Determination of the anomeric configurations of 2,3,4,6-tetra-o-acetyl-D- mannopyranosyl azide

Article abstract of DOI:10.1071/CH06157

The structures of 2,3,4,6-tetra-O-acetyl-?-d-mannopyranosyl azide and 2,3,4,6-tetra-O-acetyl-?-d-mannopyranosyl azide were determined using X-ray crystallographic and one-dimensional NOESY techniques. CSIRO 2006.

Full text of DOI:10.1071/CH06157

Communication
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2006, 59, 473–476
Determination of the Anomeric Configurations of
2,3,4,6-Tetra-O-Acetyl-d-Mannopyranosyl Azide
Kelly L. Cosgrove,A Paul V. Bernhardt,A Benjamin P. Ross,A and Ross P. McGearyA–C
A School of Molecular and Microbial Sciences, University of Queensland, Brisbane QLD 4072, Australia.
B School of Pharmacy, University of Queensland, Brisbane QLD 4072, Australia.
C Corresponding author. Email: r.mcgeary@uq.edu.au
The structures of 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl azide and 2,3,4,6-tetra-O-acetyl-β-d-mannopyranosyl
azide were determined using X-ray crystallographic and one-dimensional NOESY techniques.
Manuscript received: 10 May 2006.
Final version: 28 June 2006.
Glycosyl azides are frequently synthesized as precursors to
glycosylamines and glycosylamides, many of which are bio-
logically important.[1,2] Glycosyl azides may be reduced to
the corresponding amine and then acylated using peptide-
coupling methodologies. Alternatively, glycosyl azides can
be coupled directly to an acid chloride by means of the
Staudinger reaction.[3] The Staudinger reaction is the pre-
ferred method for the synthesis of β-glycosyl amides as it
allows reaction of the acid chloride directly with the sugar
azide, thereby eliminating the need for glycosyl amine inter-
mediates, whicharepronetoanomerizationandhydrolysis.[3]
An excellent discussion on the synthesis and structure of gly-
cosylazidescanbefoundinthereviewbyGyorgydeaketal.[4]
We have used glycosyl azides in the synthesis of a
series of glycosylamides, for a program examining the
inhibition of growth factors.[5] As part of our project
we aimed to synthesise both α- and β-mannopyranosyl
amides. This required preparation of 2,3,4,6-tetra-O-acetyl-
α-d-mannopyranosyl azide 3 and its C1 epimer 2,3,4,6-tetra-
O-acetyl-β-d-mannopyranosyl azide 4. Both 3 and 4 have
been described previously;[6–12] however, uncertainty exists
with regard to their anomeric configurations.[6,7] Despite
this uncertainty, the anomeric configurations of 3 and 4
have nevertheless been assigned quite frequently in the
literature.[6,7,10–12] In this paper we unambiguously assign
the anomeric configuration of the acetylated mannopyranosyl
azides 3 and 4, using a combination of X-ray crystallography
and NMR spectroscopy.
of mannose derivatives, then the anomeric configuration
cannot be predicted reliably from H NMR data because
1
J1,2 axial–equatorial and J1,2 equatorial–equatorial couplings
are of similar magnitude. The use of 13C chemical shifts
and carbon–proton coupling constants (1JC1,H1) are simi-
larly unreliable for establishing anomeric configuration in
mannose derivatives.[7]
We synthesized both α- and β-mannopyranosyl azides (3
and 4) and were able to assign their anomeric configurations
based on X-ray crystallography data and one-dimensional
NOESY experiments. Scheme 1 illustrates the synthesis of
3 and 4. The peracetylated sugar 1 was prepared follow-
ing the literature.[14] The general method of Pfleiderer and
Zondler[15] was used to synthesize the azides 3 and 4.
The mixture of anomers 3 and 4 was partially separated by
silica column chromatography to afford first a syrup which
was pure in one anomer, and second a syrup that contained a
mixture of anomers. The addition of hexane to this mixture
causedcrystallizationofoneanomerandallowedtheanomers
to be separated by trituration. Purification of the crystalline
anomer was achieved by recrystallization from diethyl ether.
A crystal suitable for X-ray diffraction was then grown from
methanol.Wereportherethecrystalstructureof2,3,4,6-tetra-
O-acetyl-β-d-mannopyranosyl azide 4.
Several crystals were screened before finally choosing
the one used for this structure determination. Unfortunately
the bulk sample lost crystallinity over a period of a week
or so thus preventing further examination perhaps at low
temperature. Despite compounded problems associated with
crystal twinning, instability, and disorder, the stereochem-
istry of compound 4 was unequivocally established by X-ray
crystallography (Fig. 1). All bond lengths and angles are as
expected and the azide adopts an equatorial disposition. The
carbonyl oxygen atoms and methyl carbon atom of the acetyl
group attached to C4 exhibited abnormally large anisotropic
thermal parameters during refinement and were subsequently
Typically, the anomeric configuration of a hexapyranose
1
can be readily determined from H NMR data if the pro-
ton at position 2 (H2) is axial. In that case the magnitude
of the coupling constant (J ) between H1 and H2 is rela-
tively large (J1,2 ∼ 9 Hz) if H1 is also axial. But the cou-
pling constant is comparatively small (J1,2 ∼ 2 Hz) if H1 is
equatorial, as described by the Karplus equation.[13] Unfor-
tunately, when H2 is oriented equatorially, as in the case
© CSIRO 2006
10.1071/CH06157
0004-9425/06/070473

474
K. L. Cosgrove et al.
OAc
H
OAc
O
AcO
AcO
H
H
OAc
OAc
H
H
N3
OAc
O
OAc
O
(a) 33% HBr/AcOH, CH2Cl2
5 h
(b) NaN3, DMF
3
AcO
AcO
AcO
AcO
80°C
~15 h
OAc
OAc
H
Br
OAc
1
O
2
AcO
AcO
N3
H
H
H
H
4
Scheme 1. The synthetic route for azides 3 and 4.
correlations in compound 3 indicates that H1 is on the β-face
of the pyranose ring, and that this compound is therefore the
α-anomer.
C4b2
O5b
O5a
C5b
O4a
C5a
C4a
In conclusion we have resolved the long-standing ambi-
guity surrounding the anomeric configuration of mannopyra-
nosyl azides (3 and 4). The literature previously assigned the
anomeric configuration without definitive evidence to sup-
port the stereochemistry at C1. We have presented conclusive
evidence for the anomeric configuration of 2,3,4,6-tetra-
O-acetyl-α-d-mannopyranosyl azide 3 and its C1 epimer
2,3,4,6-tetra-O-acetyl-β-d-mannopyranosyl azide 4.
C5
C5c
O1
O4b1
O3b
C4
C3
O3a
C2
C3a
C3b
C1
N3
N2
O2a
N1
C2a
O2b
Experimental
NMR experiments were recorded on a BrukerAVANCE 300 or 500 MHz
spectrometer. Chemical shifts are reported in parts per million (ppm) on
a δ scale, relative to the solvent peak (CDCl3 δH 7.24, δC 77.0; C6D6 δH
7.15). Coupling constants (J ) are reported in Hertz. All assignments
were confirmed with the aid of two-dimensional 1H, 1H (gCOSY),
1H, 13C (gHSQC), and selective ge-1D NOESY experiments. One-
dimensional NOESY experiments were recorded using C6D6 as the
solvent. For each one-dimensional NOESY experiment, 32 scans were
acquired with a Gaussian-shaped pulse and a mixing time of 500 ms.
Low- and high-resolution mass spectra were measured on a Finnigan
API-3-sprayer mass spectrometer in positive electrospray ionization
mode. Melting points were recorded on a Fischer Johns melting point
apparatus. Optical rotations were determined on a Perkin–Elmer 141
Polarimeter.
C2b
Fig. 1. ORTEP diagramof2,3,4,6-tetra-O-acetyl-β-d-mannopyranosyl
azide 4 (30% probability ellipsoids). Disorder of the acetyl group at C4
not shown for clarity.
resolved into two components that were refined with com-
plementary occupancies. The major contributor is shown in
Fig. 1. For comparison, the crystal structures of the iso-
meric 2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl azide[16]
and 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl azide[17] have
also been reported.
2,3,4,6-Tetra-O-acetyl-α-D-mannosyl Bromide 2
It could simply be assumed that the oily anomer was the
α-azide 3; however, to prove its anomeric configuration we
conducted one-dimensional NOESY experiments. For com-
parison, one-dimensional NOESY experiments were also
recorded on the β-anomer 4. As illustrated in Fig. 2a, selec-
tive irradiation of the H1 resonance of the oily anomer 3
generated an nuclear overhauser enhancement (NOE) corre-
lation with only the H2 resonance. In comparison, selective
irradiation of the H1 resonance in the crystalline β-anomer 4
(Fig. 2b) generated NOE correlations with the H2, H3, and
H5 protons. The presence of correlations with H3 and H5
in compound 4 indicates that H1 is situated on the α-face
of the pyranose ring. Conversely the absence of H3 and H5
A 33% solution of hydrogen bromide in acetic acid (30 mL) was added
to 1,2,3,4,6-penta-O-acetyl-d-mannopyranose[14] 1 (9.0 g, 23 mmol) in
CH2Cl2 (60 mL) which was stirred for 5 h at room temperature. The
reaction was quenched with cold water, and extracted with CH2Cl2. The
organic layer was washed with saturated solution of NaHCO3 and brine
before being dried (MgSO4), filtered, and evaporated under vacuum to
give the crude glycosyl bromide 2 as a yellow oil.
2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl Azide 3 and
2,3,4,6-Tetra-O-acetyl-β-D-mannopyranosyl Azide 4
2,3,4,6-Tetra-O-acetyl-d-mannopyranosyl bromide 2 (6.6 g, 16 mmol)
was dissolved in DMF (30 mL) and stirred with NaN3 (9.10 g,
140 mmole) at 80◦C for 15 h. The reaction mixture was diluted with
water and extracted with CH2Cl2. The combined organic layers were

Anomeric Configurations of 2,3,4,6-Tetra-O-Acetyl-d-Mannopyranosyl Azide
475
(a)
1D NOESY
1H NMR
H3
H2
H1
H4
H5
H6
H6Ј
5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8 4.7 4.6 4.5 4.4 4.3 4.2 4.1 4.0 3.9 3.8 3.7 ppm
(b)
1D NOESY
H4
5.6
H2
H3
H6
H6Ј
H1
H5
1H NMR
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
ppm
Fig. 2. 1H NMR and one-dimensional NOESY spectra for (a) 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl azide 3 and (b) 2,3,4,6-tetra-O-acetyl-
β-d-mannopyranosyl azide 4.
washed with brine, dried (MgSO4), filtered, and concentrated under vac-
uum to give an orange oil.∗ Partial separation of the two anomers was
achieved by column chromatography (SiO2; 2/3 EtOAc/light petroleum
bp 40–60◦C). Fractions containing a mixture of anomers were tritu-
rated with hexane. The crystalline material 4 that separated was further
purified by recrystallization from diethyl ether.
2.19, 2.09, 2.03, 1.97 (12H, 4s, 4CH3). δC (125 MHz, CDCl3) 170.6
(1 × C=O), 169.9 (2 × C=O), 169.5 (1 × C=O), 85.1 (C1), 74.7 (C5),
71.0 (C3), 69.2 (C2), 65.4 (C4), 62.3 (C6), 20.7 (2 × CH3), 20.6 (CH3),
20.5 (CH3).
Crystallography
2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl azide 3 was obtained as
a yellow oil (0.5 g, 9%). RF 0.57 (5/3/2 hexane/EtOAc/CHCl3, phospho-
molybdic acid and cerium sulfate dip). [α]D +105.2◦ (c 0.1, CHCl3;
lit[9] +103◦). m/z (ESI-MS) 396 ([M + Na]+). HRMS calculated for
[M + Na]+ 396.1019, found 396.1024. δH (500 MHz, C6D6) 5.54 (1H,
t, J 10.1, H4), 5.41 (1H, dd, J3,4 10.1, J2,3 3.4, H3), 5.24 (1H, dd, J2,3
Compound 4: C14H19N3O9, M 373.32, T 293 K, orthorhombic, space
group P212121 (no. 19), a 8.562(3), b 9.400(4), c 22.67(1) Å,V 1825(1)
Å3, Dc (Z 4) 1.359 g cm−3, F(000) 784, µ(MoKα) 0.115 mm−1, 2532
unique data (2θmax 46◦), 1434 with I > 2σ(I); R 0.0784 (obs. data), wR2
0.2182 (all data), goodness of fit 1.03.
Intensity data at 293 K were collected on an Enraf–Nonius CAD4
four-circle diffractometer using graphite monochromated MoKα radi-
ation (λ 0.71073 Å) in the ω − 2θ scan mode within the range
3 < 2θ < 46◦. A twinned specimen was chosen (from several attempts)
and lattice dimensions were determined by a least-squares fit of the
setting parameters of 20 independent reflections. Indexing was made
possible with the program DIRAX.[18] Data reduction and empirical
absorption corrections (ψ-scans) were performed with the WINGX
package.[19] The structure was solved by direct methods with SHELXS
and refined by full matrix least-squares analysis with SHELXL97.[20] All
non-H atoms were refined with anisotropic thermal parameters except
those involved in disorder (specifically the acetyl group at position 4 on
the ring). H-atoms were constrained at estimated positions. The abso-
lute configuration was assigned on the basis of the starting material.
The atomic nomenclature is defined in Fig. 1 drawn with ORTEP3.[21]
Crystallographic data in CIF format are available from the Cambridge
Crystallographic Data Base (CCDC deposition number 604525).
ꢀ
3.4, J1,2 2.0, H2), 4.66 (1H, d, J1,2 1.9, H1), 4.26 (1H, dd, J6,6 12.4,
J5,6 5.2, H6), 4.03 (1H, dd, J6,6 12.4, J5,6 2.3, H6ꢀ), 3.83 (1H, m, H5),
1.70, 1.67, 1.63, 1.58 (12H, 4s, 4 × COCH3). δH (500 MHz, CDCl3)
5.36 (1H, d, J1,2 1.9, H1), 5.28–5.21 (2H, m, H3 + H4), 5.13 (1H, dd,
ꢀ
ꢀ
ꢀ
J2,3 3.0, J1,2 2.0, H2), 4.28 (1H, dd, J6,6 12.6, J5,6 5.7, H6), 4.15 (1H, dd,
J6,6 12.6 J5,6 2.4, H6ꢀ) 4.14–4.10 (1H, m, H5), 2.14, 2.09, 2.03, 1.97
(12H, 4s, 4 × CH3). δC (125 MHz, CDCl3) 170.6, 169.8, 169.7, 169.6
(4 × C=O), 87.5 (C1), 70.7 (C5), 69.2 (C2), 68.3 (C3), 65.7 (C4), 62.2
(C6), 20.8, 20.7, 20.6, 20.6 (4 × CH3).
ꢀ
ꢀ
2,3,4,6-Tetra-O-acetyl-β-D-mannopyranosyl azide 4 was isolated as
colourless crystals (0.82 g, 14%), mp 115◦C (lit.[11] 124◦C). RF 0.43
(5/3/2 hexane/EtOAc/CHCl3, phosphomolybdic acid and cerium sul-
fate dip). [α]D −69◦ (c 0.1, CHCl3) (lit.[11] −77.0◦). m/z (ESI-MS)
396 ([M + Na]+). HRMS calculated for [M + Na]+ 396.1019, found
396.1023. δH (500 MHz, C6D6) 5.52 (1H, t, J10.2, H4), 5.44 (1H, dd,
J2,3 3.3, J1,2 1.3, H2), 4.97 (1H, dd, J3,4 10.2, J2,3 3.3, H3), 4.24 (1H,
dd, J6,6 12.5, J5,6 5.2, H6), 4.05 (1H, dd, J6,6 12.5, J5,6 2.3, H6ꢀ), 3.68
(1H, d, J1,2 1.3, H1), 3.16 (1H, m, H5), 1.69, 1.67, 1.66, 1.62 (12H, 4s,
4CH3). δH (500 MHz, CDCl3) 5.43 (1H, dd, J2,3 3.3, J1,2 1.3, H2), 5.24
(1H, t, J10.1, H4), 5.02 (1H, dd, J3,4 10.1, J2,3 3.3, H3), 4.71 (1H, d,
ꢀ
ꢀ
ꢀ
Acknowledgments
We thank Dr Tri Le for help with one-dimensional NOESY
experiments, and Mr Peter Moyle for helpful discussions.
ꢀ
ꢀ
J1,2 1.3, H1), 4.26 (1H, dd, J6,6 12.3, J5,6 5.6, H6), 4.19 (1H, dd, J6,6
12.3, J5,6 2.6, H6ꢀ), 3.74 (1H, ddd, J4,5 10.0, J5,6 5.7, J5,6 2.6, H5),
ꢀ
ꢀ
∗ Caution: While concentrating the crude product under high vacuum, a small amount of colorless liquid collected in the cold trap of the vacuum pump.
This liquid detonated the following day during disassembly of the cold trap. We suspect that the explosive liquid was diazidomethane, and we strongly
recommend that azide ion not be allowed to contact halogenated solvents, even during workup of reactions involving its use. For a discussion of the hazards
of diazidomethane, see http://pubs.acs.org/cen/safety/index.html

476
K. L. Cosgrove et al.
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