Syntheses of sugar-related trihydroxyazepanes from simple carbohydrates and their activities as reversible glycosidase inhibitors

Article abstract of DOI:10.1016/S0008-6215(00)00018-5

Five diastereomeric trideoxy-1,6-iminohexitols were synthesised, and their inhibitory activities were determined against selected glycosidases. For comparison, 1,4,5-trideoxy-1,5-imino-D-lyxo-hexitol, the 4-deoxy derivative of 1-deoxymannojirimicin, was prepared by enzymatic isomerisation of 6-azido-3,6-dideoxy-D-ribo-hexose into the corresponding 2-ulose and subsequent hydrogenation accompanied by intramolecular reductive amination. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0008-6215(00)00018-5

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Carbohydrate Research 326 (2000) 22–33
Syntheses of sugar-related trihydroxyazepanes from
simple carbohydrates and their activities as reversible
glycosidase inhibitors
Søren M. Andersen a, Christian Ekhart b, Inge Lundt a,*, Arnold E. Stu¨tz b
a Department of Organic Chemistry, Technical Uni6ersity of Denmark, Building 201, DK-2800 Lyngby, Denmark
b Institut fu¨r Organische Chemie der Technischen Uni6ersita¨t Graz, Stremayrgasse 16, A-8010 Graz, Austria
Received 8 July 1999; accepted 3 January 2000
Abstract
Five diastereomeric trideoxy-1,6-iminohexitols were synthesised, and their inhibitory activities were determined
against selected glycosidases. For comparison, 1,4,5-trideoxy-1,5-imino-
1-deoxymannojirimicin, was prepared by enzymatic isomerisation of 6-azido-3,6-dideoxy-
D
-lyxo-hexitol, the 4-deoxy derivative of
-ribo-hexose into the
D
corresponding 2-ulose and subsequent hydrogenation accompanied by intramolecular reductive amination. © 2000
Elsevier Science Ltd. All rights reserved.
Keywords: Trihydroxy azepanes; Glycosidase inhibitors; Simple carbohydrates
1. Introduction
Several sugar analogues with basic nitrogen
instead of oxygen in the ring (imino sugars)
have been discovered as natural products, and
have attracted considerable attention due to
their pronounced glycosidase inhibitory prop-
erties leading to notable biological effects [1].
Many
D
-mannosidases as well as known
a-L
-fucosidases are strongly inhibited by such
imino sugars and imino alditols structurally
related to their natural substrates. 1-Deoxy-
mannojirimycin (1) and the bicyclic alkaloid
swainsonine (2) are good mannosidase in-
hibitors, and 1-deoxyfuconojirimycin (3) is the
most powerful inhibitor of a- -fucosidases
L
known to date; thus these are important tools
for glycobiology.
Due to the similarity of the
the -fucosyl moieties, good
D
-mannosyl and
-mannosidase
-fucosi-
L
D
inhibitors frequently also inhibit a-
L
* Corresponding author. Tel.: +45-45-252133; fax: +45-
45-933968.
E-mail address: okil@pop.dtu.dk (I. Lundt)
dases and vice versa. Hence, with respect to
their potentially interesting properties as tools
0008-6215/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(00)00018-5

S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
23
and 31) and a regioisomer (compound 33)
were synthesised in order to estimate the im-
portance for biological activity of the presence
and orientation of different hydroxyl groups
on the ring. In addition, we wished to probe a
point raised by Winkler and Holan on the
minimum structural requirements of mannosi-
dase inhibitors related to 1-deoxymannojiri-
mycin, namely that the positions of the three
hydroxyl groups in swainsonine (2) are related
to the orientations of OH-2, OH-3, and OH-6
in 1-deoxymannojirimycin (1). Conveniently,
both issues could be addressed by the same
synthetic approach via easily available 6-
azido-3,6-dideoxysugars, taking advantage of
an additional enzymatic key step in the prepa-
in glycobiology and glycotechnology, selective
inhibitors of either -fucosidases or -man-
L
D
nosidases have been deemed highly desirable.
Basic structure–activity relationships in the
area of mannosidase inhibitors have been dis-
cussed in important work by Winkler and
Holan with the aid of computer-assisted
molecular modelling [2]. For example, these
workers correlated hydroxyl groups in the
powerful mannosidase inhibitor swainsonine
with hydroxyl groups at C-2, C-3, and C-6 in
1-deoxymannojirimycin. Despite their efforts,
quite a few activity-determining structural re-
quirements, such as conformational prerequi-
sites and the number and positions of
hydroxyl groups necessary for a good in-
hibitor, have remained not fully understood.
In context with our continuing interest in the
synthesis and biological evaluation of novel
ration of desired 1,4,5-trideoxy-1,5-imino- -
D
lyxo-hexitol (1,4-dideoxymannojirimycin, 35).
selective inhibitors of
well as a- -fucosidases [3–5], we became inter-
D
-mannosidases [3] as
L
2. Results and discussion
ested in seven-membered ring imino alditols as
potential inhibitors of the types of enzymes
under consideration. We hoped that due to
the flexibility of the azepane ring system, cer-
tain conformational advantages over the five-
and the six-membered ring inhibitors in terms
of fitting into the active sites of glycosidases
might emerge. As was suggested by molecular
modelling, trihydroxyazepanes bearing one
unsubstituted methylene group along the
chain might be interesting for the elucidation
of selectivities as well as structure–activity
relationships in these systems. Recently, such
a tetrahydroxyazepane (4) with C2-symmetry
was found by Wong and his group [6,7], as
well as by Le Merrer et al. [8], to exhibit
noteworthy glycosidase inhibitory properties
against quite a range of different types of
glycosidases.
This fact was rationalised by superposition-
ing the functional groups of the azepane and
those of a range of proven powerful glycosi-
dase inhibitors, which in many of the cases
reported were found to match nicely [7]. Thus,
we decided to synthesise two diastereomeric
trihydroxyazepanes, compounds 28 and 29,
which are structurally related to the estab-
Synthesis.—The 6-azido-3,6-dideoxysugars
can be easily prepared from partially pro-
tected 3-deoxysugars. Of these, the required
3-deoxy-D-ribo-hexose (5) is readily obtained
from 1,2:5,6-di-O-isopropylidene-a-
D-gluco-
furanose [9]. Other 1,2:5,6-protected 3-deoxy-
hexoses are more conveniently prepared by
conventional reduction [10] of 3-deoxy-hex-
ono-1,4-lactones [11], followed by protection
of the resulting free 3-deoxyaldoses. Regiose-
lective deprotection of O-5 and O-6 in the
1,2:5,6-di-O-isopropylidene protected 3-de-
oxyhexoses 5, 11, 17, and 23, followed by
6-O-sulfonylation, gave the primary tosylates
7, 13, 19, and 25, respectively. Subsequent
displacement with azide led to the correspond-
ing 6-azidodeoxy derivatives 8, 14, 20, and 26,
which by conventional deprotection furnished
the corresponding free 6-azido-3,6-dideoxy-
hexoses with
(21), and
D
-ribo (9),
D
-arabino (15), -xylo
D
L
-xylo (27) configurations, respec-
tively, in good yields (Scheme 1).
Catalytic reduction using hydrogen in the
presence of palladium-on-charcoal (5%) and
concomitant intramolecular reductive amina-
tion of the intermediary 6-aminodeoxy sugars
led to the desired 2,4,5-trihydroxyazepanes
28–31 (Scheme 2). The 3,4,5-trihydroxy-
lished inhibitors of
as well as -fucosidases (1 and 3). Further-
more, two additional epimers (compounds 30
D
-mannosidases (1 and 2),
L
azepane 33, a regioisomer of 29 with
D-ara-

24
S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
Scheme 1. (a) 90% AcOH (aq). (b) Tosyl chloride, pyridine. (c) NaN3, DMF, reflux 1 h. (d) Amberlite IR-120 (H+),
H2O–CH3CN, 40 °C. (e) Disiamylborane, THF. (f) Acetone, camphor sulfonic acid (cat).
bino configuration, was prepared by reduction
of the lactam 32, readily obtainable from 2,6-
in the absence of the enzyme under the slightly
basic conditions employed in this study. Cata-
lytic reduction of the ketose 34 furnished
dibromo-2,6-dideoxy- -mannolactone [12].
D
Following previous studies [13] and prelimi-
nary experiments [14] on the tolerance of an
immobilised glucose isomerase (Sweetzyme T
from Novo Nordisk A/S) for non-natural sub-
strate analogues, the azidodideoxyhexoses ob-
tained were exposed to this enzyme. Grati-
fyingly, azidodideoxysugar 9 could be con-
verted into the 6-azido-3,6-dideoxy derivative
1,4,5-trideoxy-1,5-imino- -lyxo-hexitol (1,4-
D
dideoxymannojirimycin, 35) in good yield and
with high diastereoselectivity.
Inhibition of glycosidases.—Activities of 28,
29, 30, 31, 33 and 35 were determined with a
variety of glycosidases, and the results are
listed in Table 1.
Trihydroxyazepanes 28–31 did not exhibit
any appreciable inhibitory activity against the
glycosidases employed in this study. Apart
from the lack of symmetry as compared with
active azepanes such as 4, the spatial distribu-
tion of functional groups in the preferred con-
of
D-fructofuranose 34 in more than 70% iso-
lated yield. Conversely, azidodideoxyhexoses
15, 21, and 27 did not give conversion rates
with glucose isomerase that were significantly
different from results of control experiments

S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
25
Scheme 2. (g) 5% Pd/C, H2, MeOH. (h) Glucose isomerase, H2O–CH3CN, 60 °C. (i) BH3·Me2S.
Table 1
Inhibition of glycosidases with compounds 28, 29, 30, 31, 33, and 35
Enzymes
Ki (mM) a
28
29
30
31
33
35
a-
b-
a-
a-
a-
b-
D
D
D
D
D
D
-Glucosidase (baker’s yeast)
-Glucosidase (almond)
-Galactosidase (green coffee bean)
-Galactosidase (Escherichia coli )
-Mannosidase (jack bean)
142
72
NI
NI
NI
NI
NI
NI
112
NI
NI
200
NI
NI
314
NI
NI
NI
NI
NI
NI
NI
76
13
NI
78
27
140
150
196
12
51
NI
NI
NI
NI
NI
107
800
13
NI
196
156
-Mannosidase (snail acetone powder)
b-N-Acetylglucosaminidase (jack bean)
a- -Fucosidase (bovine kidney)
72
<b306.
L
a NI: Ki\1 mM.
formations neither match the motifs of sub-
strates, nor those of proven inhibitors or pro-
posed transition states such as the putative
‘flap-up’ mannopyranosyl oxocarbonium ion.
The comparably good values found with
azepane 33 can be attributed to the fact that
this compound can adopt conformations that
superimpose to varying degrees with estab-
lished glycosidase inhibitors. Superposition of
azepane 33 with the glucosidase inhibitor 1-
deoxynojirimycin shows good alignment of
the secondary hydroxyl groups of both
molecules. The selectivity of 33 for a-glucosi-
dases can be related to the lack of a hydroxyl
group matching OH-6 of 1-deoxynojirimycin,
as it has been found that a-glucosidases, as
opposed to their b-specific counterparts, do
not require this structural feature for recogni-

26
S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
distinct deviation of the position of the ring
nitrogen. This and the lack of a hydroxy
group in the position of OH-6 seem to be
responsible for the lower activity as compared
with good inhibitors of
as 1-deoxymannojirimycin (1). The a-
D
-mannosidases such
-fucosi-
L
dase inhibitory activity exhibited by 33 was
expected, since the compound has the minimal
structural motif necessary for inhibition of
this enzyme [5]. The inhibition can then most
likely be attributed to the good fitting of the
ring nitrogen as well as all three hydroxyl
groups with the
mycin (3) structural motif (Fig. 1).
L
-fucose/1-deoxy- -fuconojiri-
L
Fig. 1. Comparison of the spatial characteristics among 1-de-
oxyfuconojirimycin (3), 4-epi-isofagomine (36) and trihydrox-
yazepane 33.
As with 1,5-dideoxy-1,5-imino- -arabinitol
D
(Ki =40 mM at pH 5 with the enzyme from
tion/binding [15]. Galactosidase inhibitory ac-
tion was found to be stronger with the b-
galactosidase probed. This might be due to the
fact that 33 exhibits a similar alignment of
functional groups as the 4-epi analogue of
isofagomine 36 (Ki=4 nM with b-galactosi-
dase from Aspergillus oryzae at pH 6.8) (Fig.
1) [16]. Mannosidase inhibitory activity of 33
was found in the same range for both the a-
and the b-specific enzyme probed. A compari-
son of Dreiding models and computer-aided
superposition with the ‘flap-up’ mannopyran-
osyl oxocarbocation ion (Fig. 2) shows excel-
lent matching of hydroxyl groups with 2-OH,
3-OH, as well as 4-OH of the latter, but a
bovine kidney) [4], the nor derivative of the
powerful inhibitor 1,5-dideoxy-1,5-imino- -
L
fucitol (3), the lack of the methyl group at
C-5, which was found to be a vital prerequisite
for pronounced inhibition, is limiting this par-
ticular activity. In Fig. 3 the superposition of
1,5-dideoxy-1,5-imino-
shown.
D-arabinitol with 33 is
The reasons for the activities found with the
4-deoxy derivative of 1-deoxymannojirimycin
(35) with glycosidases are not apparent. Its
selective b-galactosidase inhibitory activity
seems noteworthy.
In conclusion, the most active compounds
in this study, azepane 33 and piperidine 35,
were found to be more effective against
cosidases and -galactosidases than against
the other enzymes probed. This points to the
D-glu-
D
Fig. 2. Superposition of mannopyranosyl oxocarbonium ion
with compound 33.
Fig. 3. Superposition of 1,5-dideoxy-1,5-imino-D-arabinitol
with compound 33.

S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
27
fact that in the azepane series, three vicinal
hydroxyl groups appear to be essential for
basic glycosidase inhibitory activity. In the
case of piperidine 35, deoxygenation at C-4
and, consequently, improved flexibility
provide an interesting shift from selectivity for
and (6) the molecules were minimised after
sketching. Simulated annealing, for 2000 fs at
600 K, and then cooling down for 10,000 fs to
50 K in 10 separate cycles, minimised before
annealing, and cooled in a logarithmic func-
tion. The best four candidates were discrimi-
nated by the lowest potential energy after
minimisation and clustering of the simulated
annealing sampled conformations.
D
-mannosidases and a-
with the parent compound 1 to activity
against -glucosidase and b- -galactosidase.
L
-fucosidase found
D
D
General procedures
Regioselecti6e remo6al of the 5,6-O-iso-
propylidene group. The 3-deoxy-1,2:5,6-di-O-
isopropylidenehexofuranose (1 g) was stirred
at ambient temperature in 90% aq AcOH (10
mL). The reaction was followed by TLC.
When the reaction was complete (6–10 h), the
solution was neutralised and concentrated.
Regioselecti6e tosylation. To a solution of
3-deoxy-1,2-O-isopropylidenehexofuranose (1
g) in pyridine (20 mL), tosyl chloride (1.2
equiv) was added at 0 °C. The mixture was
stirred at ambient temperature for 16 h. Ice
was added, and the reaction mixture was con-
centrated to a residue that was dissolved in
CH2Cl2 (30 mL). The organic phase was
washed consecutively with 4 M HCl (10 mL)
and satd aq NaHCO3 (10 mL), dried
(MgSO4), and concentrated.
Nucleophilic substitution with azide. NaN3
(1.5 equiv) was added to a solution of 3-deoxy-
6 - O - tosyl - 1,2 - O - isopropylidenehexofura-
nose (1 g) in N,N-dimethylformamide (10
mL). The mixture was held at reflux for 1 h
with protection from light. The mixture was
then concentrated, and partitioned between
diethyl ether (40 mL) and water (10 mL). The
organic phase was washed with water (10
mL), treated with activated carbon, dried
(MgSO4), and concentrated.
3. Experimental
General methods.—1H and 13C NMR spec-
tra were obtained on Bruker instruments AC
200, AC 250 and AM 500. Chemical shifts are
reported in l (ppm), and coupling constants
are given in Hz. For NMR spectra in CDCl3,
the chloroform signal was used as reference
1
for H NMR spectra (7.27 ppm) and 13C
NMR spectra (77.0 ppm). For NMR spectra
in MeOH-d4, the MeOH signal was used as a
1
reference (3.31 ppm for the H NMR spectra
and 49.0 ppm for the 13C NMR spectra).
Melting points are uncorrected. Optical rota-
tions were measured with a Perkin–Elmer 241
polarimeter. Microanalyses were carried out
at the Institut fu¨r Physikalische Chemie der
Universita¨t Wien and the Research Institute
for Pharmacy and Biochemistry, Prague.
Thin-layer chromatography (TLC) was per-
formed on E. Merck Silica Gel 60 F254 pre-
coated plates and was visualised by spraying
with a mixture of 1.5% (w/w) NH4Mo7O24·4
H2O, 1% (w/w) Ce(SO4)2·4 H2O and 10% (v/v)
H2SO4, followed by heating. Flash chromatog-
raphy was conducted on Silica Gel 60 (E.
Merck, 40–63 mm and Grace AB Amicon,
35–70 mm). Evaporations were performed in
vacuo at temperatures below 45 °C. All sol-
vents were distilled before use. The calcula-
tions were performed using Sybyl 6.5 on an
Octane workstation by Silicon Graphics. Cal-
culations/modelling were carried out as fol-
lows: (1) charges were derived by the
Gasteiger–Hu¨ckel method; (2) the minimiser
was set to terminate after 10,000 steps or
energy convergency; (3) the BGFS-minimiser
was used; (4) the dielectric constant was set to
4; (5) the Tripos Force Field was employed,
Remo6al of the 1,2-O-isopropylidene group.
To a solution of the 6-azido-3,6-dideoxy-1,2-
O-isopropylidenehexofuranose (1 g) in CH3-
CN (10 mL) and water (10 mL), Amberlite
IR-120 [H+] (5 mL, wet) was added. The
reaction mixture was stirred at 40 °C over-
night. The ion-exchange resin was filtered off,
and the filtrate was concentrated.
Reduction of lactones.—To a solution of
borane–methyl sulfide complex (6 equiv) in
THF (10 mL), 2-methyl-2-butene (12 equiv)

28
S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
(C-6), 35.0 (C-3). Anal. Calcd for C9H16O5: C,
52.93; H, 7.90. Found: C, 53.05; H, 8.08.
was added at 0 °C under a nitrogen atmo-
sphere over a 10-min period. After 2.5 h
at ambient temperature, the 3-deoxyhexo-
nolactone (1 g) was added as a solid over 30
min and stirred overnight under a nitrogen
atmosphere. Water (3 mL) was slowly added,
and the mixture was refluxed for 4 h. The
solution was concentrated and extracted with
water (2×20 mL). The aqueous phase was
consecutively washed with CH2Cl2 (20+10
mL) and diethyl ether (20 mL), and concen-
trated.
Di-O-isopropylidene protection. To a sus-
pension of 3-deoxyhexose (1 g) and acetone
(40 mL), camphorsulfonic acid (0.1 g) was
added. The mixture was refluxed in a Soxhlet
apparatus overnight. The solution was neu-
tralised with NaHCO3. The mixture was con-
centrated and partitioned between ethyl
acetate (20 mL) and water (10 mL). The or-
ganic phase was washed with water (10 mL),
treated with activated carbon, dried (MgSO4),
and concentrated.
6-Azido-3,6-dideoxy-h/i- -ribo-hexose (9)
D
3-Deoxy-1,2-O-isopropylidene-6-O-tosyl-h-
-ribo-hexofuranose (7). Regioselective O-to-
D
sylation of 3-deoxy-1,2-O-isopropylidene-a- -
D
ribo-hexofuranose (6) (2.77 g, 13.6 mmol),
following the general procedure, gave a pale
syrup (5.31 g), which after chromatography
(1:1 hexane–EtOAc) yielded product 7 (3.82
1
g, 78%) as a syrup. H NMR (CDCl3, 500
MHz): l 5.74 (d, 1 H, J1,2 3.5 Hz, H-1), 4.70
(dd, 1 H, J2,3b 4.5 Hz, H-2), 3.9–4.2 (complex,
4 H, H-4, H-5, H-6a, H-6b), 2.06 (dd, 1 H,
J3a,3b 13.5, J3a,4 4.5 Hz, H-3a), 1.76 (ddd, 1 H,
J3b,4 10.5 Hz, H-3b). 13C NMR (CDCl3, 62.9
MHz): l 105.0 (C-1), 80.0 and 77.2 (C-2 and
C-4), 70.8 (C-6), 69.8 (C-5), 33.8 (C-3).
6-Azido-3,6-dideoxy-1,2-O-isopropylidene-h-
D
-ribo-hexofuranose (8). Reaction of 3-deoxy-
1,2-O-isopropylidene-6-O-tosyl-a- -ribo-hexo-
D
furanose (7) (3.58 g, 9.99 mmol) with NaN3
according to the general procedure gave com-
pound 8 (1.81 g, 79%) as a pale-yellow syrup.
1H NMR (CDCl3, 200 MHz): l 5.79 (d, 1 H,
J1,2 3.5 Hz, H-1), 4.75 (dd, 1 H, J2,3b 3.5 Hz,
H-2), 4.17 (ddd, 1 H, J3a,4 4.5, J3b,4 10.5, J4,5
4.5 Hz, H-4), 3.96 (ddd, 1 H, J5,6a 4.5, J5,6b 6.5
Hz, H-5), 3.39 (dd, 1 H, J6a,6b 13.0 Hz, H-6a),
3.31 (dd, 1 H, H-6b), 2.07 (dd, 1 H, J3a,3b 13.5
Hz, H-3a), 1.84 (ddd, 1 H, H-3b). 13C NMR
(CDCl3, 50.3 MHz): l 105.2 (C-1), 80.4 and
78.5 (C-2 and C-4), 70.8 (C-5), 53.2 (C-6), 33.2
(C-3).
Hydrogenation. To a solution of the hexose
(200 mg) in MeOH (20 mL), a suspension of a
catalytic amount of 5% Pd/C in MeOH (10
mL) (Caution! extreme fire hazard!) was
added. The mixture was hydrogenated at 3
MPa and ambient temperature for 16 h. The
catalyst was filtered off, and the solvent was
evaporated.
3-Deoxy-1,2-O-isopropylidene-h- -ribo-
D
hexofuranose (6).—Regioselective deprotec-
tion of 3-deoxy-1,2:5,6-di-O-isopropylidene-a-
D-ribo-hexofuranose (5, 805 mg, 3.30 mmol),
6-Azido-3,6-dideoxy-h/i-
6-Azido-3,6-dideoxy-1,2-O-isopropylidene-a-
-ribo-hexofuranose (8, 880 mg, 3.84 mmol)
D-ribo-hexose (9).
following the general procedure, gave a pale-
yellow syrup (1.07 g), which after chromatog-
D
raphy
(1:1
hexane–EtOAc)
afforded
was hydrolysed to a yellow syrup that was
purified by chromatography (EtOAc) to yield
compound 9 as a semi-crystalline compound
(520 mg, 71%). By addition of diethyl ether, 9
crystallised (260 mg, 36%), mp 109.5–
110.5 °C, [h]D +31.5° (c 1.1, H2O). NMR
(D2O) showed an anomeric mixture of the
furanose and pyranose forms. 13C NMR
(D2O, 50.3 MHz): l 102.6 (C-1, b-furanose),
98.7 (C-1, a-furanose), 97.3 (C-1, b-pyranose),
91.7 (C-1, a-pyranose). Anal. Calcd for
C6H11N3O4: C, 38.09; H, 5.87; N, 22.21.
Found: C, 38.12; H, 5.89; N, 21.96.
compound 6 (570 mg, 85%) as a syrup that
crystallised slowly, mp 80–82 °C. Recrystalli-
sation from Et2O afforded a pure sample (322
mg, 48%), mp 81–82 °C, [h]D −18.5° (c 1.2,
1
CHCl3). H NMR (MeOH-d4, 500 MHz): l
5.78 (d, 1 H, J1,2 4.0 Hz, H-1), 4.8 (H-2), 4.20
(ddd, 1 H, J3a,4 5.0, J3b,4 10.5, J4,5 5.0 Hz,
H-4), 3.70 (ddd, 1 H, J5,6a 4.5, J5,6b 6.0 Hz,
H-5), 3.59 (dd, 1 H, J6a,6b 11.5 Hz, H-6a), 3.51
(dd, 1 H, H-6b), 2.05 (dd, 1 H, J3a,3b 13.5 Hz,
13
H-3a), 1.83 (ddd, 1 H, J2,3b 5.0 Hz, H-3b). C
NMR (MeOH-d4, 125.8 MHz): l 106.9 (C-1),
81.9 and 79.9 (C-2 and C-4), 73.9 (C-5), 64.6

S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
29
H-4), 2.68 (d, 1 H, OH), 2.34 (ddd, 1 H, J3a,3b
14.5 Hz, H-3a), 2.13 (ddd, 1 H, H-3b). 13C
NMR (CDCl3, 125.8 MHz): l 106.4 (C-1),
80.3 and 80.0 (C-2 and C-4), 71.7 (C-6), 70.5
(C-5), 32.6 (C-3). Anal. Calcd for C16H22O7S:
C, 53.62; H, 6.19. Found: C, 53.61; H, 6.22.
6-Azido-3,6-dideoxy-1,2-O-isopropylidene-
3-Deoxy-1,2-O-isopropylidene-6-O-tosyl-i-
-arabino-hexofuranose (13)
D
3-Deoxy-1,2:5,6-di-O-isopropylidene-i-
D-
arabino-hexofuranose (11). 3-Deoxy- -ara-
D
bino-hexono-1,4-lactone (10) [11] (5.66 g, 34.9
mmol) was reduced following the general pro-
cedure and subsequently protected to give 11
as a pale-yellow syrup (2.34 g, 27%). 1H NMR
(CDCl3, 200 MHz): l 5.75 (d, 1 H, J1,2 4.0 Hz,
H-1), 4.73 (ddd, 1 H, J2,3a 1.5, J2,3b 5.5 Hz,
H-2), 4.31 (ddd, 1 H, J4,5 9.0, J5,6a 5.5, J5,6b 5.5
Hz, H-5), 4.11 (dd, 1 H, J6a,6b 9.0 Hz, H-6a),
3.99 (ddd, 1 H, J3a,4 3.0, J3b,4 8.0 Hz, H-4),
3.91 (dd, 1 H, H-6b), 2.31 (ddd, 1 H, J3a,3b
14.5 Hz, H-3a), 2.17 (ddd, 1 H, H-3b). 13C
NMR (CDCl3, 50.3 MHz): l 106.5 (C-1), 82.0
and 80.7 (C-2 and C-4), 77.0 (C-5), 67.8 (C-6),
33.9 (C-3).
i-
D
-arabino-hexofuranose (14).—Reaction of
3-deoxy-1,2-O-isopropylidene-6-O-tosyl-b-
D-
arabino-hexofuranose (13, 870 mg, 2.43 mmol)
with NaN3 furnished a crystalline residue (450
mg, 81%), which by addition of pentane gave
crystalline compound 14 (202 mg, 36%), mp
67–68 °C. Recrystallisation from pentane af-
forded an analytical sample: mp 67–68 °C,
[h]D +31.6° (c 1.1, CHCl3). 1H NMR
(CDCl3, 500 MHz): l 5.78 (d, 1 H, J1,2 4.0 Hz,
H-1), 4.75 (ddd, 1 H, J2,3a 1.0, J2,3b 6.0 Hz,
H-2), 4.05 (complex, 2 H, H-4, H-5), 3.59 (dd,
1 H, J5,6a 3.0, J6a,6b 12.5 Hz, H-6a), 3.42 (dd, 1
H, J5,6b 6.0 Hz, H-6b), 2.46 (d, 1 H, JOH,5 4.5
Hz, OH), 2.34 (broad d, 1 H, J3a,3b 14.5 Hz,
3-Deoxy-1,2-O-isopropylidene-i-D-arabino-
hexofuranose (12). Regioselective deprotection
of 3-deoxy-1,2:5,6-di-O-isopropylidene-b- -
D
arabino-hexofuranose (11, 2.02 g, 8.27 mmol)
gave a crude material that was purified by
chromatography (EtOAc) to furnish com-
13
H-3a), 2.17 (ddd, 1 H, J3b,4 8.0 Hz, H-3b). C
NMR (CDCl3, 62.9 MHz): l 106.4 (C-1), 81.2
and 80.5 (C-2 and C-4), 71.7 (C-5), 54.2 (C-6),
32.7 (C-3). Anal. Calcd for C6H15N3O4: C,
47.15; H, 6.60; N, 18.33. Found: C, 47.17; H,
6.49; N, 18.06.
1
pound 12 as a syrup (1.13 g, 67%). H NMR
(MeOH-d4, 250 MHz): l 5.74 (d, 1 H, J1,2 4.0
Hz, H-1), 4.75 (ddd, 1 H, J2,3a 1.0, J2,3b 6.0 Hz,
H-2), 3.95 (ddd, 1 H, J3a,4 2.5, J3b,4 8.0, J4,5 9.0
Hz, H-4), 3.82 (ddd, 1 H, J5,6a 3.0, J5,6b 6.0 Hz,
H-5), 3.73 (dd, 1 H, J6a,6b 11.0 Hz, H-6a), 3.50
(dd, 1 H, H-6b), 2.28 (ddd, 1 H, J3a,3b 14.0 Hz,
H-3a), 2.12 (ddd, 1 H, H-3b). 13C NMR
(MeOH-D4, 62.9 MHz): l 108.0 (C-1), 82.0
(C-2 and C-4), 74.1 (C-5), 64.8 (C-6), 34.1
(C-3).
6-Azido-3,6-dideoxy-h,i-
(15).—Deprotection of 6-azido-3,6-dideoxy-
1,2-O-isopropylidene-b- -arabino-hexofur-
D
-arabino-hexose
D
anose (14, 430 mg, 1.88 mmol) gave a crude
product (243 mg, 68%) that was purified by
chromatography (EtOAc) to give free sugar 15
as a syrup (222 mg, 62%). NMR (D2O)
showed an anomeric mixture of furanose and
3-Deoxy-1,2-O-isopropylidene-6-O-tosyl-i-
13
D
-arabino-hexofuranose (13). Regioselective
O-tosylation of 3-deoxy-1,2-O-isopropylidene-
b- -arabino-hexofuranose (12, 467 mg, 2.29
pyranose forms. C NMR (D2O, 50.3 MHz):
l 104.4 (C-1, b-furanose), 97.7 (C-1, a-fura-
nose), 97.2 (C-1, b-pyranose), 96.1 (C-1, a-
pyranose).
D
mmol) gave a crystalline residue (602 mg,
73%). By addition of EtOAc–hexane, product
13 crystallised (353 mg, 43%), mp 98–100 °C.
Recrystallisation from EtOAc–hexane gave
an analytical sample: mp 101 °C, [h]D +52.5°
3-Deoxy-1,2:5,6-di-O-isopropylidene-h-
xylo-hexofuranose (17). Following the general
procedure, 3-deoxy- -xylo-hexono-1,4-lactone
D
-
D
(16, 5.19 g, 32.0 mmol) was converted into
crude crystalline compound 17 (3.49 g, 44%).
By addition of hexane, 17 crystallised (2.17 g,
27%), mp 71–76 °C. Several recrystallisations
from hexane afforded an analytical sample:
1
(c 1.3, CHCl3). H NMR (CDCl3, 500 MHz):
l 5.73 (d, 1 H, J1,2 4.0 Hz, H-1), 4.70 (ddd, 1
H, J2,3a 1.0, J2,3b 6.0 Hz, H-2), 4.29 (dd, 1 H,
J5,6a 2.5, J6a,6b 10.0 Hz, H-6a), 4.11 (dddd, J4,5
8.5, J5,6b 6.5, J5,OH 5.0 Hz, H-5), 4.05 (dd, 1 H,
H-6b), 4.02 (ddd, 1 H, J3a,4 2.5, J3b,4 8.5 Hz,
1
mp 76–78 °C, [h]D −29.5° (c 1.1, CHCl3). H
NMR (CDCl3, 250 MHz): l 5.78 (d, 1 H, J1,2

30
S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
6-Azido-3,6-dideoxy-1,2-O-isopropylidene-h-
-xylo-hexofuranose (20). Reaction of 3-de-
4.0 Hz, H-1), 4.70 (broad dd, 1 H, J2,3a 5.0 Hz,
H-2), 4.41 (ddd, 1 H, J4,5 8.5, J5,6a 7.0, J5,6b 7.0
Hz, H-5), 4.08 (ddd, 1 H, J3a,4 8.5, J3b,4 3.5 Hz,
H-4), 4.02 (dd, 1 H, J6a,6b 8.0 Hz, H-6a), 3.58
(dd, 1 H, H-6b), 2.19 (ddd, 1 H, J3a,3b 14.5 Hz,
D
oxy-1,2-O-isopropylidene-6-O-tosyl-a- -xylo-
D
hexofuranose (19, 1.81 g, 5.05 mmol) with
NaN3, following the general procedure, gave
azidodeoxy sugar 20 as a colourless syrup (820
mg, 71%). By addition of EtOAc–hexane,
compound 20 crystallised (380 mg, 33%), mp
45–46 °C. Recrystallisation from EtOAc–hex-
ane gave an analytical sample: mp 45–46 °C,
[h]D −34.0° (c 1.0, CHCl3). 1H NMR
(CDCl3, 200 MHz): l 5.80 (d, 1 H, J1,2 4.0 Hz,
H-1), 4.75 (ddd, 1 H, J2,3a 6.0, J2,3b 1.5 Hz,
H-2), 4.18 (ddd, 1 H, J3a,4 8.5, J3b,4 3.0, J4,5 7.5
Hz, H-4), 3.93 (m, 1 H, H-5), 3.37 (dd, 1 H,
J5,6a 4.0, J6a,6b 13.0 Hz, H-6a), 3.26 (dd, 1 H,
J5,6b 6.0 Hz, H-6b), 2.23 (ddd, 1 H, J3a,3b 14.5
Hz, H-3a), 2.02 (ddd, 1 H, H-3b). 13C NMR
(CDCl3, 50.3 MHz): l 106.1 (C-1), 81.3 and
80.4 (C-2 and C-4), 71.8 (C-5), 53.0 (C-6), 33.2
(C-3). Anal. Calcd for C9H15N3O4: C, 47.16;
H, 6.60; N, 18.33. Found: C, 47.44; H, 6.86;
N, 18.62.
13
H-3a), 1.79 (broad dd, 1 H, H-3b). C NMR
(CDCl3, 62.9 MHz): l 106.3 (C-1), 81.4 and
80.3 (C-2 and C-4), 77.5 (C-5), 65.9 (C-6), 33.4
(C-3). Anal. Calcd for C12H20O5: C, 59.00; H,
8.26. Found: C, 58.76; H, 8.20.
3-Deoxy-1,2-O-isopropylidene-h-
hexofuranose (18). Regioselective deprotection
of 3-deoxy-1,2:5,6-di-O-isopropylidene-a-
D
-xylo-
D
-
xylo-hexofuranose (17, 5.50 g, 22.5 mmol)
gave a yellow syrup (5.6 g). Purification by
chromatography (EtOAc) yielded product 18
(2.20 g, 48%) as a syrup, which crystallised
upon addition of EtOAc–Et2O to give pure
material (1.30 g, 28%), mp 96–97 °C. Recrys-
tallisation from EtOAc gave an analytical
sample: mp 96–97 °C, [h]D −30.4° (c 1.1,
1
CHCl3). H NMR (CDCl3, 250 MHz): l 5.87
(d, 1 H, J1,2 4.0 Hz, H-1), 4.83 (ddd, 1 H, J2,3a
6.0, J2,3b 1.0 Hz, H-2), 4.30 (ddd, 1 H, J3a,4 8.0,
J3b,4 3.0, J4,5 8.0 Hz, H-4), 3.94 (ddd, 1 H, J5,6a
3.5, J5,6b 5.0 Hz, H-5), 3.81 (dd, 1 H, J6a,6b
12.0 Hz, H-6a), 3.62 (dd, 1 H, H-6b), 2.28
(ddd, 1 H, J3a,3b 14.5 Hz, H-3a), 2.11 (ddd, 1
H, H-3b). 13C NMR (CDCl3, 62.9 MHz): l
105.9 (C-1), 80.7 and 80.3 (C-2 and C-4), 72.4
(C-5), 63.0 (C-6), 33.1 (C-3). Anal. Calcd for
C9H16O5: C, 52.93; H, 7.90. Found: C, 52.97;
H, 8.15.
6 - Azido - 3,6 - dideoxy - h/i -
(21). Deprotection of 6-azido-3,6-dideoxy-1,2-
O-isopropylidene-a- -xylo-hexofuranose (20,
D
- xylo - hexose
D
260 mg, 1.13 mmol) gave a pale-yellow syrup
of free aldose 21 (155 mg, 73%), which was
purified by chromatography (EtOAc) to fur-
nish a colourless syrup (121 mg, 57%). NMR
(D2O) showed an anomeric mixture of fura-
nose and pyranose forms. 13C NMR (D2O,
50.3 MHz): l 104.7 (C-1, b-furanose), 100.7
(C-1, b-furanose), 97.5 (C-1, b-pyranose), 96.1
(C-1, a-pyranose).
6-Azido-3,6-dideoxy-1,2-O-isopropylidene-h-
-xylo-hexofuranose (20)
D
3-Deoxy-1,2:5,6-di-O-isopropylidene-h-
xylo-hexofuranose (23).—Reduction and pro-
tection of 3-deoxy- -xylo-hexono-1,4-lactone
L
-
3-Deoxy-1,2-O-isopropylidene-6-O-tosyl-h-
-xylo-hexofuranose (19). Regioselective O-to-
D
L
sylation of 3-deoxy-1,2-O-isopropylidene-a- -
D
(22, 5.80 g, 35.8 mmol), according to the
general procedures given above, yielded a
crystalline residue (3.15 g, 36%). By addition
of hexane, deoxy sugar 23 crystallised (1.31 g,
15%), mp 73–76 °C. Several recrystallisations
from hexane afforded an analytical sample:
mp 76–77.5 °C, [h]D +29.3° (c 1.0, CHCl3).
xylo-hexofuranose (18, 550 mg, 2.69 mmol)
1
gave syrupy compound 19 (670 mg, 70%). H
NMR (CDCl3, 200 MHz): l 5.76 (d, 1 H, J1,2
4.0 Hz, H-1), 4.73 (ddd, 1 H, J2,3a 6.0, J2,3b 1.5
Hz, H-2), 4.22 (ddd, 1 H, J3a,4 8.5, J3b,4 3.5,
J4,5 6.0 Hz, H-4), 4.07 (two dd, 2 H, J5,6a 7.0,
J5,6b 4.0, J6a,6b 12.0 Hz, H-6a, H-6b), 3.93
(dddd, 1 H, J5,OH 4.5 Hz, H-5), 3.01 (d, 1 H,
OH), 2.23 (ddd, 1 H, J3a,3b 14.5 Hz, H-3a),
2.05 (ddd, 1 H, H-3b). 13C NMR (CDCl3, 50.3
MHz): l 106.0 (C-1), 80.3 and 80.2 (C-2 and
C-4), 70.1 (C-5 and C-6), 32.9 (C-3).
NMR data as for the
D
-xylo isomer 17. Anal.
Calcd for C12H20O5: C, 59.00; H, 8.26. Found:
C, 58.74; H, 7.96.
3 - Deoxy - 1,2 - O - isopropylidene - h - - xylo-
L
hexofuranose (24).—Regioselective deprotec-
tion of 3-deoxy-1,2:5,6-di-O-isopropylidene-a-

S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
31
(C-1), 38.8 (C-3). HRFABMS. Calcd for
C6H14NO3: 148.0974. Found: [M+H]+
148.0979.
L
-xylo-hexofuranose (23, 3.20 g, 13.1 mmol)
gave a yellow syrup that was purified by chro-
matography (EtOAc) to give crude compound
24 (1.10 g, 41%). By addition of Et2O, 24
crystallised (544 mg, 20%), mp 95–97 °C. Re-
crystallisation from EtOAc–Et2O gave an an-
alytical sample: mp 96–97 °C, [h]D +30.6° (c
1.0, CHCl3). NMR data were identical with
values for the
for C9H16O5: C, 52.93; H, 7.90. Found: C,
52.95; H, 7.67.
1,3,6-Trideoxy-1,6-imino-
(29).—Hydrogenation of 6-azido-3,6-dideoxy-
a,b- -arabino-hexose (15, 97 mg, 0.51 mmol)
D
-arabino-hexitol
D
gave product 29 (63 mg, 84%) as a pale-yellow
syrup. Purification by chromatography (5%
NH3 in 1:1 MeOH–CH2Cl2) furnished an ana-
lytical sample. 1H NMR (MeOH-d4, 250
MHz): l 4.08 (ddd, 1 H, J3a,4 9.5, J3b,4 2.5, J4,5
2.5 Hz, H-4), 4.01 (dddd, 1 H, J1a,2 4.5, J1b,2
6.0, J2,3a 4.5, J2,3b 6.0 Hz, H-2), 3.85 (ddd, 1 H,
J5,6a 4.0, J5,6b 6.0 Hz, H-5), 3.10 (dd, 1 H, J1a,1b
14.0 Hz, H-1a), 2.95 (dd, 1 H, J6a,6b 14.0 Hz,
H-6a), 2.86 (dd, 1 H, H-6b), 2.73 (dd, 1 H,
H-1b), 2.29 (ddd, 1 H, J3a,3b 14.5 Hz, H-3a),
1.75 (ddd, 1 H, H-3b). 13C NMR (MeOH-d4,
62.9 MHz): l 74.6 (C-5), 70.4 (C-4), 67.8
(C-2), 55.5 (C-6), 52.1 (C-1), 38.1 (C-3). HR-
FABMS. Calcd for C6H14NO3: 148.0974.
Found: [M+H]+ 148.0959.
D-xylo isomer 18. Anal. Calcd
3-Deoxy-1,2-O-isopropylidene-6-O-tosyl-h-
-xylo-hexofuranose (25).—Regioselective O-
L
tosylation of 3-deoxy-1,2-O-isopropylidene-a-
L
-xylo-hexofuranose (24, 0.93 g, 4.55 mmol)
gave sulfonate 25 as a syrup (1.63 g, quant).
NMR data were identical with those for the
D
-xylo isomer 19.
6-Azido-3,6-dideoxy-1,2-O-isopropylidene-h-
L
-xylo-hexofuranose (26).—Reaction of 3-de-
oxy-1,2-O-isopropylidene-6-O-tosyl-a- -xylo-
L
hexofuranose (25, 1.57 g, 4.38 mmol) with
NaN3 gave product 26 (680 mg, 68%) as a
syrup. By addition of EtOAc–hexane, com-
pound 26 crystallised (320 mg, 32%), mp 45–
46 °C. Recrystallisation from EtOAc–hexane
afforded an analytical sample: mp 45–46 °C,
[h]D +34.3° (c 0.9, CHCl3). NMR data were
1,3,6 - Trideoxy - 1,6 - imino -
(30).—Hydrogenation of 6-azido-3,6-dideoxy-
-xylo-hexose (21, 46 mg, 0.24 mmol)
D
- xylo - hexitol
a,b-D
gave a pale-yellow syrup of imino alditol 30
(30 mg, 85%). Purification by chromatography
(5% NH3 in 1:1 MeOH–CH2Cl2) gave an
identical with those for the
6 - Azido - 3,6 - dideoxy - h/i-
(27). —Deprotection of 6-azido-3,6-dideoxy-
-xylo-hexofuranose
D
-xylo isomer 20.
1
analytical sample. H NMR (MeOH-d4, 250
L
-xylo-hexose
MHz): l 4.01 (dddd, 1 H, J1a,2 5.0, J1b,2 6.5,
J2,3a 6.5, J2,3b 3.5 Hz, H-2), 3.87 (ddd, 1 H,
J3a,4 3.0, J3b,4 8.0, J4,5 6.5 Hz, H-4), 3.55 (ddd,
1 H, J5,6a 4.0, J5,6b 6.5 Hz, H-5), 3.05 (dd, 1 H,
J1a,1b 14.0 Hz, H-1a), 3.05 (dd, 1 H, J6a,6b 14.0
Hz, H-6a), 2.74 (dd, 1 H, H-6b), 2.65 (dd, 1
H, H-1b), 2.05 (ddd, 1 H, J3a,3b 14.5 Hz,
H-3a), 1.95 (ddd, 1 H, H-3b). 13C NMR
(MeOH-d4, 62.9 MHz): l 76.6 (C-5), 71.5
(C-4), 67.6 (C-2), 56.4 (C-6), 52.9 (C-1), 39.9
(C-3). HRFABMS: Calcd for C6H14NO3:
148.0974. Found: [M+H]+ 148.0974.
1,2-O-isopropylidene-a-
L
(26, 400 mg, 1.75 mmol) gave compound 27
(270 mg, 82%) as a pale-yellow syrup. NMR
data were identical with those for the
isomer 21.
D
-xylo
1,3,6 - Trideoxy - 1,6 - imino -
D
- ribo - hexitol
(28).—Hydrogenation of 6-azido-3,6-dideoxy-
a,b- -ribo-hexose (9, 93 mg, 0.49 mmol) gave
D
compound 28 (72 mg, 100%) as a pale-yellow
syrup. Purification by chromatography (5%
NH3 in 1:1 MeOH–CH2Cl2) yielded an ana-
lytical sample. 1H NMR (MeOH-d4, 250
MHz): l 3.8–3.9 (complex, 3 H, H-2, H-4,
H-5), 2.97 (dd, 1 H, J 4.5, J 14.0 Hz, H-6 or
H-1), 2.88 (complex, 2 H, H-1 and/or H-6),
2.80 (dd, 1 H, J 6.5, J 14.0 Hz, H-6 or H-1),
2.17 (ddd, 1 H, J3a,3b 14.0, J 8.5, J 10.0 Hz,
H-3a), 1.93 (ddd, 1 H, J 4.0, J 4.0 Hz, H-3b).
13C NMR (MeOH-d4, 62.9 MHz): l 73.9 (C-
5), 71.6 (C-4), 68.7 (C-2), 56.0 (C-6), 52.2
1,3,6 - Trideoxy - 1,6 - imino -
(31).—Hydrogenation of 6-azido-3,6-dideoxy-
a,b- -xylo-hexose (27, 130 mg, 0.67 mmol)
L
- xylo - hexitol
L
gave a pale-yellow syrup of product 31 (54
mg, 55%). Purification by chromatography
(5% NH3 in 1:1 MeOH–CH2Cl2) gave an
analytical sample. NMR data as for enan-
tiomer 30. HRFABMS: Calcd for C6H14NO3:
148.0974. Found: [M+H]+ 148.0972.

32
S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
foam that was purified by chromatography to
give ketose 34 (64 mg, 70% overall). From H
1,2,6-Trideoxy-1,6-imino-
(33).—6-Amino-2,6-dideoxy-
D
-arabino-hexitol
-arabino-hexo-
1
D
nolactam (32, 1.0 g, 6.2 mmol) was suspended
in CH3CN (10 mL), and a mixture of hexa-
methyldisilazane (4.30 mL, 19.6 mmol) and
chlorotrimethylsilane (0.17 mL, 0.10 mmol)
was added. After stirring at 80 °C for 1 h, the
mixture was filtered, and the filtrate was con-
centrated to a syrup. The residue was dis-
solved dioxane (35 mL), and under an
atmosphere of N2 BH3·Me2S (10 M, 3.1 mL,
31 mmol) was added. The mixture was then
stirred for 5 h at 100 °C and allowed to stand
at ambient temperature overnight. Hydrochlo-
ric acid (1 M, 26 mL) was added, and the
mixture was kept under reflux for 1 h, filtered,
concentrated, and co-concentrated with 1%
v/v acetyl chloride–MeOH (3×) to yield a
crude residue (1.13 g, \100%). Purification
by chromatography (5% NH3 in 1:1 MeOH–
NMR spectra (MeOH-d4) a mixture of fura-
nose forms and the open-chain ketose were
identified.
1,4,5 - Trideoxy - 1,5 - imino -
(35).—Hydrogenation of 6-azido-3,6-dideoxy-
-erythro-hex-2-ulose (34, 64 mg, 0.34
D
- lyxo - hexitol
a,b-D
mmol) gave a pale-yellow syrup (52 mg,
100%) that could be crystallised from acetone
to give imino alditol 35 (19 mg, 37%), mp
1
140–150 °C, [h]D −29.1° (c 1.6, MeOH). H
NMR (MeOH-d4, 250 MHz): l 3.74 (ddd, 1
H, J1a,2 3.0, J1b,2 1.5, J2,3 3.0 Hz, H-2), 3.66
(ddd, 1 H, J3,4a 6.0, J3,4b 11.0 Hz, H-3), 3.55
(dd, 1 H, J5,6a 4.5, J6a,6b 13.5 Hz, H-6a), 3.51
(dd, 1 H, J5,6b 5.0 Hz, H-6b), 3.06 (dd, 1 H,
J1a,1b 13.5 Hz, H-1a), 2.70 (dd, 1 H, H-1b),
2.60 (ddd, 1 H, J4a,5 5.0 Hz, H-5), 1.65 (com-
plex, 1 H, H-4a), 1.56 (dd, 1 H, J4a,4b 12.5 Hz,
H-4b). 13C NMR (MeOH-d4, 62.9 MHz): l
71.0 (C-2), 68.6 (C-3), 66.0 (C-6), 57.7 (C-5),
50.8 (C-1), 32.2 (C-4). HRFABMS: Calcd for
C6H14NO3: 148.0974. Found: [M+H]+
148.0960.
1
CH2Cl2) gave an analytical sample. H NMR
(MeOH-d4, 250 MHz): l 3.94 (ddd, 1 H, J4,5
2.5, J5,6a 6.5, J5,6b 2.5 Hz, H-5), 3.82 (ddd, 1 H,
J2a,3 3.0, J2b,3 7.5, J3,4 7.5 Hz, H-3), 3.60 (dd, 1
H, H-4), 2.95 (dd, 1 H, J6a,6b 14.0 Hz, H-6a),
2.88 (complex, 2 H, H-1a and H-1b), 2.83 (dd,
1 H, H-6b), 1.98 (dddd, 1 H, J1,2a 4.5 and 6.0,
J2a,2b 15.0 Hz, H-2a), 1.72 (dddd, 1 H, J1,2b 6.0
Inhibition studies. a-
baker’s yeast, b- -glucosidase from almonds,
a- -galactosidase from green coffee beans, b-
-galactosidase from Escherichia coli, a-
mannosidase from jack bean, b- -manno-
sidase from snail acetone powder, b-N-acetyl-
-fu-
D
-Glucosidase from
D
D
13
and 8.0 Hz, H-2b). C NMR (MeOH-d4, 62.9
D
D-
MHz): l 9.3 (C-4), 71.6 and 71.4 (C-3 and
C-5), 48.7 (C-6), 43.5 (C-1), 34.2 (C-2). HR-
FABMS: Calcd for C6H14NO3: 148.0974.
Found: [M+H]+ 148.0960.
D
glucosaminidase from jack beans and a-
L
cosidase from bovine kidney were purchased
6-Azido-3,6-dideoxy-h/i-
ulose (34).—A solution of 6-azido-3,6-
dideoxy-a,b- -ribo-hexose (9, 110 mg, 0.58
D
-erythro-hex-2-
from Sigma Chemical Co. Ki determinations
were performed at 25 °C for a-
dase, b- -galactosidase, a- -mannosidase and
at 35 °C for a- -glucosidase, b- -glucosidase,
b- -mannosidase, a- -fucosidase, b-N-acetyl-
D-galactosi-
D
D
D
mmol), water (1 mL) and 1% aq MgSO4 (one
drop) was adjusted to pH 8.5 by the addition
of solid Na2CO3. Sweetzyme T (immobilised
glucose isomerase EC. 5.3.1.5, 110 mg) was
added, and the mixture was stirred at 40 °C
for 3 days. The brown residue was filtered and
concentrated to a pale-yellow syrup (130 mg)
that was purified by chromatography (EtOAc)
to give a 1:9 mixture of 9 and 34 (110 mg) as
a clear syrup. This mixture (100 mg, 0.54
mmol) was dissolved in water (8.5 mL) and
BaCO3 (380 mg) and Br2 (180 mg, 1.1 mmol)
were added. After 20 min, air was bubbled
through the mixture to remove excess Br2.
Filtration and concentration gave a colourless
D
D
D
L
glucosaminidase using the corresponding p-ni-
trophenyl-a-(or b)-glycoside at various final
concentrations ranging from 0.7×10−3 to
2.7×10−3 mol L−1 (for b-N-acetylgluco-
saminidase: 0.16×10−3 to 0.66×10−3 mol
L
−1) at pH 6.8 (0.12 mol L−1 phosphate
buffer). For the inhibition studies, inhibitors
were incorporated into the buffer to give
a final concentration of 1.7×10−5 mol L−1
.
The enzymes were incorporated into the
buffer to give a final concentration of
0.17 units/mL. Dissociation constants for

S.M. Andersen et al. / Carbohydrate Research 326 (2000) 22–33
33
[2] D.A. Winkler, G. Holan, J. Med. Chem., 32 (1989)
2084–2089.
[3] I. Lundt, R. Madsen, S. Al Daher, B. Winchester, Tetra-
hedron, 50 (1994) 7513–7520.
[4] G. Legler, A.E. Stu¨tz, H. Immich, Carbohydr. Res., 272
(1995) 17–30.
inhibition were calculated from a Hanes plot
([S]/w against [S]) from the rates of substrate
hydrolysis in the absence and presence of in-
hibitor.
[5] M. Godskesen, I. Lundt, R. Madsen, B. Winchester,
Bioorg. Med. Chem., 4 (1996) 1857–1865.
[6] F. Moris-Varas, X.-H. Qian, C.-H. Wong, J. Am. Chem.
Soc., 118 (1996) 7647–7652.
Acknowledgements
[7] X. Qian, F. Moris-Varas, M.C. Fitzgerald, C.-H. Wong,
Bioorg. Med. Chem., 4 (1996) 2055–2069.
[8] Y. Le Merrer, L. Poitout, J.-C. Depezay, I. Dosbaa, S.
Geoffroy, M.J. Foglietti, Bioorg. Med. Chem., 5 (1997)
519–533.
[9] S. Iacono, J.R. Rasmussen, Org. Synth., 64 (1986) 57–62.
[10] K. Bock, I. Lundt, C. Pedersen, Carbohydr. Res., 90 (1981)
7–16.
One of us (A.E.S.) thanks the Technical
University of Denmark (DTU) for a guest
professorship. We thank Novo Nordisk A/S
for a gift of immobilised glucose isomerase as
well as Dr M. Godskesen and L. Kastrup for
determination of the inhibition data, and J.G.
Rasmussen for her assistance.
[11] K. Bock, I. Lundt, C. Pedersen, Acta Chem. Scand., Sect.
B, 35 (1981) 155–162.
[12] K. Bock, I. Lundt, C. Pedersen, Acta Chem. Scand., Sect.
B, 41 (1987) 435–441.
[13] K. Bock, M. Meldal, B. Meyer, L. Wiebe, Acta Chem.
Scand., Sect. B, 37 (1983) 101–108.
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