Synthesis of Unsaturated Aminopyranosides as Possible Transition State Mimics for Glycosidases

Article abstract of DOI:10.1081/CAR-120026469

Four unsaturated aminopyranosides have been prepared as possible transition-state mimics targeted towards carbohydrate processing enzymes. The conformations of the protonated aminosugars have been investigated by molecular modelling and their ability to i

Full text of DOI:10.1081/CAR-120026469

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Synthesis of Unsaturated Aminopyranosides as
Possible Transition State Mimics for Glycosidases
Allan M. Jordan , Helen M. I. Osborn , Petra M. Stafford , George Tzortzis &
Robert A. Rastall
To cite this article: Allan M. Jordan , Helen M. I. Osborn , Petra M. Stafford , George Tzortzis
& Robert A. Rastall (2003) Synthesis of Unsaturated Aminopyranosides as Possible Transition
State Mimics for Glycosidases , Journal of Carbohydrate Chemistry, 22:7-8, 705-717, DOI:
10.1081/CAR-120026469
To link to this article: http://dx.doi.org/10.1081/CAR-120026469
Published online: 23 Feb 2007.
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Date: 09 September 2015, At: 23:03

JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, Nos. 7 & 8, pp. 705–717, 2003
Synthesis of Unsaturated Aminopyranosides as Possible
Transition State Mimics for Glycosidasesy
1
Allan M. Jordan,1 Helen M. I. Osborn,1, Petra M. Stafford,
*
George Tzortzis,2 and Robert A. Rastall2
2
1School of Chemistry, and School of Food Biosciences,
University of Reading, Whiteknights Reading, UK
ABSTRACT
Four unsaturated aminopyranosides have been prepared as possible transition-state
mimics targeted towards carbohydrate processing enzymes. The conformations of the
protonated aminosugars have been investigated by molecular modelling and their
ability to inhibit a- and b-glucosidases and an a-mannosidase have been probed.
Two targets proved moderate inhibitors of a-glucosidases from Brewer’s yeast and
Bacillus stearothermophilus.
Key Words: Glycosyl processing enzyme; Oligosaccharide; Inhibitor.
yThis paper is dedicated to Professor Ge´rard Descotes on the occasion of his 70th birthday.
*
Correspondence: Helen M. I. Osborn, School of Chemistry, University of Reading, Whiteknights,
Reading, RG6 6AD, UK; Fax: +44 (0) 118 931 6331; E-mail: h.m.i.osborn@rdg.ac.uk.
705
DOI: 10.1081/CAR-120026469
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

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Jordan et al.
INTRODUCTION
As progress in molecular biology and cellular biochemistry has developed, it has
become increasingly apparent that the glycoproteins expressed on cell surfaces play
fundamental roles in a vast array of biological processes and interactions, including
cell-cell recognition, cell growth and development.[1–3] More recently, it has been
found that a major pathway towards the malignancy of tumour cells is the decoration of
these proteins with aberrant oligosaccharides.[4,5] These altered carbohydrates appear to
confer a variety of properties on the cancerous cells, such as their ability to grow
unchecked, evade the immune system and metastasise to secondary sites around the
body.[6,7] Clearly, therefore, inhibiting the formation of these altered oligosaccharides
offers an important method for therapeutic intervention. A multitude of natural and
unnatural inhibitors of the carbohydrate processing enzymes have been reported, and
their therapeutic properties are currently being assessed.[8]
During glycosidase mediated oligosaccharide processing, a half chair transition
state with substantial sp2 character at the anomeric position is formed and it has been
postulated that compounds capable of mimicking this flattened transition state are good
candidates for inhibition studies.[9–11] For example, unsaturated derivatives of the
natural pyranoses are often good inhibitors of glycosidases in bacterial and fungal
systems with Ki values several hundred times smaller than for their saturated
analogues.[12,13] This is believed to be a result of unsaturation in the pyranose ring
forcing them to adopt the half-chair conformation. The incorporation of amine
substituents within monosaccharide derivatives has also been shown to further enhance
inhibition.[14,15] For example, glycosylamines derived from D-glucose and D-mannose
display affinites for their respective hydrolases up to 1000 times higher than the parent
aldoses.[16] Similar findings have also been reported for D-galactosylamines.[17]
Furthermore, it is worth noting that the amine need not be directly attached to the
sugar ring, as b-D-glucosylmethylamine has also been shown to demonstrate moderate
enzymatic inhibitory activity.[18]
RESULTS AND DISCUSSION
As part of a programme directed towards the identification of novel inhibitors for
the glycoside processing enzymes, we were interested to determine whether unsaturated
aminopyranosides would be of use as mimics of glycosidase transition states. Four
compounds 5, 12, 15 and 18 that contained both endocyclic double bonds and amine
functionalities were identified as synthetic targets (Figure 1).
At the beginning of our programme, MOPAC molecular modelling studies were
performed to probe the lowest energy conformations of the protonated targets and in
each case this was found to be the half-chair conformation. Encouraged by these
results, synthetic routes to 5, 12, 15 and 18 were devised. The partially protected
mannopyranoside 1[19] proved a key intermediate for entry to target 5 (Scheme 1).
Oxidation of the alcohol using pyridinium chlorochromate (PCC) in toluene at reflux
allowed one-pot entry to the required enal 2 in 58% yield.[20] Installation of the amine
moiety at C-6 of enal 2 to afford intermediate 3 was achieved by reductive amination.

Synthesis of Unsaturated Aminopyranosides
707
Figure 1.
The 6-hydroxy derivative 4 was also formed as a by-product in 10% yield. Removal of
the benzoate groups from the C-2 and C-3 hydroxyl groups of 3 was achieved using
K2CO3 or NaOMe in MeOH to afford the required target 5 in good yield.
Glycal 6[21] proved to be a convenient precursor to target 12 via a Ferrier
glycosylation[22] with methanol (Scheme 2).
Optimum results for the Ferrier reaction were obtained using either BF3 ꢀ OEt2 or
SnCl4 as catalyst, affording yields and ratios of methyl glycosides[23] 7 and 7 of 43%
(8:1) and 49% (16:3) respectively. However, separation of the anomers proved impo-
ssible by column chromatography. The mixture of enopyranosides 7 and 7 thus
formed was therefore treated with LiAlH4 to induce reductive elimination of the ace-
tates,[24] to afford diol 8.[25] The reaction presumably proceeds via the formation of the
intermediate allylic ketone and the highly stereoselective reduction of the ketone, as a
result of hydride approach from the less hindered face of the molecule. Thus for the
required methyl-a-glycoside 7 , hydride attack occurs from the beta face. Separation of
the anomers of 8 was possible at this stage, affording the a-D-erythro derivative 8 in
61% yield.
In order to introduce the amine moiety at C-6, diol 8 was converted to tosylate 9
in good yield via treatment with p-toluenesulfonyl chloride and triethyamine. Optimum
Scheme 1. i) PCC, silica gel, toluene, 58%; ii) NH4OAc, NaBH3CN, MeOH, 49% 3 and 10% 4;
iii) MeOH, K2CO3, 74%.

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Jordan et al.
Scheme 2. i) MeOH, BF3ꢀOEt2, 7 :7 8: 1 43%; ii) LiAlH4, THF, 61%; iii) p-TsCl, Et3N, DCM,
73%; iv) PPh3, PhCO2H, DEAD, 90%; v) NaN3, DMF, 42%; vi) LiAlH4, Et2O, 83%.
yields were obtained by performing the reaction under high dilution, but some of the
di-tosylated derivative was consistently formed. Prior to introduction of the amine
moiety at C-6, the hydroxyl group at C-2 was inverted via a Mitsunobu reaction, under
standard reaction conditions, to afford the required a-D-threo derivative 10 in excellent
yield. Pleasingly, no allylic rearrangement was observed, and the inversion of confi-
guration at C-2 was confirmed by the reduced value of the J1,2 coupling constant (0.8
Hz in 10 versus 4.4 Hz in 9). Indirect introduction of the amine moiety to C-6 was
achieved by displacement of the C-6 tosylate with sodium azide in DMF, at reflux, to
afford azide 11 in moderate yield. Finally, reduction of the azide and deprotection of
the C-2 benzoate ester was achieved in one-pot using lithium aluminium hydride in
ether at reflux, to afford target 12 in an excellent 83% yield over two steps.
Synthetic entry to the 1-aminomethyl 15 and 3-amino 18 targets was possible from
3,4,6-tri-O-acetyl-D-glucal (Schemes 3 and 4). Lewis acid mediated reaction of 3,4,6-tri-
O-acetyl glucal with trimethylsilylcyanide afforded the acetylated derivative 13 as a
mixture of anomers.[26] Column chromatography allowed isolation of the required
a-anomer, 13 , in moderate yield. Removal of the acetate groups was achieved via acid
mediated hydrolysis, to afford diol 14 in 76% yield. Subsequent reduction of the nitrile
moiety using lithium aluminium hydride in ether, at reflux, gave the amine derivative
15 in excellent yield.
Scheme 3. i) TMSCN, BF3ꢀOEt2, DCM, 38%; ii) p-TsOH, MeOH, 76%; iii) LiAlH4, Et2O, 80%.

Synthesis of Unsaturated Aminopyranosides
709
Scheme 4. i) TMSN3, BF3ꢀOEt2, MeCN; ii) MeOH, Na, 36% over 2 steps; iii) LiAlH4, Et2O,
52%.
The synthesis of the C-3 aminoglycal 18 was achieved by Lewis acid mediated
reaction of 3,4,6-tri-O-acetyl-D-glucal with trimethylsilylazide.[27] Purification was best
achieved after the following deacetylation step, affording the azidoglucal 17[27] in 36%
yield over 2 steps. Selective reduction of the azide, in the presence of the glycal double
bond, to afford the aminoglycal 18[27] was then achieved employing lithium aluminium
hydride, in ether, at reflux.
Enzyme Inhibition Studies
The abilities of compounds 5, 12, 15 and 18 to inhibit a range of glycosidases were
analysed. Their activities against two a-glucosidases (EC 3.2.1.20 from brewers yeast
and from Bacillus stearothermophilus), a b-glucosidase (EC 3.2.1.21 from almonds)
and an a-mannosidase (EC 3.2.1.24 from Jack beans) were assessed spectrophotomet-
rically using a p-nitrophenyl glycoside as substrate.[28,29] No inhibition of a-man-
nosidase or b-glucosidase was found with any of the inhibitors tested. Amine 15
displayed very weak inhibition of the a-glucosidases; a 4.1 mM concentration resulted
in 16% reduction of activity of the B. stearothermophilus enzyme. Stronger inhibition
of a-glucosidases from B. stearothermophilus and yeast was seen with target 5 (IC50
values of 27 mM and 1.1 mM respectively).
CONCLUSIONS
Synthesis of four unsaturated amine-containing carbohydrate derivatives has been
achieved and preliminary glycosidase inhibition data have been collected. This has
illustrated that targets 5 and 15 displayed moderate inhibitory activity against a-
glucosidases from B. stearothermophilus and yeast.
EXPERIMENTAL
General methods. All NMR spectra were recorded on a Bruker WM250 or
Bruker AMX400 spectrometer, using CHCl3 as an internal standard unless stated
1
otherwise (7.26 ppm for H NMR, 77.0 ppm for 13C NMR). Coupling constants are
expressed in Hz. 13C spectra were recorded using Distortionless Enhancement by
Polarisation Transfer. Mass spectra were recorded on a Fisons VG Autospec. Infrared

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Jordan et al.
spectra were recorded on a Perkin-Elmer Paragon 1000 FT-IR spectrometer. Optical
activities were determined using a Perkin-Elmer 341 polarimeter. Melting points were
determined using an Electrothermal digital melting point apparatus, and are
uncorrected. All chemicals and materials were obtained from the Sigma-Aldrich
Chemical Company, the B.D.H. Chemical Company or Lancaster Chemicals and were
used as received. Anhydrous tetrahydrofuran was distilled from sodium and benzo-
phenone immediately prior to use. Other anhydrous solvents were purchased and used
as received. Molecular modeling was carried out using CS Chem3D Pro 4.0, on a Dell
XPS T700r workstation. The semi-empirical MOPAC molecular computation
application was employed, with PM3 (Parameterised Model revision 3) as the force-
field. The structures discussed are the lowest energy conformations found after mini-
mization. Compounds 3-azido-4,6-di-O-acetyl-3-deoxy-D-glucal 16, 3-azido-3-deoxy-D-
glucal 17 and 3-amino-3-deoxy-D-glucal 18 were all prepared as previously described
in the literature.[27]
Methyl 2,3-di-O-benzoyl-4-deoxy-6-aldehydo- -L-erythro-hex-4-enopyranoside
(2). A dry 250 mL 3-necked round bottom flask was charged with Merck silica gel
60 (ꢁ5 g). The flask was fitted with an efficient reflux condenser and a magnetic
stirring bead, sealed and the system was flushed with nitrogen. A solution of 1[19] (6.43
g, 12.7 mmol) dissolved in anhydrous toluene (100 mL) was added to the reaction
vessel via syringe, followed by a further 60 mL anhydrous toluene. PCC (4.18 g, 19.4
mmol) was added portion-wise and the reaction mixture was heated at reflux for 36
h.[20] After cooling to rt the solution was filtered through a Celite1 pad. The crude
residue was washed with toluene (3 ꢂ 30 mL) and solvents removed in vacuo to yield
a brown oil. After purification by flash column chromatography (hexane: Et2O, 1:1, v/
20
v) the desired product 2 was obtained as a white foam (2.82 g, 58%). [a]D + 133.3 (c
1, chloroform); Rf 0.32 (hexane: Et2O, 1:1, v/v); nmax (liquid film)/cmꢃ1 3071 (C-
Harom), 2936 (br, C-H), 2848 (w), 1729 (s, C O), 1647 (w), 1602 and 1585 (C-
Harom), 1452 (m), 1411 (w), 1316 (m), 1272 (s), 1177 (m), 1138 (m), 1113 (m), 1070
1
(m), 1026 (w), 989 (w), 915 (m), 709 (m); H NMR (250 MHz; CDCl3) d 3.51 (3H, s,
OCH3), 5.26 (1H, d, J 2.8, H-1), 5.63 (1H, dd, J 4.4 and 1.7, H-2), 5.96 (10H, PhH,
benzoyl), 9.24 (1H, s, H-6); 13C NMR (62.8 MHz; CDCl3) d 57.3 (OCH3), 64.8 (C-3),
65.4 (C-2), 99.2 (C-1), 118.6 (C-4), 128.7–130.5 (CHarom, Cquart, benzoyl), 133.9
and 134.0 (C O, benzoyl), 149.8 (C-5), 186.3 (C-6); m/z (CI), 383 ([M + H]+, 11%),
261 (100), 231 (69), 139 (24), 105 (74), 94 (7), 77 (16). HRMS (CI) Calcd for
C21H19O7: 383.1131. Found: [M + H]+ 383.1162.
Methyl 2,3-di-O-benzoyl-4,6-dideoxy-6-amino- -L-erythro-hex-4-enopyranoside
(3) and methyl 2,3-di-O-benzoyl-4-deoxy- -L-erythro-hex-4-enopyranoside (4). To
a stirred solution of ammonium acetate (1.46 g, 18.9 mmol), sodium cyanoborohydride
˚
(82.7 mg, 1.32 mmol) and 3 A activated molecular sieves in anhydrous methanol (35
mL), was added aldehyde 2 (0.72 g, 1.88 mmol) dissolved in anhydrous methanol (5
mL). The reaction was stirred at rt for 14.5 h and then filtered through a Celite1 pad,
washed with methanol (2 ꢂ 20 mL) and solvents removed in vacuo. The crude residue
was taken up in a minimum of water (ꢁ100 mL) and extracted with chloroform
(6 ꢂ 30 mL). The combined organic layers were dried (Na2SO4), filtered and solvents

Synthesis of Unsaturated Aminopyranosides
711
removed in vacuo. Purification by flash column chromatography (hexane: Et2O, 1:1,
20
v/v) gave the desired product 3 as a pale orange foam (207 mg, 28%). [a]D + 129.2
(c 1, chloroform); Rf 0.26 (Et2O); nmax (liquid film)/cmꢃ1 3065 (C-Harom), 2928 (br,
C-H), 2842 (w), 1728 (s, C O), 1681 (w), 1603 and 1586 (C-Harom), 1449 (m), 1313
(m), 1273 (s), 1173 (m), 1141 (m), 1116 (s), 1069 (m), 1026 (w), 978 (w), 915 (m),
1
711 (m); H NMR (250 MHz; CDCl3) d 3.31 (2H, s, 2 ꢂ H-6), 3.48 (3H, s, OCH3),
4.98 (1H, d, J 2.3, H-1), 5.13 (1H, d, J 4.9, H-4), 5.46–5.50 (1H, m, H-3), 5.79–5.83
(1H, m, H-2), 7.27–7.43 and 7.83–7.94 (10H, m, PhH, benzoyl); 13C NMR (62.8
MHz; CDCl3) d 50.4 (C-6), 56.9 (OCH3), 65.3 (C-2), 67.0 (C-3), 96.9 (C-1), 99.1 (C-
4), 128.7–133.7 (CHarom, Cquart, benzoyl), 152.1 (C-5), 166.0 and 166.2 (C O,
benzoyl); m/z (CI), 384 ([M + H]+, 16%), 232 (7), 140 (5), 105 (100), 78 (30). HRMS
(CI) Calcd for C21H22NO6: 384.1447. Found: [M + H]+ 384.1444.
20
Further elution gave 4 (71 mg, 10%) as a yellow semi-solid. [a]D + 117.6 (c 1,
chloroform); Rf 0.51 (Et2O); nmax (liquid film)/cmꢃ1 3510 (br, O-H), 3062 (w, C-
Harom), 2938 (br, C-H), 2841 (w), 1728 (s, C O), 1601 and 1588 (w, C-Harom),
1450 (w), 1315 (m), 1274 (s), 1177 (w), 1116 (m), 1071 (m), 1027 (w), 913 (w), 710
1
(m); H NMR (250 MHz; CDCl3) d 3.51 (3H, s, OCH3), 4.09 (2H, s, 2 ꢂ H-6), 5.09
(1H, d, J 2.5, H-1), 5.15 (1H, d, J 5.0, H-4), 5.48–5.51 (1H, m, H-3), 5.82 (1H, dd, J
4.4 and 3.2, H-2), 7.29–7.37 and 7.85–8.01 (10H, m, PhH, benzoyl); 13C NMR (62.8
MHz; CDCl3) d 57.0 (OCH3), 62.7 (C-6), 65.2 (C-2), 67.1 (C-3), 95.9 (C-1), 99.1 (C-
4), 128.7–133.7 (CHarom, Cquart, benzoyl), 153.4 (C-5), 165.9 and 166.2 (C O,
benzoyl); m/z (CI), 385 ([M + H]+, 11%), 263 (15), 231 (14), 141 (23), 122 (10), 105
(100), 77 (13).
Methyl 4,6-dideoxy-6-amino- -L-erythro-hex-4-enopyranoside (5). To a solu-
tion of hex-4-enopyranoside 3 (43 mg, 0.16 mmol) in absolute methanol (25 mL) was
added anhydrous potassium carbonate (51 mg, 0.37 mmol). The solution was stirred at
rt until TLC analysis showed an absence of any starting material (ca. 2.5 h). Solvents
were removed in vacuo to yield the crude product and residual K2CO3, purification by
flash column chromatography (EtOH: ammonium hydroxide, 9:1, v/v) furnished the
20
target compound 5 as a yellow foam (14 mg, 48%). [a]D + 87.0 (c 1, methanol); Rf
0.33 (EtOH: ammonium hydroxide, 9:1, v/v); nmax (liquid film)/cmꢃ1 3420 (br, O-H),
2943 (m, C-H), 2852 (w, O-CH3), 1648 (m, -NH2), 1447 (w), 1347 (w), 1261 (w), 1182
1
(w), 1149 (m), 1065 (m), 1026 (m), 990 (m); H NMR (400 MHz; CD3OD) d 3.16–
3.26 (2H, m, 2 ꢂ H-6), 3.53 (3H, s, OCH3), 3.67 (1H, dd, J 3.5 and 0.8, H-3), 4.20–
4.23 (1H, m, H-2), 4.84 (1H, d, J 3.2, H-4), 4.86 (1H, d, J 0.9, H-1); 13C NMR (100
MHz; CD3OD) d 50.6 (C-6), 56.8 (OCH3), 63.9 (C-2), 68.7 (C-3), 101.8 (C-1), 102.3
(C-4), 150.3 (C-5); m/z (CI), 102 (34), 85 (100).
Methyl 2,4,6-tri-O-acetyl-3-deoxy- -D-erythro-hex-2-enopyranoside (7).[23] To
a stirred solution of 2,3,4,6-tetra-O-acetyl-D-glucal 6[21] (5.10 g, 15.4 mmol) in
anhydrous toluene (100 mL) was added anhydrous methanol (1.25 mL, 30.9 mmol),
followed by boron trifluoride diethyl etherate (3.80 mL, 30.9 mmol).[22] After stirring
under an inert atmosphere for 25 min the reaction was quenched by the dropwise
addition of satd aq NaHCO3 soln. (100 mL). The resulting solution was extracted with
ethyl acetate (3 ꢂ 100 mL). The combined organic layers were washed with brine

712
Jordan et al.
(2 ꢂ 100 mL), dried (MgSO4), filtered and the solvents removed in vacuo to yield a
yellow oil. Purification by flash column chromatography (Et2O) furnished the hex-2-
enopyranoside 7 as a colourless syrup (2.01 g, 43%, inseparable anomeric mixture; 9:2,
a:b). Characterisation for this mixture was in agreement with that reported in the
literature.[23]
Methyl 3,4-dideoxy- -D-erythro-hex-3-enopyranoside (8).[25] To a stirred sus-
pension of LiAlH4 (0.41 g, 10.8 mmol) in anhydrous THF (20 mL) cooled to rt was
added a solution of hex-2-enopyranoside 7 (1.16 g, 3.85 mmol) in anhydrous THF (10
mL)[24] After stirring at 0°C under an inert atmosphere for 35 min the reaction was
quenched by the dropwise addition of a sat aq potassium sodium tartrate solution (ꢁ20
mL). The insoluble solids were filtered off through a Celite1 pad and washed with
EtOAc (100 mL). The resulting filtrate was dried (MgSO4), filtered and solvents
removed in vacuo to yield a crude mixture of anomers. Purification by flash column
chromatography (EtOH: ammonium hydroxide, 9:1, v/v) allowed separation of the
anomers, furnishing methyl-3,4-dideoxy-a-D-erythro-hex-3-enopyranoside 8 as a
colourless semi-solid (373 mg, 61%) whose data was in agreement with that reported
in the literature.[25]
Methyl 3,4-dideoxy-6-O-tosyl- -D-erythro-hex-3-enopyranoside (9). To a solu-
tion of hex-3-enopyranoside 8 (0.75 g, 4.68 mmol) in anhydrous DCM (50 mL) cooled
to 0°C was added dropwise TEA (1.30 mL) followed by a solution of p-toluenesulfonyl
chloride (recrystallised from hot ether) (893 mg, 4.68 mmol) in anhydrous DCM (10
mL). The reaction was stirred under an inert atmosphere and allowed to warm to rt
overnight. After 48 h the reaction mixture was quenched by the addition of water (100
mL) and extracted with DCM (3 ꢂ 100 mL). The combined organic extracts were dried
(MgSO4), filtered and solvents removed in vacuo. Purification by flash column
chromatography (toluene: EtOAc, 5:1, v/v) furnished the hex-3-enopyranoside 9 as a
20
colourless syrup (788 mg, 73%). [a]D + 0.9 (c 1, chloroform); Rf 0.32 (toluene:
EtOAc, 5:1, v/v); nmax (liquid film)/cmꢃ1 3509 (br, O-H), 3044 (w, C-Harom), 2935
(m, C-H), 2833 (m, O-CH3), 1596 (w), 1450 (w), 1402 (w), 1358 (m, S O), 1174 (s,
1
O), 1096 (m), 1049 (m), 1032 (m), 970 (m), 899 (w), 814 (w), 661 (m); H NMR
S
(250 MHz; CDCl3) d 1.92 (br, s, OH), 2.38 (3H, s, CH3, tosyl), 3.38 (3H, s, OCH3),
3.92–4.01 (2H, m, H-6), 4.03–4.06 (1H, m, H-2), 4.21–4.23 (1H, m, H-5), 4.73 (1H,
d, J 4.4, H-1), 5.54 (1H, dddd, J 10.6, 1.0, 1.0 and 1.0, H-4), 5.68–5.73 (1H, dm, J
10.6, H-3), 7.26–7.29 and 7.70–7.75 (4H, m, PhH, tosyl); 13C NMR (62.8 MHz;
CDCl3) d 22.1 (CH3, tosyl), 56.6 (OCH3), 64.3 (C-2), 66.7 (C-5), 71.3 (C-6), 98.3
(C-1), 125.0 (C-4), 128.4 (CHarom, tosyl), 130.0 (C-3), 130.2 (CHarom, tosyl), 133.3
and 145.4 (Cquart, tosyl); m/z (CI), 332 ([M + NH4]+, 22%), 300 (31), 143 (11), 111
(51), 82 (100), 54 (24). HRMS (CI) Calcd for C14H22NO6S : 332.1168. Found:
332.1169.
Methyl 2-O-benzoyl-3,4-dideoxy-6-O-tosyl- -D-threo-hex-3-enopyranoside (10).
To a solution of hex-3-enopyranoside 9 (0.70 g, 2.23 mmol), triphenylphosphine (2.63 g,
10.0 mmol) and benzoic acid (0.82 g, 6.68 mmol) in anhydrous THF (15 mL) cooled to
0°C was added diethyl azodicarboxylate (DEAD) (1.94 g, 1.76 mL, 10.9 mmol) dropwise.
The reaction mixture was stirred at 0°C under an inert atmosphere for 30 min, then

Synthesis of Unsaturated Aminopyranosides
713
concentrated in vacuo. The resulting residue was diluted with diethyl ether (100 mL) and
washed successively with satd aq NaHCO3 soln. (3 ꢂ 100 mL) and brine (2 ꢂ 100 mL).
The organic extract was dried (MgSO4), filtered and solvents removed in vacuo to yield an
orange/pink syrup. Purification by flash column chromatography (toluene: Et2O, 3:1, v/v)
20
furnished the benzoate 10 as a pale pink oil (835 mg, 90%). [a]D + 73.7 (c 1,
chloroform); Rf 0.24 (toluene: Et2O, 3:1, v/v); nmax (liquid film)/cmꢃ1 3051 (w, C-
Harom), 2976 (m, C-H), 2935 (m, C-H), 2833 (w, O-CH3), 1759 (s, C O), 1715 (s), 1599
(m), 1450 (m), 1365 (s, S O), 1314 (m), 1252 (m), 1174 (s, S O), 1110 (m), 1069 (m),
977 (w), 811 (w), 712 (m); 1H NMR (400 MHz; CDCl3) d 2.42 (3H, s, CH3, tosyl), 3.46
(3H, s, OCH3), 4.16–4.25 (2H, m, H-6), 4.34–4.44 (1H, m, H-5), 4.87 (1H, d, J 0.8, H-1),
5.13–5.16 (1H, m, H-2), 5.98 (1H, dd, J 11.8 and 1.38, H-4), 6.01–6.06 (1H, m, H-3),
7.29–7.33, 7.44–7.48, 7.71–7.83 and 8.02–8.06 (9H, m, PhH, tosyl and benzoyl); 13C
NMR (100 MHz; CDCl3) d 21.6 (CH3, tosyl), 56.1 (OCH3), 65.3 (C-2), 65.9 (C-5), 70.5
(C-6), 98.6 (C-1), 122.9 (C-3), 129.6 (C-4), 127.9–134.3 (CHarom, tosyl and benzoyl),
144.3 and 152.3 (Cquart, tosyl), 165.7 (Cquart, benzoyl), 169.1 (C O, benzoyl); m/z (CI),
436 ([M + NH4]+, 20%), 387 (90%, M+-OMe), 353 (8), 295 (15), 264 (14), 233 (18), 177
(6), 155 (7), 105 (100), 77 (43). HRMS (CI) Calcd for C21H26NO7S: 436.1430. Found:
436.1425.
Methyl 2-O-benzoyl-3,4,6-trideoxy-6-azido- -D-threo-hex-3-enopyranoside (11).
A solution of hex-3-enopyranoside 10 (813 mg, 1.94 mmol) and NaN3 (1.26 g, 19.4
mmol) in anhydrous N,N-dimethylformamide (20 mL) was stirred under an inert
atmosphere at rt overnight. The progress of the reaction was studied by TLC analysis.
After 44 h TLC analysis (hexane: EtOAc, 3:1, v/v) indicated that no change had
occurred. The reaction mixture was then heated at 75–80°C for 2 h. The solution was
cooled to rt and solvents removed in vacuo. The resulting residue was taken up in Et2O
(50 mL) and washed with water (3 ꢂ 50 mL). The organic extract was dried (MgSO4),
filtered and solvents removed in vacuo to yield a yellow syrup. Purification by flash
column chromatography (hexane: EtOAc, 3:1, v/v) yielded the azide 11 as a colourless
20
oil (235 mg, 42%). [a]D + 262.7 (c 1, chloroform); Rf 0.35 (hexane: EtOAc, 3:1, v/
v); nmax (liquid film)/cmꢃ1 3058 (w, C-Harom), 2928 (m, C-H), 2833 (w, O-CH3),
2096 (s, N3), 1718 (s, C O), 1599 (m), 1534 (w), 1443 (m), 1314 (m), 1263 (m), 1106
1
(m), 1062 (m), 1025 (w), 998 (w), 950 (w), 712 (m); H NMR (400 MHz; CDCl3) d
3.25 (1H, dd, J 11.9 and 6.0, H-6), 3.43 (1H, dd, J 11.9 and 4.0, H-6), 3.44 (3H, s,
OCH3), 4.31–4.36 (1H, m, H-5), 4.88 (1H, d, J 0.8, H-1), 5.09–5.12 (1H, m, H-2),
5.91 (1H, dd, J 10.9 and 1.1, H-4), 5.95–6.00 (1H, m, H-3), 7.31–7.38, 7.45–7.48 and
7.98–8.02 (5H, m, PhH, benzoyl); 13C NMR (100 MHz; CDCl3) d 54.5 (C-6), 55.2
(OCH3), 62.1 (C-2), 66.4 (C-5), 99.8 (C-1), 123.4 (C-3), 132.5 (C-4), 129.3–139.0
(CHarom, Cquart, benzoyl), 166.8 (C O, benzoyl); m/z (CI), 290 ([M + H]+, 9%), 258
(81), 202 (10), 105 (100), 77 (11). HRMS (CI) Calcd for C14H16N3O4: 290.1141.
Found: 290.1134.
Methyl 3,4,6-trideoxy-6-amino- -D-threo-hex-3-enopyranoside (12). LiAlH4
(65 mg, 1.80 mmol) was dissolved in anhydrous Et2O (8 mL) and brought to reflux.
A solution of azide 11 (55 mg, 0.19 mmol) dissolved in anhydrous diethyl ether (4 mL)
was added via syringe to the refluxing suspension of LiAlH4. The reaction mixture was
heated at reflux for 2.5 h. After cooling to rt the reaction was quenched by the

714
Jordan et al.
dropwise addition of satd aq potassium sodium tartrate soln. (ꢁ10 mL). The insoluble
solids were filtered off through a Celite1 pad and washed with warm methanol (ꢁ50
mL). The resulting filtrate was dried (MgSO4), filtered and solvents removed in vacuo
to yield a yellow oil. Purification by flash column chromatography (EtOH: ammonium
hydroxide, 9:1, v/v) gave the target compound 12 as a pale yellow oil (25 mg, 83%).
20
[a]D + 8.5 (c 1, methanol); Rf 0.52 (EtOH: ammonium hydroxide, 9:1, v/v); nmax
(liquid film)/cmꢃ1 3392 (br, O-H), 2934 (m, C-H), 2843 (w), 1642 (m, -NH2), 1567
(m), 1488 (m), 1384 (w), 1322 (w), 1195 (w), 1124 (m), 1065 (m), 970 (w), 905 (w);
1H NMR (400 MHz; Acetone-d6) d 3.29–3.43 (2H, m, H-6), 3.39 (3H, s, OCH3),
3.62–3.66 (1H, m, H-2), 4.35–4.40 (1H, m, H-5), 4.66 (1H, d, J 0.8, H-1), 5.86 (1H,
dd, J 10.3 and 2.0, H-4), 5.95–6.01 (1H, m, H-3); 13C NMR (100 MHz; Acetone-d6) d
54.8 (C-6), 55.7 (OCH3), 64.3 (C-2), 69.8 (C-5), 103.1 (C-1), 127.2 (C-3), 131.7 (C-4);
m/z (CI), 160 ([M + H]+, 100%, ), 128 (38), 112 (50), 81 (53), 43 (18). HRMS (CI)
Calcd for C7H14NO3: 160.0974. Found: 160.0977.
2,3-Dideoxy-4,6-di-O-acetyl- -D-erythro-hex-2-enopyranosyl cyanide (13).[26] To
a stirred solution of 3,4,6-tri-O-acetyl-D-glucal (0.2 g, 0.73 mmol) in anhydrous
dichloromethane (3.5 mL) was added trimethylsilylcyanide (0.08 g, 0.1 mL 0.8 mmol),
followed by boron trifluoride diethyl etherate (1 drop)[26] After stirring for five minutes,
the solution was poured into diethyl ether (20 mL), washed with saturated sodium
bicarbonate solution (5 mL) and water (10 mL) and dried (magnesium sulfate). After
filtration through a short pad of silica gel, the solution was concentrated in vacuo and
purified by column chromatography (hexane:ethyl acetate, 4:1 v/v) to give the
a-cyanoglycal 13 as a white powder (0.31 g, 38%). Data for 13 agreed with that re-
ported previously in the literature.[26]
2,3-Dideoxy- -D-erythro-hex-2-enopyranosyl cyanide (14). To a stirred solution
of 13 (0.2 g, 0.89 mmol) in anhydrous methanol (10 mL) was added p-toluenesulfonic
acid (0.04 g) and the solution was stirred overnight. After neutralisation with barium
carbonate, the solution was filtered and concentrated in vacuo. Column chromatography
20
on silica gel afforded the diol 14 as a colourless oil (0.05 g, 40%). [a]D ꢃ 50.0 (c
0.25, MeOH), Rf 0.19 (ethyl acetate:hexane 2:1 v/v); nmax (KBr disc)/cmꢃ1 3419, 2928,
1
1714, 1684, 1243, 1186, 1089, 971, 901; H NMR (400 MHz, CDCl3) d 3.62 (1H, m,
H-5), 3.75 (1H, dd, J 5.9, H-6), 3.94 (1H, d, J 12.0, H-6), 4.12 (1H, d, J 8.8, H-4), 5.25
(1H, br s, H-3), 5.91 (1H, d, J 9.9, H-2), 6.09 (1H, d, J 9.9, H-1); 13C NMR (100 MHz,
CDCl3) d 62.3 (CH2), 62.5 (CH), 63.7 (CH), 81.6 (CH), 117.8 (C), 123.7 (CH), 134.8
(CH); m/z (CI) 173 ([M + NH4]+, 15%), 129 (25), 111 (20), 81 (1), 68 (100), 55 (5), 43
(30). HRMS (CI) Calcd for C7H9NO3: 173.0926. Found: 173.0919.
1-Aminomethyl-2,3-dideoxy- -D-erythro-hex-2-enopyranose (15). A solution of
glycal 14 (1.0 g, 4.16 mmol) in anhydrous diethyl ether (3 mL) was added dropwise to
a suspension of LiAlH4 in anhydrous diethyl ether (5 mL) at reflux. After heating at
reflux for 2.5 hours, the solution was cooled and crushed ice was added until
effervescence ceased. The insoluble solids were filtered off and washed with warm
methanol. The combined organics were concentrated to dryness and submitted to
column chromatography (ethanol:ammonium hydroxide 9:1 v/v) to afford the
aminoglycal 15 as a colourless oil (0.53 g, 80 %); [a]D20+ 53 (c 0.4, chloroform);

Synthesis of Unsaturated Aminopyranosides
715
Rf 0.19 (silica, ethanol:ammonium hydroxide 9:1 v/v); nmax (KBr disc)/cmꢃ13420 (br),
1
2924, 1700, 1558, 1040; H NMR (400 MHz, CDCl3) d 2.63 and 2.78 (2H, 2 ꢂ br s,
CH2NH2), 3.39 (1H, br t, J 7.0, H-6), 3.51 (1H, dd, J 12.1, 7.0, H-5), 3.75 (2H, m, H-4
and H-6) 4.06 (1H, br d, J 9.2, H-1), 5.67 (1H, d, J 10.6, H-2), 5.76 (1H, d, J 10.6, H-
3); 13C NMR (100 MHz, CDCl3) d 63.1 (CH2), 64.0 (CH), 75.7 (CH), 128.8 (CH),
131.5 (CH); m/z (CI) 160 ([M + H]+, 80%), 142 (5), 112 (15), 94 (10), 81 (100).
HRMS (CI) Calcd for C7H17NO3: 160.0973. Found: 160.0970.
Enzyme Inhibition Studies
These studies were conducted essentially as described in the literature.[28,29]
Enzymes were obtained from Sigma, and diluted in 0.02 M sodium acetate buffer,
except for Jack bean mannosidase, which was diluted in 0.05 M sodium acetate buffer
containing 0.1 mM zinc sulfate. a-Mannosidase and b-glucosidase experiments were
conducted at pH 5.0 whilst a-glucosidase experiments were conducted at pH 6.8.
Enzyme assays were incubated over a period of 30 min. a-Mannosidase inhibition
assays employed p-nitrophenol a-D-mannopyranoside and other enzymes were assayed
using the corresponding conjugate at 5 mM concentration in an appropriate buffer.
Absorbances at 400 nm were recorded using a Perkin Elmer Lambda Bio UV/Visi-
ble spectrophotometer.
ACKNOWLEDGMENTS
We gratefully acknowledge the BBSRC (Post-doctoral fellowship to A.M.J.) and
the Royal Society for their financial support of this work.
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Received January 27, 2003
Accepted July 24, 2003