Synthesis of C-3 nitrogen-containing derivatives of N-acetyl-α,β-D-mannosamine as substrates for N-acetylneuraminic acid aldolase

Article abstract of DOI:10.1016/S0008-6215(01)00091-X

The synthesis of 3-azido-3-deoxy, 3-amino-3-deoxy and 3-N-tert-butyloxycarbonyl-3-deoxy derivatives of 2-acetamido-2-deoxy-α,β-D-mannose (N-acetyl-α,β-D-mannosamine, ManNAc), is presented. The 3-azido-3-deoxy- and 3-N-tert-butyloxycarbonyl compounds were

Full text of DOI:10.1016/S0008-6215(01)00091-X

www.elsevier.nl/locate/carres
Carbohydrate Research 332 (2001) 133–139
Synthesis of C-3 nitrogen-containing derivatives of
N-acetyl-a,b-
D
-mannosamine as substrates for
N-acetylneuraminic acid aldolase
Gaik B. Kok,a Michael Campbell,a Brendan L. Mackey,a Mark von Itzsteina,b,*
aDepartment of Medicinal Chemistry, Monash Uni6ersity (Park6ille Campus), 381 Royal Parade, Park6ille 3052,
Victoria, Australia
bCentre for Biomolecular Science and Drug Disco6ery, Griffith Uni6ersity (Gold Coast Campus),
PMB 50 Gold Coast Mail Centre, Queensland 9726, Australia
Received 25 January 2001; accepted 22 March 2001
Abstract
The synthesis of 3-azido-3-deoxy, 3-amino-3-deoxy and 3-N-tert-butyloxycarbonyl-3-deoxy derivatives of 2-acet-
amido-2-deoxy-a,b-
D
-mannose (N-acetyl-a,b-D-mannosamine, ManNAc), is presented. The 3-azido-3-deoxy- and
3-N-tert-butyloxycarbonyl compounds were further characterised as their peracetates. A preliminary study has found
that these C-3 nitrogen-substituted derivatives of ManNAc not to be substrates for Neu5Ac aldolase. © 2001
Elsevier Science Ltd. All rights reserved.
Keywords: ManNAc derivatives; Neu5Ac aldolase; Sialic acid; N-Acetylneuraminic acid
1. Introduction
ars).2 Imino sugars such as 1-deoxynojirimycin
and 2,5-anhydro-2,5-imino- -mannitol, a class
D
Aminodeoxy sugars have received consider-
able attention owing to their diverse structure
and biological properties. Many differing
structural types of such sugars frequently oc-
cur in antibiotics glycosidically linked to com-
plex aglycon units or as simple derivatives in
fermentation products.1 As well as having
therapeutic potential,1 some aminodeoxy sug-
ars are important synthetic intermediates to
more elaborate systems. For example,
aminodeoxy sugars have been employed as
reaction intermediates in the synthesis of
imino sugars (sometimes referred to as azasug-
of sugar mimics with a nitrogen in the ring,
are inhibitors of a number of glycohydro-
lases.3 It is not surprising, therefore, that effi-
cient preparation of aminodeoxy sugars of
natural and unnatural products has been of
longstanding interest to organic and medicinal
chemists.
Over the last decade, numerous papers deal-
ing with the synthesis of derivatives of the
aminodeoxy sugar, 2-acetamido-2-deoxy-a,b-
D
-mannose (N-acetyl- -mannosamine, Man-
D
NAc) have appeared, particularly with respect
to their use as substrates in the enzyme-
catalysed construction of higher 3-deoxy-2-
ulosonic acid derivatives.4 Thus, as shown in
Scheme 1, Neu5Ac aldolas e catalyses the re-
versible aldol reaction of ManNAc and pyru-
vate, which results in the formation of
* Corresponding author. Tel.: +61-7-55528232; fax: +61-
7-55528098.
E-mail address: m.vonitzstein@mailbox.gu.edu.au (M. von
Itzstein).
0008-6215/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(01)00091-X

134
G.B. Kok et al. / Carbohydrate Research 332 (2001) 133–139
Scheme 1.
N-acetylneuraminic acid (Neu5Ac, 1), a nine-
carbon ulosonic acid. Ulosonic acids such as 1
and their derivatives are involved in a range of
important biological processes such as molec-
ular recognition5 (Scheme 1). Besides Man-
NAc itself, Neu5Ac aldolase also accepts as
substrates a range of ManNAc derivatives
substituted at the 4- and 6-position.4,6 This has
provided an attractive enzymatic route to a
number of natural and unnatural neuraminic
acid derivatives. Studies employing various
D
-
mannoses substituted at C-3 have found that
the hydroxyl group at this position is essential
for successful Neu5Ac aldolase-catalysed con-
densation.7 To the best of our knowledge, no
C-3 substituted derivatives of ManNAc have
been investigated as potential substrates of
Neu5Ac aldolase. Here, we attempt to delin-
eate the reactivity of several C-3 substituted
ManNAc derivatives as substrates of Neu5Ac
aldolase. In particular, we have focussed on
the C-3 nitrogen-substituted derivatives (2–4).
Such ManNAc derivatives, if accepted by the
enzyme, have the potential to generate the
nitrogen isostere of Neu5Ac such as 5. A
16-step total chemical synthesis of 5 starting
2. Results and discussion
Two fundamental protocols for the synthe-
sis of vicinal diamines are known: fission of
epimino-derivatives by nucleophiles, e.g.,
azide ion,9 and opening of an epoxide ring
with a nitrogen-containing nucleophile.10 In
the latter case, the resulting alcohol is then
converted into the diamino derivative via di-
rect nucleophilic substitution by azide or an
amino equivalent through intermediate sulfo-
nate esters.
from
D
-glucose has previously been reported8
by Baumberger and Vasella, and this com-
pound was found to be a modest inhibitor of
sialidase from Vibrio cholerae. The synthesis
of the ManNAc derivatives (2–4) themselves,
per se, is also of interest, as this has not been
previously reported. These compounds will al-
low us, for the first time, to investigate the
substrate specificity of Neu5Ac aldolase for
C-3 substituted ManNAc derivatives. Prelimi-
nary results of these experiments are
presented.
Retrosynthetic analysis of the ManNAc
derivatives (2–4) suggested that the pyranose
with an azido group at C-2, methyl 2-azido-
4,6-O-benzylidene-2-deoxy-a- -altropyra-
D
noside (6),11 was an obvious precursor
(Scheme 2). Following previously published
procedures,11 this compound was conveniently
prepared from the commercially available
methyl a- -glucopyranoside in four steps.
D
It was anticipated that direct SN2 displace-
ment with azide at C-3 via the methanesul-

G.B. Kok et al. / Carbohydrate Research 332 (2001) 133–139
135
Scheme 2. Reagents and conditions: (i) H2, PtO2, Boc2O, rt, 40 h; (ii) Tf2O, pyridine, −30 °C“0 °C; (iii) n-Bu4NN3, rt, 24 h;
(iv) 4:1 HOAc–water, 100 °C, 24 h; 5 N HCl, 100 °C, 3 days; (v) Ac2O, pyridine, MeOH, rt, 30 min; (vi) H2, PtO2, rt, 24 h; (vii)
Boc-ON, rt, 48 h; (viii) Ac2O, pyridine.
fonate ester of the 2,3-trans-diaxial 2-N-acet-
date. Following the one-pot catalytic hy-
drogenolysis of the azide 6 over platinum
oxide and Boc-protection, the 2-N-(tert-bu-
toxycarboxy)amine 7 was obtained in 90%
yield.
The altro-configured alcohol 7 was then
converted into the 3-O-trifluoromethanesul-
fonate 8 that underwent displacement with
azide (tetrabutylammonium azide) in benzene
to give, by inversion of configuration at C-3,
the 3-azido-3-deoxy mannoside 9 in 57% yield
(two steps). All of the protecting groups asso-
ciated with mannoside 9 were then removed.
amido-2-deoxy altroside, methyl 2-acetamido-
4,6-O-benzylidene-2-deoxy-a- -altropyranoside
D
(readily prepared via sequential reduction and
acetylation of 6) would lead to epimine forma-
tion.12 Thus with the reacting groups being
trans-diaxial, adjustment of the N-nucleo-
philicity at C-2 was necessary in order to
favour intermolecular nucleophilic displace-
ment over the competing intramolecular reac-
tion. After some experimentation, it was
found that
a
(tert-butoxycarboxy)amino
group at this position was a suitable candi-

136
G.B. Kok et al. / Carbohydrate Research 332 (2001) 133–139
the acceptor substrate is a necessary require-
ment for successful Neu5Ac aldolase-catalysed
reactions. These conclusions are consistent
with results from a previous study7 that em-
Thus, the benzylidene protecting group was
removed by treating the mannoside 9 with
aqueous acetic acid (4:1 HOAc–water) at
100 °C for 24 h. The volatiles were then re-
moved under diminished pressure, and the
residue was filtered through a short plug of
silica gel. The product was isolated from the
column using a combination of ethyl acetate
and methanol as eluant. Conventional O-deg-
lycosidation of the isolated methyl glycoside
in refluxing 5 N HCl for 3 days resulted in the
concomitant conversion of the 2-acetamido
functionality to the corresponding amine hy-
drochloride 10. Subsequent acetylation of 10
in pyridine with Ac2O in the presence of
methanol as a cosolvent gave, after chromato-
graphic purification on silica gel, the corre-
sponding 2-acetamide 2 as an amorphous
solid in 63% yield (based on the mannoside 9).
To complete the synthesis, under catalytic
hydrogenolysis conditions, the 3-azido-3-de-
oxy compound 2 was converted into the
amine 3, and 3 in turn was converted into the
ployed C-3-modified -mannose derivatives as
D
possible substrates for Neu5Ac aldolase.
3. Experimental
General.—1H NMR (300 MHz) and 13C
NMR (75 MHz) spectra (in l (ppm) downfield
from Me4Si) were recorded on a Bruker AMX
300 spectrometer at 303 K and were refer-
1
enced using residual H resonances in the sol-
vent.
J-values
are
in
hertz
(Hz).
Low-resolution (LR) ESI mass spectra (cone
voltage 30 V) were obtained using a Micro-
mass Platform II electrospray spectrometer
and high-resolution (HR) mass spectra were
recorded on a Bruker BIO-APEX II FT ion
cyclotron resonance mass spectrometer with
an ‘Analytica’ ESI source. Specific optical ro-
tations [h]D, quoted in 10−1 deg cm2/g, were
measured at rt using a JASCO DIP-370 polar-
imeter with a path length 50 mm. Concentra-
tions are quoted in 10−2 g/mL. Elemental
analyses were performed either by the Chemi-
cal and Microanalytical Service, Essendon,
Victoria or by Microanalytical Service, De-
partment of Chemistry, University of Queens-
land, Brisbane, Queensland. IR spectra were
recorded using a Hitachi 270-30 spectrophoto-
meter. Column chromatography was per-
formed on E. Merck Silica Gel 60
(0.040–0.063 mm). Reactions were monitored
by TLC on Kieselgel 60 F254 plates (E. Merck
5554) and visualised under ultraviolet (UV)
irradiation and by spraying with 95% aq
EtOH containing 5% H2SO4 and charring for
several minutes at 180 °C. Hexane refers to
the fraction of petroleum ether that boils in
the range 60–80 °C and was distilled before
use.
corresponding
3-N-(tert-butoxycarbonyl)-
amine (4) with 2-(tert-butoxycarbonyloxy-
imino)-2-phenylacetonitrile (Boc-ON), in good
overall yield (54%, two steps). Confirmation
of the structures of both 2 and 4 was provided
by the conversion of these compounds into
their corresponding peracetylated derivatives
(11 and 12, respectively) with Ac2O and pyri-
dine.
Finally, the substrate specificity of Neu5Ac
aldolase for compounds 2–4 was investigated.
In separate experiments, according to pub-
lished procedures,4,13 each of the compounds
was incubated at pH 7.5 in the presence of
excess pyruvate and Neu5Ac aldolase in a
membrane-enclosed enzyme reactor (dialysis
bag) at 37 °C, and the reaction was monitored
1
by both TLC and H NMR spectroscopy.
Preliminary investigations have found that
none of these compounds to be substrates of
the enzyme.
Preparation of methyl 2-azido-4,6-O-benzyl-
In conclusion, through a simple series of
idene-2-deoxy-h- -altropyranoside (6).—The
D
functional group transformations of
D
-gluc-
known title compound 6 was prepared accord-
ing to the literature procedure.11
ose, we have prepared three hitherto un-
known C-3 nitrogen-containing derivatives of
ManNAc, 2–4. These compounds were not
substrates for Neu5Ac aldolase, and this out-
come suggests that a hydroxyl group at C-3 of
Methyl
carbonyl)amino-2-deoxy-h-
(7).—A solution of the azide 6, methyl 2-
azido-4,6-O-benzylidene-2-deoxy-a- -altro-
4,6-O-benzylidene-2-(tert-butoxy-
D
-altropyranoside
D

G.B. Kok et al. / Carbohydrate Research 332 (2001) 133–139
137
pyranoside11 (11.2 g, 36.5 mmol) in EtOH
(300 mL) was degassed (15 mm Hg/rt) before
the addition of platinum oxide (50 mg) and
di-tert-butyl dicarbonate (9.12 g, 41.8 mmol).
The mixture was subjected to hydrogenolysis
conditions (atmospheric H2) for 40 h at rt.
After filtration through a short plug of Celite,
the clear solution was concentrated in vacuo,
and the resulting amorphous solid was recrys-
tallised (1:1 EtOAc–hexane) to give the title
compound 7 as colourless needles (9.73 g,
70%): mp 170–171 °C; [h]D +49° (c 3.49,
CHCl3); Rf 0.27 (1:2 EtOAc–hexane). 1H
NMR (CDCl3): l 1.46 (s, 9 H, C(CH3)3), 2.88
(br s, 1 H, OH), 3.44 (s, 3 H, OCH3), 3.67
(m,1 H, H-4), 3.78 (dd, 1 H, J6,5 10.1, J6,6%
10.1, H-6), 4.08–4.18 (m, 2 H, H-2, H-5), 4.25
(m, 1 H, H-3), 4.34 (dd, 1 H, J6%,5 5.1, H-6%),
4.64 (br s, 1 H, H-1), 4.78 (br, 1 H, NH), 5.63
(s, 1 H, H-7), 7.35–7.53 (m, 5 H, Ph). 13C
NMR (CDCl3): l 28.3 (C(CH3)3), 53.0 (C-2),
55.7 (OCH3), 58.3 (C-3), 68.1 (C-5), 69.0 (C-
6), 76.8 (C-4), 80.3 (C(CH3)3), 101.3 (C-1),
102.3 (C-7), 126.2, 128.2, 129.1, 137.1 (aro-
matic carbons), 154.5 (carbonyl). LRMS: 382
(MH+, 13%), 326 (30), 294 (100); HRMS for
C19H28NO7 [M++H] requires 382.1866,
found 382.1855. Anal. Calcd for C19H27NO7:
C, 59.8; H, 7.1. Found: C, 59.9; H, 7.2.
stirred vigorously at rt for 15 h. The reaction
mixture was diluted with hexane (400 mL) and
washed with water (50 mL). The organic
phase was isolated and washed again with
water (20 mL) before drying and concentrat-
ing to provide the crude azide 7 (6.01 g) as a
straw-coloured foam. After column chro-
matography (1:8“1:6 EtOAc–hexane), pure
azide 9 was obtained as an off-white solid
(5.23 g, 70%); a small sample was recrys-
tallised from hexane: mp 123–124 °C (colour-
less needles); [h]D +25° (c 1.26, CHCl3); Rf
0.60 (1:2 EtOAc–hexane). IR (wmax, NaCl):
1
2103 cm−1 (N3). H NMR (CDCl3): l 3.38 (s,
1 H, OCH3), 3.66 (dd, 1 H, J4,3 9.7, J4,5 9.7,
H-4), 3.79 (dd, 1 H, J6,5 9.9, J6,6% 9.9, H-6),
3.89 (ddd, 1 H, J5,6% 4.4, H-5), 4.09 (dd, 1 H,
J3,2 4.2, H-3), 4.19 (m, 1 H, H-2), 4.28 (dd, 1
H, H-6%), 4.69 (br s, 1 H, H-1), 4.73 (d, 1 H,
JNH,2 7.5, NH), 5.61 (s, 1 H, H-7), 7.35–7.49
(m, 5 H, Ph). 13C NMR (CDCl3): l 28.4
(C(CH3)3, 53.1 (C-2), 55.1 (OCH3), 58.3 (C-3),
63.6 (C-5), 66.7 (C-6), 78.1 (C-4), 80.5
(C(CH3)3), 101.0, 102.0 (C-1, C-7), 126.1,
128.3, 129.1, 137.0 (aromatic carbons), 155.4
(carbonyl). LRMS: 407 (MH+, 16%), 381
(82), 351 (50), 325 (100); HRMS for
C19H27N4O6 [M++H] requires 407.1931;
found 407.1932. Anal. Calcd for C19H26N4O6:
C, 56.15; H, 6.45. Found: C, 55.8; H, 6.3.
Methyl 3-azido-4,6-O-benzylidene-2-(tert-
2-Acetamido-3-azido-2,3-dideoxy-h,i- -
D
butoxycarbonyl)amino - 2,3 - dideoxy - h -
D
-
mannopyranose (2).—A mixture of methyl 3-
azido-4,6-O-benzylidene-2-(tert-butoxycarbo-
mannopyranoside (9).—A stirring solution of
the alcohol 7 (7.00 g, 18.4 mmol) and pyridine
(2.24 mL, 27.7 mmol) in dry CH2Cl2 (100 mL)
was cooled to −24 °C (CCl4 –dry ice) prior to
the addition of trifluoromethanesulfonic anhy-
dride (3.24 mL, 19.2 mmol) dropwise over a
period of 30 min. When the addition was
complete, the cold bath was replaced with an
ice bath, and stirring was continued for an-
other 3 h. Et2O (400 mL) was then added to
the orange solution, and the resulting solids
were filtered off. Evaporation of the orange
solution under reduced pressure gave the cor-
responding 3-O-triflate 8 (9.13 g, 97%), which
was used in the subsequent step without fur-
ther purification: Rf 0.45 (1:2 EtOAc–hexane).
A mixture of the crude 3-O-triflate 8 (9.13
g, 17.8 mmol), sodium azide (2.34 g, 36.0
mmol), tetrabutylammonium hydrogensulfate
(6.50 g, 19.1 mmol) and benzene (150 mL) was
nyl)amino-2,3-dideoxy-a- -mannopyranoside
D
(9) (4.00 g, 9.85 mmol) and 4:1 HOAc–water
(100 mL) was heated under reflux for 24 h and
cooled. The volatiles were then evaporated
under reduced pressure, and the residue was
filtered through a short plug of silica gel.
Elution with a combination of EtOAc and
MeOH gave, after solvent removal, a straw-
coloured foam (2.53 g).
A solution of the foam in 5 N HCl (100
mL) was heated for 3 days at 100 °C. The
solution was then cooled, and the volatiles
were evaporated in vacuo. The resulting
residue was washed with Et2O (3×20 mL)
and dried under vacuum, leaving the corre-
sponding amine hydrochloride 10 as a straw-
coloured foam (2.34 g).
To a solution of the crude amine hydrochlor-
ide 10 (2.34 g) in MeOH (16 mL) and pyrid-

138
G.B. Kok et al. / Carbohydrate Research 332 (2001) 133–139
requires 373.1359; found 373.1372. Anal.
Calcd for C14H20N4O8: C, 45.16; H, 5.41.
Found: C, 44.9; H, 5.3.
ine (8 mL) at rt, was added Ac2O (8 mL)
dropwise over 5 min. After 30 min, the
volatiles were removed by co-distillation (×2)
with toluene. After drying under high vacuum,
the residue was purified by column chro-
matography (85:12:3 EtOAc–MeOH–water),
which gave the title compound as a straw-
coloured foam (1.95 g, 82%): Rf 0.20 (85:12:3
EtOAc–MeOH–water). IR (wmax, NaCl): 2112
2-Acetamido-3-(tert-butoxycarbonyl)amino-
2,3-dideoxy-h,i-
lution of the azide, 2-acetamido-3-azido-2,3-
dideoxy-a,b- -mannopyranoside (2) (2.00 g,
D
-mannopyranose (4).—A so-
D
8.1 mmol) in MeOH (50 mL) was degassed
(15 mm Hg/rt) before the addition of Pt2O (30
mg), and the mixture was subjected to hy-
drogenolysis conditions (atmospheric H2) until
TLC analysis showed the disappearance of
starting material. After filtration, the solution
was concentrated in vacuo, which gave the
corresponding amine 3 as a straw-coloured
foam (1.76 g, 98%).
1
cm−1 (N3). H NMR (D2O): l 2.05, 2.09 (s, 3
H, NHCOCH3(a,b)), 3.55–3.95 (m, 6 H, H-2,
H-3, H-4, H-5, H-6, H-6%), 5.05, 5.11 (each d,
1 H, J1,2 1.4 and 1.7, H-1(a,b)). 13C NMR
(D2O): l 24.6 (NHCOCH3), 54.3, 54.9 (C-
2(a,b)), 63.0 (C-6(a,b)), 64.0, 66.9, 68.0, 68.2,
74.7, 79.7 (C-3, C-4, C-5), 95.2, 95.7 (C-1(a,b)),
177.1, 178.1 (carbonyls). LRMS: 247 (MH+,
100%). 229 (89).
To a stirring solution of the amine 3 (1.76 g,
8.0 mmol) in 1:1 CH3NO2–water (10 mL) was
added NEt3 (2.23 mL, 16.0 mmol) and Boc-
ON (2.96 g, 12.0 mmol). After 2 days at rt, the
white precipitate was filtered off, and the solu-
tion was concentrated under reduced pressure.
The remaining residue was washed with Et2O
(2×20 mL) and then applied onto a silica gel
column. Elution with 10:2:1 EtOAc–2-PrOH–
water gave the title compound 4 as a colour-
less amorphous solid (1.40 g, 54%): Rf 0.20
(85:12:3 EtOAc–MeOH–water). 1H NMR
(D2O): l 1.37 (s, 9 H, C(CH3)3), 1.97, 2.01 (s,
3 H, NHCOCH3(a,b)), 3.40–3.95 (m, 5 H),
4.25–4.38 (m, 1 H), 5.04 (br s, 1 H, H-1).
2-Acetamido-4,5,6-tri-O-acetyl-3-azido-2,3-
dideoxy-h,i-
D
-mannopyranose (11).—To
a
stirring solution of 2-acetamido-3-azido-2,3-
dideoxy-a,b- -mannopyranose (2) (100 mg,
D
0.41 mmol) in pyridine (1 mL) at rt was added
Ac2O (0.5 mL). After 3 h, the reaction mixture
was concentrated to dryness, and the residue
was purified by column chromatography
(1:2“1:1 EtOAc–hexane) to give 11 as an
a/b anomeric mixture (110 mg, 73%), a:b=
4:1 (by 1H NMR spectroscopy): Rf 0.58
(EtOAc). A small sample was recrystallised
from 1:1 EtOAc–hexane, which gave the a
anomer,
azido-2,3-dideoxy-a-
2-acetamido-4,5,6-tri-O-acetyl-3-
-mannopyranose (11) as
Selected 13C NMR (D2O):
(NHCOCH3), 30.8 (C(CH3)3), 54.7, 55.5 (C-
(a,b)), 63.6 (C-6), 67.3, 67.4 (C-3(a,b)), 75.5,
l
24.9
D
fine colourless needles: mp 162–163 °C; [h]D
+64° (c 1.31, CHCl3). IR (wmax, NaCl): 2108
cm−1 (N3). 1H NMR for the a anomer
(CDCl3): l 1.54 (s, 9 H, C(CH3)3), 2.09, 2.10,
2.15, 2.17 (s, each 3 H, OCOCH3 ×3), 3.99
(ddd, 1 H, J5,4 9.8, J5,6 2.6, J5,6% 5.1, H-5),
4.10–4.35 (m, 2 H, H-3, H-6), 4.26 (dd, 1 H,
J6%,6 12.4, H-6%), 4.62 (ddd, 1 H, J2,1 1.7, J2,3
4.4, J2,NH 9.1, H-2), 5.07 (dd, 1 H, J4,3 10.0,
H-4), 5.68 (d, 1 H, NH), 6.04 (d, 1 H, H-1).
2
80.6 (C-4, C-5), 95.8, 96.2 (C-1(a,b)), 81.0
(C(CH3)3).
2-Acetamido-4,5,6-tri-O-acetyl-3-(tert-but-
oxycarbonyl)amino - 2,3 - dideoxy - h,i -
D
-
mannopyranose (12).—A solution of 2-acet-
amido - 3 - (tert - butoxycarbonyl)amino - 2,3-
dideoxy-a,b- -mannopyranose (4) (100 mg,
D
0.313 mmol) in pyridine (1.0 mL) was treated
with Ac2O (0.5 mL) at rt for 16 h and then
concentrated in vacuo. The resulting residue
was dissolved in CH2Cl2, and the solution was
washed with water (2×10 mL) and then
dried. After evaporation of the solvent, the
crude product (130 mg) was purified by
column chromatography (2:1 EtOAc–hexane)
to give first, the a anomer of the title com-
1
Selected H NMR data for the b anomer
(CDCl3): l 5.02 (dd, 1 H, J4,3 9.6, J4,5 9.6,
H-4), 5.78 (d, 1 H, J1,2 2.1, H-1). 13C NMR
(CDCl3): l 20.6, 20.7, 23.1 (OCOCH3 ×3),
49.8 (C-5), 59.2 (C-2), 62.3 (C-6), 66.9 (C-4),
70.0 (C-3), 91.5 (C-1), 167.9, 169.7, 170.4 (car-
bonyls). LRMS: 373 (MH+, 29%), 347 (61),
313 (100); HRMS for C14H21N4O8 [M+ +H]

G.B. Kok et al. / Carbohydrate Research 332 (2001) 133–139
139
Acknowledgements
pound 12 as a colourless crystalline solid (95
mg), followed by the b anomer (24 mg), giving
a combined total yield of 85%. Physicochemi-
cal data for the a anomer: mp 183–184 °C;
[h]D +46° (c 1.00, CHCl3); Rf 0.27 (3:1
We thank GlaxoSmithKline, Australia and
the Australian Research Council for financial
support.
1
EtOAc–hexane). H NMR (CDCl3): l 1.42 (s,
9 H, C(CH3)3), 2.07, 2.08, 2.10, 2.16 (s, each 3
H, NHCOCH3, OCOCH3 ×3), 4.04 (dd, 1 H,
J6,5 2.0, J6,6% 12.0, H-6), 4.08 (m, 1 H, H-5),
4.26–4.36 (m, 2 H, J6%,5 4.3, H-3, H-6%), 4.54
(m, 1 H, H-2), 4.81 (d, 1 H, JNH,3 9.0, NHa),
4.90 (dd, 1 H, J4,3 10.0, J4,5 10.0, H-4), 5.75 (d,
1 H, JNH,2 9.1, NHb), 5.95 (br s, 1 H, H-1). 13C
NMR l 20.5, 20.6, 20.8 (OCOCH3 ×3), 23.2
(NHCOCH3), 28.2 (C(CH3)3), 49.5 (C-3), 50.6
(C-2), 62.5 (C-6), 67.1 (C-4), 70.4 (C-5), 80.0
(C(CH3)3), 91.7 (C-1), 155.4, 168.3, 170.5,
170.6, 170.9 (carbonyls). LRMS: 447 (MH+,
4%), 402 (12), 380 (35), 347 (10), 331 (100),
280 (65), 239 (12), 60 (25). Anal. Calcd for
C19H30N2O10: C, 51.12; H, 6.77; N, 6.27.
Found: C, 51.0; H, 6.9; N, 6.2.
References
1. Hauser, F. M.; Ellenberger, S. R. Chem. Re6. 1986, 86,
35–67.
2. (a) See for example: Tadano, K.; Morita, M.; Hotta, Y.;
Ogawa, S. J. Org. Chem. 1988, 53, 5209–5215;
(b) Fleet, G. W. J.; Smith, P. W. Tetrahedron 1987, 43,
971–978.
3. Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J.
Tetrahedron: Asymmetry 2000, 11, 1645–1680.
4. Auge´, C.; Gautheron-Le Narvor, C. In Preparati6e Car-
bohydrate Chemistry; Hannesian, S., Ed.; Marcel Dekker:
New York, 1997; pp. 469–484 and references cited
therein.
5. (a) See for example: Biological Roles of the Sialic Acids;
Rosenberg, A., Ed.; Plenum Press: New York, 1995;
(b) von Itzstein, M.; Thomson, R. J. Curr. Med. Chem.
1997, 4, 185–210.
6. (a) Auge´, C.; Gautheron, C. J. Chem. Soc., Chem. Com-
mun. 1987, 857–860;
Physicochemical data for the b anomer: Rf
(b) Auge´, C.; David, S.; Gautheron, C.; Malleron, A.;
Cavaye, B. New J. Chem. 1988, 12, 733–744;
(c) Auge´, C.; Bouxom, B.; Cavaye, B.; Gautheron, C.
Tetrahedron Lett. 1989, 30, 2217–2220;
(d) Schrell, A.; Whitesides, G. M. Liebigs Ann. Chem.
1990, 1111–1114.
1
0.18 (3:1 EtOAc–hexane). H NMR (CDCl3):
l 1.39 (s, 9 H, C(CH3)3), 2.03, 2,04, 2.06, 2.13
(s, each 3 H, NHCOCH3, OCOCH3 ×3), 3.82
(m, 1 H, H-5), 3.98–4.12 (m, 2 H, H-3, H-6),
4.32 (m, 1 H, J6%,5 5.0, J6%,6 12.3, H-6%), 4.64 (m,
1 H, H-2), 4.82 (dd, 1 H, J4,3 10.4, J4,5 10.4,
H-4), 4.91 (br d, 1 H, JNH,3 7.0, NHa), 5.81 (br
7. Fitz, W.; Schwark, J.-R.; Wong, C.-H. J. Org. Chem.
1995, 60, 3663–3670.
8. Baumberger, F.; Vasella, A. Hel6. Chim. Acta 1988, 71,
429–445.
9. Guthrie, R. D.; Murphy, D. J. Chem. Soc. 1965, 3828–
3834.
13
s, 1 H, H-1), 6.32 (d, 1 H, JNH,2 9.2, NHb). C
NMR:
l
20.7
(OCOCH3 ×3),
23.5
10. Guthrie, R. D.; Murphy, D. J. Chem. Soc. 1963, 5288–
5294.
11. Ali, Y.; Richardson, A. C. Carbohydr. Res. 1967, 5,
441–448.
12. See for example: Buss, D. H.; Hough, L.; Richardson, A.
C. J. Chem. Soc. 1963, 5295–5301.
13. Bednarski, M. D.; Chenault, H. K.; Simon, E. S.;
Whitesides, G. M. J. Am. Chem. Soc. 1987, 109, 1283–
1285.
(NHCOCH3), 28.2 (C(CH3)3), 51.2 (C-3), 53.0
(C-2), 62.1 (C-6), 66.9 (C-4), 74.3 (C-5), 80.3
(C(CH3)3), 91.9 (C-1), 155.3, 168.3, 170.5,
170.6, 171.5 (carbonyls). LRMS: 447 (MH+,
3%), 380 (22), 347 (32), 331 (35), 280 (65), 239
(34), 141 (27), 60 (100).
.