
methanol-d4), 61.7, 61.8 (C-6¢), 64.0, 64.2, 71.4, 72.0, 74.6, 75.3,
76.1, 77.0 (C-2¢, C-3¢, C-4¢, C-5¢), 94.5, 98.6 (C-1¢), 125.6 (d, JC–F
271.3 Hz, CF3), 126.6, 127.4, 129.3, 129.6, 130.2, 130.3, 130.7,
(62 mg, 100%) as a white solid, mp 95–96 ◦C; nmax (thin film) 3233
-1
=
(br, OH), 1749 (m, C C) cm ; dH (400 MHz, DMSO-d6) (a : b,
=
=
133.3, 133.5 (C CH, ArCH, ArC), 145.1, 145.2 (C CH); m/z
(ES+) 773 (2M + Na+, 100), 751 (2M + H+, 40), 398 (M + Na+,
75), 376 (M + H+, 85%); HRMS (ES+) Calcd. for C15H16N3O5F3
(MNa+) 398.0934. Found 398.0933.
1 : 1), 0.83 (5.4H, t, J 6.7 Hz, CH3-a, CH3-b), 1.22–1.26 (18H, m,
5 ¥ CH2-a, 5 ¥ CH2-b), 1.55–1.57 (3.8H, m, CH2-a, CH2-b), 2.58
(3.8H, m, CH2-a, CH2-b), 2.98 (1.8H, m, H-6¢a, H-6¢b), 3.07–4.14
(2.8H, m, H-2b, H-6¢¢a, H-6¢¢b), 3.32 (0.8H, dd, J1,2 3.6 Hz, J2,3
9.5 Hz, H-2¢a), 3.81–3.87 (1.8H, m, H-5¢a, H-5¢b), 3.84 (1H, at, J
9.5 Hz, H-3¢b), 4.09 (0.8H, at, J 9.5 Hz, H-3¢a), 4.21–4.25 (1.8H,
m, H-4¢a, H-4¢b), 4.53 (1H, d, J1,2 7.9 Hz, H-1¢b), 5.07 (0.8H, d,
1,2,3,6-Tetra-O-acetyl-4-isobutylamido-4-deoxy-D-glucopyra-
noside (8a). Azide 4a (100 mg, 0.27 mmol) was dissolved in dry
DCM (20 mL) under an atmosphere of argon. PBu3 (50 mg,
0.27 mmol) and isobutyryl chloride (0.03 mL, 0.27 mmol) were
added, and the reaction mixture was allowed to stir at reflux. After
48 h, t.l.c. (petrol–ethyl acetate, 5 : 4) indicated the formation
of a single product (Rf 0.25) and complete consumption of
the starting material (Rf 0.6). The solution was concentrated
in vacuo and purified by flash column chromatography (petrol–
ethyl acetate, 6 : 4) to give isobutyl amide 8a (52 mg, 37%) as a
=
=
J 3.6 Hz, H-1¢a), 7.75 (1H, s, C CH), 7.78 (0.8H, s, C CH); dC
(100.6 MHz, DMSO-d6) 13.9, 14.0 (CH3), 20.7, 22.0, 28.4, 28.5,
28.6, 28.7, 28.8 (CH2), 60.0, 60.1, 62.0, 62.2, 69.7, 69.9, 72.6, 73.5,
74.3, 75.1 (C-2¢, C-3¢, C-4¢, C-5¢, C-6¢), 92.3, 96.7 (C-1¢), 122.2,
+
+
=
=
122.6 (C CH), 146.3, 146.5 (C CH); m/z (ES ) 687 (2M + H ,
80), 398 (M + Na+, 25), 344 (M + H+, 100%); HRMS (ES+)
Calcd. for C16H29N3O5 (MNa+) 366.1999 Found 366.1998.
=
white solid; nmax (thin film) 3519 (br, NH), 1748 (br, C O), 1654
-1
4-Pyridin-2-yl-1-(4¢-deoxy-D-glucopyranose)-1,2,3-triazole (7c).
Acetylated triazole 6c (40 mg, 0.08 mmol) was dissolved in
methanol (5 mL). Freshly prepared NaOMe (0.01 mL of a 1 M
solution) was added portion-wise. The reaction was monitored by
t.l.c. (DCM–methanol, 4 : 1) which upon completion indicated the
formation of a single product (Rf 0.6). Amberlight IR-120 (H+) was
added until the solution reached pH 7; the solution was filtered and
concentrated in vacuo to afford de-protected triazole 7c (22 mg,
=
(br, NC O) cm ; dH (400 MHz, CDCl3) (a : b, 17 : 1), 1.11
(6.4H, m, CH(CH3)2-a, CH(CH3)2-b), 2.02 (3H, s, OCCH3-a),
2.03 (0.2H, s, COCH3-b), 2.04 (0.2H, s, COCH3-b), 2.05 (3H, s,
COCH3-a), 2.09 (0.2H, s, COCH3-b), 2.10 (3H, s, COCH3-a), 2.12
(0.2H, s, COCH3-b), 2.17 (3H, s, COCH3-a), 3.73–3.76 (0.1H,
m, H-5b), 4.00 (1H, dt, J 10.8 Hz, J 3.4 Hz, H-5a), 4.09–4.13
(0.2H, m, H-6b, H-6¢b), 4.17 (2H, m, H-6a, H-6¢a), 4.21–4.29
(1.1H, m, H-4a, H-4b), 4.98–5.03 (0.1H, m, H-2b), 5.11 (1H,
dd, J1,2 3.6 Hz, J2,3 10.2 Hz, H-2a), 5.14–5.16 (0.1H, m, H-3b),
5.35 (1H, at, J 10.4 Hz, H-3a), 5.53 (1.1H, m, NH-a, NH-b),
5.68 (0.1H, d, J1,2 8.2 Hz, H-1b), 6.37 (1H, d, J 3.6 Hz, H-
1a); dC (100.6 MHz, CDCl3) a (major) anomer only: 19.3, 19.3
((CH3)2CH), 20.5, 20.7, 20.8, 20.9 (COCH3), 35.7 ((CH3)2CH),
49.7 (C-4), 62.6 (C-6), 69.3 (C-2), 69.3 (C-3), 71.8 (C-5), 89.3 (C-1),
168.7, 169.6, 170.9, 171.5, 177.0 (COCH3, CONH); m/z (ES+)
100%) as a colourless foam; nmax (thin film) 3356 (br, OH), 1667
-1
=
=
(m, C C), 1560 (s, C N) cm ; dH (400 MHz, methanol-d4) (a : b,
1 : 1), 3.23–3.29 (3H, m, H-2¢b, H-6¢a, H-6¢b), 3.34–3.38 (3H,
m, H-2¢a, H-6¢¢a, H-6¢¢b), 3.72–3.81 (1H, m, H-5¢b), 4.10–4.18
(2H, m, H-5¢a, H-3¢a), 4.43 (1H, at, J 9.1 Hz, H-3¢b), 4.52–4.63
(2H, m, H-4¢a, H-4¢b), 4.76 (1H, d, J1,2 7.9 Hz, H-1¢b), 5.30 (1H,
d, J1,2 3.5 Hz, H-1¢a), 7.19–7.22 (2H, m, ArH), 7.35–7.38 (2H,
857.2 (2M + Na+, 100), 852.3 (2M + NH4 , 95), 440 (M + Na+,
+
=
m, ArH), 7.89–7.93 (2H, m, ArH), 8.47, 8.49 (C CH), 8.56–8.61
30), 418 (M + H+, 25%); HRMS (ES+) Calcd. for C18H27NO10
(MNa+) 440.1527. Found 440.1524.
(2H, m, ArH); dC (100.6 MHz, methanol-d4) 61.7, 61.8 (C-6¢),
64.1, 64.2, 64.2, 72.1, 74.4, 75.5, 75.9, 76.9 (C-2¢, C-3¢, C-4¢, C-5¢),
94.3, 98.5 (C-1¢), 121.6, 124.0, 125.3, 125.3, 125.4, 138.9 (ArCH,
1,2,3,6-Tetra-O-acetyl-4-deoxy-4-hexamido-D-glucopyranoside
(8b). Azide 4a (240 mg, 0.64 mmol) was dissolved in dry DCM
(20 mL) under an atmosphere of argon. PPh3 (170 mg, 0.64 mmol)
and hexanoyl chloride (0.09 mL, 0.64 mmol) were then added, and
the reaction mixture was allowed to stir at reflux. After 38 h,
t.l.c. (petrol–ethyl acetate, 5 : 4) indicated formation of single
product (Rf 0.25) and complete consumption of starting material
(Rf 0.6). The solution was concentrated in vacuo and purified by
+
+
=
=
ArC, C CH), 148.1, 148.2 (C CH); m/z (ES ) 639 (2M + Na ,
100), 331 (M + Na+, 100), 309 (M + H+, 40%); HRMS (ES+)
Calcd. for C13H16N4O5 (MNa+) 331.1013. Found 331.1015.
4-Trifluoromethylphen-2-yl-1-(4¢-deoxy-D-glucopyranose)-1,2,3-
triazole (7d). Trifluoromethylphenyl triazole 6d (96 mg,
0.17 mmol) was dissolved in methanol (5 mL). Freshly prepared
NaOMe (0.02 mL of a 1 M solution) was then added portion-wise.
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flash column chromatography (petrol–ethyl acetate, 6 : 4) to give
acetate, 3 : 2) indicated the formation of a single product (Rf 0.3)
and complete consumption of the starting material (Rf 0.5).
The solution was then concentrated in vacuo and purified by
flash column chromatography (petrol–ethyl acetate, 3 : 2) to
give trifluoromethylbenzamide 8d (38 mg, 61%) as a colourless
hexamide 8b (94 mg, 33%) as a colourless oil; nmax (thin film) 3742
-1
=
(br, NH), 1641 (br, NC O) cm ; dH (400 MHz, CDCl3) (a : b,
10 : 1), 0.88–0.90 (3.3H, m, CH3), 1.22–1.35 (4.4H, m, 2 ¥ CH2),
2.08–2.13 (2.2H, m, CH2), 1.54–1.60 (2.2H, m, CH2), 2.02 (3H, s,
COCH3-a), 2.04 (0.3H, s, COCH3-b), 2.05 (0.3H, s, COCH3-b),
2.06 (3H, s, COCH3-a), 2.09 (0.3H, s, COCH3-b), 2.10 (3H, s,
COCH3-a), 2.14 (0.3H, s, COCH3-b), 2.17 (3H, s, COCH3-a),
3.73–3.76 (0.1H, m, H-5b), 3.99 (0.1H, d at, J 4.1 Hz, J4,5 10.7 Hz,
H-5a), 4.08–4.20 (2.2H, m, H-6a, H-6a, H-6b, H-6b), 4.21–4.29
(1.1H, m, H-4a, H-4b), 5.03–5.07 (0.1H, m, H-2b), 5.11 (1H, dd,
J1,2 3.5 Hz, J2,3 10.1 Hz, H-2a), 5.13–5.15 (0.1H, m, H-3b), 5.33
(1H, at, J 10.4 Hz, H-3a), 5.50–5.52 (1.1H, m, NH-a, NH-b),
5.68 (0.1H, d, J1,2 7.9 Hz, H-1b), 6.36 (1H, d, J 3.5 Hz, H-1a); dC
(100.6 MHz, CD3OH) a (major) anomer only: 13.9 (CH3), 20.5,
20.7, 20.8, 20.9 (COCH3), 22.3, 25.2, 31.3, 36.6 (CH2), 49.9 (C-4),
62.6 (C-6), 69.3 (C-2), 69.4 (C-3), 71.7 (C-5), 89.3 (C-1) 168.7,
169.6, 170.9, 171.5, 173.2 (COCH3, CONH); m/z (ES+) 913.3
=
foam; nmax (thin film) 3520 (br, NH), 1652 (br, NC O), 1360 (s,
C-F) cm-1; dH (400 MHz, CDCl3) (a : b, 5 : 1), 2.01 (0.6H, s,
COCH3-b), 2.02 (3H, s, COCH3-a), 2.04 (3H, s, COCH3-a),
2.07 (0.6H, s, COCH3-b), 2.08 (0.6H, s, COCH3-b), 2.10 (3H, s,
COCH3-a), 2.13 (0.6H, s, COCH3-b), 2.18 (3H, s, COCH3-a),
3.87–3.91 (0.2H, m, H-5b), 4.10–4.14 (1H, ddd, J5-4 10.7 Hz, J5–6
7.6 Hz, J
2.8 Hz, H-5a), 4.24–4.26 (2.4H, m, H-6a, H-6¢a,
5–6¢
H-6b, H-6b), 4.39–4.49 (1.2H, m, H-4a, H-4b), 5.20 (1H, dd,
J2–3 10.4 Hz, J2-1 3.8 Hz, H-2a), 5.23–5.26 (0.2H, m, H-3b), 5.30
(0.2H, at, J 10.4 Hz, H-2b), 5.48 (1H, at, J 10.4 Hz, H-3a), 5.74
(0.2H, d, J1,2 8.3 Hz, H-1b), 6.41 (1H, d, J1,2 3.5 Hz, H-1a),
6.44 (1H, d, J 9.1 Hz, NH-a), 6.63 (0.2H, d, J 9.1 Hz, NH-b),
7.70–7.72 (2.4H, m, 2 ¥ ArH-a, 2 ¥ ArH-b), 7.83–7.85 (2.4H,
m, 2 ¥ ArH-a, 2 ¥ ArH-b); dC (100.6 MHz, CDCl3) a (major)
anomer only: 20.5, 20.7, 20.8, 20.9 (COCH3), 50.9 (C-4), 62.9
(C-6), 69.2 (C-2), 69.5 (C-3), 71.7 (C-5), 89.3 (C-1) 125.8, 127.5
(ArCH), 133.5, 136.5 (ArC), 166.1, 168.7, 169.6, 171.0, 172.0
+
(2M + Na+, 80), 908 (2M+ NH4 , 100), 855 (2M + H+, 50%), 468
(M + Na+, 30), 463 (M + NH4 , 40), 446 (M + H+, 50%) HRMS
+
(ES+) Calcd. for C20H31NO10 (MNa+) 468.1840. Found 468.1825.
1,2,3,6-Tetra-O-acetyl-4-benzamido-4-deoxy-D-glucopyranoside
(8c). Azide 4a (35 mg, 0.09 mmol) was dissolved in dry DCM
(2 mL) under an atmosphere of argon. PPh3 (25 mg, 0.09 mmol)
and benzoyl chloride (22 mL, 0.18 mmol) were then added, and
the reaction mixture was allowed to stir at reflux. After 5 days,
t.l.c. (petrol–ethyl acetate, 1 : 1) indicated the formation of a
single product (Rf 0.25) and complete consumption of the starting
material (Rf 0.6). The solution was diluted with DCM (5 mL),
washed with sodium bicarbonate (5 mL of a saturated aqueous
solution), brine, dried (MgSO4), filtered and concentrated in vacuo.
The residue was purified by flash column chromatography (petrol–
(COCH3, CONH); m/z (ES+) (M + NH4 , 100%); HRMS (ES+)
+
Calcd. for C22H24F3NO10 (MNa+) 542.1245. Found 542.1244.
4-Isobutylamido-4-deoxy-D-glucopyranose
(9a). Acetylated
amide 8a (70 mg, 0.17 mmol) was dissolved in methanol (5 mL).
Freshly prepared NaOMe (0.01 mL of a 1 M solution) was
then added portion-wise. The reaction was monitored by t.l.c.
(DCM–methanol, 4 : 1) which upon completion indicated the
formation of a single product (Rf 0.7). Amberlight IR-120 (H+)
was then added until the solution was at pH 7; the solution was
filtered and concentrated in vacuo to afford de-protected amide
ethyl acetate, 1 : 1) to give benzamide 8c (25 mg, 59%) as a white
9a (47 mg, 100%) as a white solid; mp 169–170 ◦C; nmax (thin
-1
-1
=
=
=
solid; nmax (thin film) 3630 (br, NH), 1644 (br, NC O) cm ; dH
film) 3320 (br, OH), 1745 (br, C O), 1658 (br, NC O), cm ; dH
(400 MHz, methanol-d4) (a : b, 1 : 1), 1.16 (12H, m, (CH3)2CH-b,
(CH3)2CH-a), 2.52 (2H, m, (CH3)2CH-a, (CH3)2CH-b), 3.16–
3.24 (1H, m, H-2b), 3.44 (1H, dd, J1,2 3.5 Hz, J2,3 9.1 Hz, H-2a),
3.48–3.52 (2H, m, H-4a, H-4b), 3.54–3.60 (5H, m, H-5b, H-6a,
H-6¢a, H-6b, H-6¢b, ), 3.66–3.72 (2H, m, H-3a, H-3b), 3.79–3.83
(3H, m, H-5a, NH-a, NH-b), 4.48 (1H, d, J1,2 7.6 Hz, H-1b)
5.18 (1H, d, J 3.5 Hz, H-1a); dC (100.6 MHz, methanol-d4) 19.7,
19.9 (CH3), 36.4, 37.2 ((CH3)2CH), 53.0, 53.1, 63.0, 63.1, 71.9,
72.4, 74.6, 75.2, 77.1, 77.4 (C-2, C-3, C-4, C-5, C-6), 94.1, 98.3
(C-1), 181.2, 181.3 (CONH); m/z (ES+) 521 (2M + Na+, 50), 272
(M + Na+, 35), 250.1 (M + H+, 10%); HRMS (ES+) Calcd. for
C10H19NO6 (MNa+) 272.1105. Found 272.1101.
(400 MHz, CDCl3) (a : b, 9 : 1), 2.0 (0.4H, s, COCH3-b), 2.01
(3H, s, COCH3-a), 2.04 (3H, s, COCH3-a), 2.06 (0.4H, s, COCH3-
b), 2.07 (0.4H, s, COCH3-b), 2.09 (3H, s, COCH3-a), 2.13 (0.4H, s,
COCH3-b), 2.18 (3H, s, COCH3-a), 4.11 (1H, d at, J 3.1 Hz, J4,5
10.1 Hz, H-5a), 4.20–4.27 (0.4H, m, H-5b), 4.25 (2.3H, m, H-6a,
H-6¢a, H-6b, H-6¢b), 4.46–4.48 (1.1H, m, H-4a, H-4b), 5.05–5.11
(0.1H, m, H-2b), 5.08 (1H, dd, J1,2 3.6 Hz, J2,3 10.3 Hz, H-2a),
5.33 (0.1H, at, J 10.3 Hz, H-3b), 5.48 (1H, at, J 10.3 Hz, H-3a),
5.74 (0.1H, d, J1,2 8.1 Hz, H-1b), 6.29 (1H, d, J 9.2 Hz, NH-a),
6.34 (0.1H, d, J 9.4 Hz, NH-b), 6.41 (1H, d, J 3.6 Hz, H-1a),
7.41–7.46 (2.3H, m, ArH), 7.49–7.54 (1.1H, m, ArH-a, ArH-b),
7.70–7.72 (2.3H, m, 2 ¥ ArH-a, 2 ¥ ArH-b); dC (100.6 MHz,
CDCl3) a (major) anomer only: 20.5, 20.7, 20.8, 20.9 (COCH3),
50.8 (C-4), 62.9 (C-6), 69.3 (C-3), 69.4 (C-2), 71.8 (C-5), 89.3 (C-
1), 126.9, 128.8, 130.8, 132.1, 133.3 (ArCH), 167.3, 168.8, 169.6,
170.1, 171.9 (COCH3, CONH); m/z (ES+) 925 (2M + Na+, 30),
474 (M + Na+, 60%); HRMS (ES+) Calcd. for C21H25NO10 (MNa+)
474.1371. Found 474.1368.
4-Deoxy-4-hexamido-D-glucopyranose (9b). Protected amide
8b (69 mg, 0.15 mmol) was dissolved in methanol (5 mL).
Freshly prepared NaOMe (0.016 mL of a 1 M solution) was then
added portion-wise. The reaction was monitored by t.l.c. (DCM–
methanol, 4 : 1) which upon completion indicated the formation
of a single product (Rf 0.7). Amberlight IR-120 (H+) was then
added until the solution was at pH 7; the solution was filtered
and concentrated in vacuo to afford de-protected amide 9b (43 mg,
1,2,3,6-Tetra-O-acetyl-4-(4-trifluoromethyl)-benzamido-4-deoxy-
D-glucopyranoside (8d). Azide 4a (45 mg, 0.12 mmol) was
dissolved in dry DCM (5 mL) under an atmosphere of argon.
PBu3 (36 mL, 0.14 mmol) and 4-trifluoromethyl benzoyl chloride
(22 mL, 0.14 mmol) were then added, and the reaction mixture
was allowed to stir at reflux. After 48 h, t.l.c. (petrol–ethyl
100%) as a white solid, mp 136–138 ◦C; nmax (thin film) 3443 (br,
-1
=
OH), 1643 (s, NC O) cm ; dH (400 MHz, methanol-d4) (a : b,
1 : 1), 0.93–0.96 (3.3H, m, CH3-a, CH3-b), 1.34–1.40 (4.4H, m, 2 ¥
CH2-a, 2 ¥ CH2-b), 1.62–1.68 (2.2H, m, CH2-a, CH2-b), 2.24–2.28
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(2.2H, m, CH2-a, CH2-b), 3.15–3.21 (0.1H, m, H-2b), 3.43
(1H, dd, J1,2 3.5 Hz, J2,3 9.14 Hz, H-2a), 3.46–3.54 (1.1H, m,
H-4a, H-4b), 3.56–3.62 (2.2H, m, H-6a, H-6¢a, H-6b, H-6¢b),
3.68–3.74 (1.1H, m, H-3a, H-3b), 3.77–3.82 (2.2H, m, H-5a, H-
5b, NH-a, NH-b), 4.48 (0.1H, d, J1,2 7.6 Hz, H-1b), 5.17 (1H, d,
J 3.5 Hz, H-1a); dC (100.6 MHz, methanol-d4) 14.3 (2 ¥ CH3),
23.5, 26.8, 26.8, 26.9, 32.6, 37.2 (CH2), 53.3, 53.4, 63.1, 63.2, 72.0,
72.3, 74.6, 75.1, 77.0, 77.3 (C-2, C-3, C-4, C-5, C-6), 94.0, 98.2
(C-1), 177.4, 177.5 (CONH); m/z (ES+) 577.3 (2M + Na+, 100),
300.1 (M + Na+, 70), 278.2 (M + H+, 20%); HRMS (ES+) Calcd.
for C12H23O6N (MNa+) 300.1418. Found 300.1421.
guinalis (DIGSA, large crab grass) were sown in pots containing
sandy loam soil (post-emergence) and sand (pre-emergence). After
cultivation for either one day (pre-emergence), or for 8 days (post-
emergence) under controlled conditions in a glasshouse, the plants
were sprayed at a rate of 2500 L/ha with an aqueous spray solution
derived from the formulation of the tested compound (1000 g/ha)
in an acetone–water (50 : 50) solution containing 0.5% Tween 20.
The test plants were t◦hen grown in a glasshouse under controlled
conditions (at 24/16 C, day/night; 14 hours per day light; 65%
humidity), and watered twice daily. The results were evaluated as
percentage herbicide effect (100% = total damage to plant; 0% =
no damage to plant) 13 days after herbicide application for both
pre- and post-emergence tests.
4-Benzamido-4-deoxy-D-glucopyranose (9c). Acetylated ben-
zamide 8c (100 mg, 0.22 mmol) was dissolved in methanol (5 mL).
Freshly prepared NaOMe (0.023 mL of a 1 M solution) was then
added portion-wise. The reaction was monitored by t.l.c. (DCM–
methanol, 4 : 1) which upon completion indicated the formation
of a single product (Rf 0.7). Amberlight IR-120 (H+) was then
added until the solution was at pH 7; solution was filtered and
concentrated in vacuo to afford de-protected benzamide 9c (63 mg,
Cell expansion assays
Solutions of compounds for testing were dissolved in 100%
DMSO. Solutions of the control inhibitors thiazolidinone 10 and
Orazylin 11 and were made up at concentrations of 50 ppm, 5 ppm
and 0.5 ppm, by ten-fold serial dilution. Solutions of compounds
6c, 7c, 8b, 9b, 8c and 9c, were made up at 500 ppm, 167 ppm, and
56 ppm by three-fold serial dilution. Compounds 4b and 6d were
made up at concentrations of 500 ppm, 250 ppm, 125 ppm, and
62.5 ppm by two-fold serial dilution. Freshly split BY2 tobacco
cells (1 ml) were then treated with 10 ml aliquots of either the test
compound or control inhibitor solutions, and the cells were then
incubated. Cell morphology was examined after 7 days with a ¥20
microscopic lens.
100%) as a colourless solid; mp 140–141 ◦C; nmax (thin film) 3645
-1
=
(br, NH), 1632 (br, C O) cm ; dH (400 MHz, methanol-d4) (a : b,
1 : 1), 3.25–3.28 (1H, m, H-2b), 3.50–3.54 (2H, m, H-2a, H-4b),
3.58–3.71 (6H, m, H-4a, H-5b, H-6a, H-6¢a, H-6b, H-6¢b), 3.89–
4.02 (5H, m, H-3a, H-3b, H-5a, NH-a, NH-b), 4.55 (1H, d, J1,2
7.6 Hz, H-1b), 5.22 (1H, d, J1,2 3.8 Hz, H-1a), 7.87 (5H, m, 5 ¥
ArH), 7.47–7.51 (5H, m, 5 ¥ ArH); dC (100.6 MHz, methanol-d4)
54.0, 54.1, 63.2, 63.3, 72.1, 72.3, 74.6, 75.1, 77.2, 77.3 (C-2, C-3,
C-4, C-5, C-6), 94.1, 98.3 (C-1), 128.5, 128.6, 129.5, 132.8, 132.9,
135.6 135.6 (ArC, ArCH), 171.3 171.4 (CONH). m/z (ES+) (2M +
Na+, 90), (M + H+, 75%); HRMS (ES+) Calcd. for C13H17NO6
(MNa+) 306.0948. Found 306.0946.
Acknowledgements
The authors gratefully thank Professor Chris Hawes and Anne
Kearns (Oxford Brookes University, UK) for kindly providing
BY2 cells.
(4-Trifluoromethyl)-benzamido-4-deoxy-D-glucopyranose (9d).
Protected trifluoromethylbenzamide 8d (69 mg, 0.15 mmol)
was dissolved in methanol (5 mL). Freshly prepared NaOMe
(0.016 mL of a 1 M solution) was then added portion-wise. The
reaction was monitored by t.l.c. (DCM–methanol, 4 : 1) which
upon completion indicated the formation of a single product (Rf
0.7). Amberlight IR-120 (H+) was then added until the solution
was at pH 7; the solution was filtered and concentrated in vacuo to
afford de-protected trifluorobenzamide 9d (43 mg, 100%) as a pale
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The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1097–1105 | 1105
©