Rapid and clean microwave-assisted synthesis of N-acetylneuraminic acid methyl ester and its β-methyl glycoside

Article abstract of DOI:10.1016/j.tetlet.2012.09.017

The effect of microwave irradiation on the synthesis of N-acetylneuraminic acid methyl ester and its β-methyl glycoside is investigated. On a 1 g scale, a high yield (94%) of the methyl ester was obtained after 15 min at 80 °C. Acceleration of the glycosidation reaction was achieved, with the β-methyl glycoside isolated in good yield following optimisation of reaction conditions to 15 min at 12°C.

Full text of DOI:10.1016/j.tetlet.2012.09.017

Tetrahedron Letters 53 (2012) 6254–6256
Contents lists available at SciVerse ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Rapid and clean microwave-assisted synthesis of N-acetylneuraminic acid
methyl ester and its b-methyl glycoside
⇑
Pradeep Chopra, Robin J. Thomson, I. Darren Grice, Mark von Itzstein
Institute for Glycomics, Griffith University Gold Coast Campus, Queensland 4222, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 June 2012
Revised 30 August 2012
Accepted 6 September 2012
Available online 14 September 2012
The effect of microwave irradiation on the synthesis of N-acetylneuraminic acid methyl ester and its b-
methyl glycoside is investigated. On a 1 g scale, a high yield (94%) of the methyl ester was obtained after
15 min at 80 °C. Acceleration of the glycosidation reaction was achieved, with the b-methyl glycoside iso-
lated in good yield following optimisation of reaction conditions to 15 min at 120 °C.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Sialic acid
Microwave
N-Acetylneuraminic acid methyl ester
b-Methyl glycoside
Sialic acids comprise a family of over 50 nine-carbon acidic
monosaccharides, with the most prevalent member being N-acet-
1
ylneuraminic acid (Neu5Ac, 1). They are widely distributed in nat-
ure, typically glycosidically linked to the non-reducing end of a
range of glycoprotein- and glycolipid-associated glycans that are
2
–4
Scheme 1.
present on cell surfaces.
These carbohydrates are intimately in-
volved in a wide range of biological processes, ranging from cell–
cell interactions, cell differentiation and tumour metastasis, to
Microwave irradiation has been used in some thermally driven
organic reactions as an alternative source of heat energy to accel-
5–7
host–pathogen recognition phenomena.
In order to investigate
1
1,12
the requirements of sialic acid recognizing proteins, in particular
the enzymes involved in sialic acid metabolism such as CMP-sia-
late synthase (CMAS), sialidases and Neu5Ac aldolase, extensive
manipulations have been carried out on Neu5Ac, resulting in mod-
erate chemical transformations.
Some of the major advantages
of using microwave-assisted synthesis include reduced reaction
1
3
times, increased purity and higher yields. The use of microwave
irradiation in chemical manipulations of carbohydrates is still rel-
8
14
ification at each carbon. In this context, we recently reported the
atively in its infancy, although some well known reactions such
1
5
synthesis of C-9 oxidised derivatives of
sides of N-acetylneuraminic acid methyl ester.
a
- and b- (2) methyl glyco-
as Fischer glycosylation have been reported. To date, micro-
wave-assisted synthesis has been poorly explored in the chemistry
9
1
6
The functionalisation of N-acetylneuraminic acid (1) typically
commences with the methyl esterification of the anomeric carboxy
group to provide Neu5Ac1Me (3) and is followed by, in specific in-
stances, glycosidation of the anomeric hydroxy group. Both the b-
methyl glycoside of N-acetylneuraminic acid methyl ester
of sialic acids. Herein we report on the optimisation of the rapid
and facile synthesis of both 2 and 3 using microwave irradiation.
To examine the effect of microwave irradiation on the synthesis
Ò
of the methyl ester of 1 and its b-methyl glycoside using Dowex
+
50x8 (H ) resin in anhydrous methanol, initial screening was car-
1
7
[
Neu5Acb1,2Me
2
(2)] and methyl ester 3 itself, are useful interme-
ried out to optimise microwave irradiation time (Scheme 2).
We employed a reaction temperature of 100 °C at a maximum
microwave power of 100 W for a period of 5, 10, 15 and 20 min,
10
diates in sialic acid chemistry. Kuhn et al. first reported the syn-
thesis of 2 in 1966, which involved refluxing 1 with dry Dowex
5
Ò
+
18
0x8 (H ) resin in anhydrous methanol for 24 h (Scheme 1). This
respectively (Table 1).
method is still favoured today despite the long reaction time.
The progress of the reaction was monitored by thin-layer chro-
1
18
matography (TLC) and H NMR spectroscopy.
TLC analysis
showed that after 5 min complete consumption of starting mate-
1
⇑
Corresponding author. Tel.: +61 7 5552 7025; fax: +61 7 5552 9040.
E-mail address: m.vonitzstein@griffith.edu.au (M. von Itzstein).
rial 1 had occurred. Investigation by H NMR spectroscopy (in
2
D O) indicated that the major product formed was Neu5Ac1Me
0
040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.09.017

P. Chopra et al. / Tetrahedron Letters 53 (2012) 6254–6256
6255
Scheme 2. Reagents and conditions: (i) MeOH (anhyd), DowexÒ 50Wx8 (H ) resin, 100 °C, maximum power 100 W.
+
Table 1
Table 3
a
a
Microwave-assisted synthesis of 2 and 3 at 100 °C
Optimisation of the reaction conditions: time at 120 °C
Entry
Time (min)
Ratio (2:3)b
Entry
Reaction time (min)
Ratio (2:3)b
1
2
3
4
5
10
15
20
0.06:1.0
0.83:1.0
1.0:0.70
1.0:1.0
1
2
3
4
5
10
12
15
18
20
1.0:0.25
1.0:0.25
1.0:0.20
1.0:0.22
1.0:0.20
a
Reaction was carried out on 50 mg (0.16 mmol) of 1 at 100 °C under microwave
18
a
irradiation.
Reactions carried out on 50 mg (0.16 mmol) of 1 at 120 °C under microwave
18
b
Ratio of reaction products (2:3) in the reaction mixture as monitored by 1
H
)
irradiation.
b
NMR (300 MHz, D
2
O, ppm): d 2.42 (dd, 1H, J3eq,3ax 12.9 Hz, J3eq,4 5.1 Hz, H-3eq
Composition of the reaction products (2:3) in the reaction mixture as monitored
by 1H NMR (300 MHz, D
O, ppm): d 2.42 (dd, 1H, J3eq,3ax 12.9 Hz, J3eq,4 5.1 Hz, H-3eq
Neu5Acb1,2Me (2), d 2.40 (dd, 1H, J3eq,3ax 12.9 Hz, J3eq,4 5.1 Hz ,H-3eq) Neu5Ac1Me
3).
Neu5Acb1,2Me (2), d 2.40 (dd, 1H, J3eq,3ax 12.9 Hz, J3eq,4 5.1 Hz, H-3eq) Neu5Ac1Me
3).
2
2
)
(
2
(
(
3
3). This was established by the presence of a broad singlet at d
.75 corresponding to COOMe. Also formed as a very minor compo-
nent was Neu5Acb1,2Me (2), as indicated by the presence of a sin-
of 2 was then carried out by exploring five reaction times between
0 and 20 min (Table 3). The optimum reaction time for the syn-
thesis of 2 at this temperature was found to be 15 min.
1
2
1
glet at d 3.30, corresponding to the anomeric OMe of 2. H NMR
analysis of the 10 min reaction, revealed the clear presence of a
doublet of doublets at d 2.42 and two singlets at d 3.30 and d
It was of interest to determine if methyl ester 3 could be formed
as the sole reaction product under the microwave irradiation con-
Ò
ditions. It was found that by decreasing the amount of Dowex
5
3
.86, corresponding to H-3eq, OMe and COOMe, respectively, of 2,
+
18
0x8 (H ) resin catalyst (from 0.05 to 0.01 g of resin per 0.05 g
confirming the formation of 2, although 3 was still the predomi-
nant reaction product in a ratio of 0.83:1.0 (2:3). Interestingly, irra-
diation of the reaction mixture for 15 min resulted in the dominant
formation of 2 in the ratio of 1.0:0.70, as indicated by the integra-
tion of the respective H-3eq signals in the H NMR spectrum of the
reaction mixture (see Table 1).
of 1) and decreasing the reaction concentration, using the condi-
tions of 80 °C and 15 min reaction time, only ester 3 was formed,
1
19
as indicated by TLC and H NMR spectroscopy.
1
To investigate the effect of microwave irradiation on the scale of
synthesis of 2 and 3 we increased the mass of starting material 1 by
2
0
2
0-fold to 1.0 g. The optimised microwave irradiation conditions
for the preparation of 2 (15 min at 120 °C at maximum power of
00 W) afforded the desired compound 2 in 63% yield, with 3 also
A progressive increase in the quantity of the b-methyl glycoside
was obtained with increasing microwave irradiation from 5 to 10
2
1
to 15 min. However, irradiating for 20 min resulted in decomposi-
tion of the carbohydrate. Based on this observation, 15 min of
microwave exposure was selected as the optimum reaction time.
We then proceeded to optimise the reaction temperature, employ-
ing temperatures ranging from 80 to 180 °C (Table 2). Tempera-
tures below 100 °C favoured the formation of Neu5Ac1Me (3),
while above 120 °C increasing levels of decomposition were
observed. It is evident from Table 2 that the optimum reaction
temperature for the synthesis of 3 is 80 °C. The optimum reaction
temperature for the synthesis of 2 was 120 °C. While maintaining
the temperature at 120 °C, further optimisation for the synthesis
2
0
isolated in 15% yield [(2:3 ratio, 1.0:0.23)]. The determined opti-
mised reaction conditions for the preparation of ester 3 provided
the target compound in 94% isolated yield.
2
1
In conclusion, we have developed and optimised a rapid and
convenient method employing microwave irradiation for a facile
access to N-acetylneuraminic acid methyl ester (3) in excellent
yield and its b-methyl glycoside (2). Although the yield of 2 was
comparable to the conventional method of synthesis, 1
0,21
the ma-
jor advantage of this approach was that the reaction time was
greatly reduced from 24 h to 15 min with microwave assistance.
Additionally, there is a significant reduction in the amount of sol-
vent and acidic resin required under our conditions. Scale-up syn-
thesis for both 2 and 3 was demonstrated to be feasible and with
advancement in microwave reactors the often required large-scale
production of grams to kilograms of product would appear to be an
available option, with little or no further procedural modifications
required.
Table 2
a
Optimisation of the reaction conditions: temperature
Entry
Temp (°C)
Ratio (2:3)b
1
2
3
4
5
6
7
8
9
80
90
0.10:1.0
0.15:1.0
0.46:1.0
1.0:0.50
1.0:0.20
1.0:0.58
1.0:0.47
0.24:1.0
0.20:1.0
100
110
120
130
140
160
180
Acknowledgments
P.C. thanks Griffith University (Research Scholarship) and the
Institute for Glycomics for financial support. M.v.I. is grateful to
the Australian Research Council and the National Health and Med-
ical Research Council for financial support.
a
Reaction was carried out on 50 mg (0.16 mmol) of 1 for 15 min under micro-
18
wave irradiation.
b
Composition of the reaction products (2:3) in the reaction mixture as monitored
O, ppm): d 2.42 (dd, 1H, J3eq,3ax 12.9 Hz, J3eq,4 5.1 Hz, H-3eq
(2), d 2.40 (dd, 1H, J3eq,3ax 12.9 Hz, J3eq,4 5.1 Hz, H-3eq) Neu5Ac1Me
References and notes
by 1H NMR (300 MHz, D
Neu5Acb1,2Me
3).
)
2
2
1.
(a) Schauer, R. Angew. Chem., Int. Ed. 1973, 12, 127–138; (b) Angata, T.; Varki, A.
Chem. Rev. 2002, 102, 439–469.
(

6
256
P. Chopra et al. / Tetrahedron Letters 53 (2012) 6254–6256
was decanted off. The resin was then similarly washed with MeOH
2
.
.
.
.
.
Varki, A. Glycobiology 1992, 2, 25–40.
Schauer, R. Glycoconjugate J. 2000, 17, 485–499.
Chen, X.; Varki, A. ACS Chem. Biol. 2010, 5, 163–176.
Varki, A. Trends Mol. Med. 2008, 14, 351–360.
Hedlund, M.; Padler-Karavani, V.; Varki, N. M.; Varki, A. Proc. Natl. Acad. Sci.
U.S.A. 2008, 105, 18936–18941.
3
4
5
6
(2 Â 200 mL) and finally with anhydrous MeOH (200 mL). The washed resin
was dried on a rotary evaporator, then under high vacuum for 16 h, and was
2
finally stored under N or Ar.
19. Optimised conditions for the synthesis of Neu5Ac1Me (3) from N-
acetylneuraminic acid (1) (0.05 g, 0.16 mmol), that consistently gave high
yields (>92%), used less of the freshly dried resin (0.01 g) and an increased
solvent volume (3 mL), with reaction at 80 °C for 15 min.
7
8
.
.
Inoue, S.; Sato, C.; Kitajima, K. Glycobiology 2010, 20, 752–762.
(a) Zbiral, E. In Carbohydrates—Synthetic Methods and Applications in Medicinal
Chemistry; Ogura, H., Hasegawa, A., Suami, T., Eds.; VCH: Weinheim, 1992; pp
20. To explore larger scale syntheses of 2 and 3 a mixture of N-acetylneuraminic
Ò
+
3
04–339; (b) von Itzstein, M.; Thomson, R. J. Curr. Med. Chem. 1997, 4, 185–210.
acid (1) (1.0 g, 3.24 mmol) and dry Dowex 50x8 (H ) resin (1.0 g for the
synthesis of 2 or 0.2 g for the synthesis of 3) in anhydrous MeOH (25 mL) was
irradiated for 15 min at either 120 °C (for the synthesis of 2) or 80 °C (for the
synthesis of 3). After completion of the holding time, the reaction mixture was
cooled to room temperature, the resin was removed by filtration over Celite,
and the filtrate was concentrated under reduced pressure to give a syrup.
9
.
Kiefel, M. J.; Chopra, P.; Madge, P. D.; Szyczew, A.; Thomson, R. J.; Grice, I. D.;
von Itzstein, M. Tetrahedron Lett. 2011, 52, 98–100.
1
1
0. Kuhn, R.; Lutz, P.; MacDonald, D. L. Chem. Ber. 1966, 99, 611–617.
1. Gedye, R.; Smith, F.; Westaway, K.; Ali, H.; Baldisera, L.; Laberge, L.; Rousell, J.
Tetrahedron Lett. 1986, 27, 279–282.
1
1
1
2. Giguere, R. J.; Bray, T. L.; Duncan, S. M.; Majetich, G. Tetrahedron Lett. 1986, 27,
2
Synthesis of Neu5Acb1,2Me (2): The crude product mixture was dissolved in a
4
945–4948.
small volume of EtOAc–MeOH, adsorbed onto silica, and purified using a
Ò
3. (a) Kappe, C. O. Angew. Chem., Int. Ed. 2004, 43, 6250–6284; (b) Caddick, S.;
Fitzmaurice, R. Tetrahedron 2009, 65, 3325–3355.
4. (a) Corsaro, A.; Chiacchio, U.; Pistarà, V.; Romeo, G. Curr. Org. Chem. 2004, 8,
Reveleris flash chromatography system [40 g column; flow rate 30 mL/min;
eluent: 100% EtOAc to 4:1 EtOAc–MeOH] to provide Neu5Acb1,2Me
2
(2)
(0.68 g, 63%) and Neu5Ac1Me (3) (0.15 g, 15%).
f 2 3
= 0.55 (EtOAc–MeOH–H OD): d 1.62
O 7:2:1); 1H NMR (CD
5
11–538; (b) Corsaro, A.; Chiacchio, U.; Pistarà, V.; Romeo, G., 2nd ed. In
Microwaves in Organic Synthesis; Loupy, A., Ed.; Wiley-VCH, 2006; Vol. 1, pp
79–614; (c) Richel, A.; Laurent, P.; Wathelet, B.; Wathelet, J.-P.; Paquot, M. C.
Compound 2: R
(1H, dd, J3ax, 3eq 12.9 Hz, J3ax, 4 11.4 Hz, H-3ax), 1.99 (3H, s, NAc), 2.33 (1H, dd,
3eq, 3ax 12.9 Hz, J3eq, 4 5.1 Hz, H-3eq), 3.25 (3H, s, OMe), 3.49 (1H, d, J6,5 9.0 Hz,
H-6), 3.64 (1H, dd, J9A,9B 12.0 Hz, J9A,8 6.3 Hz, H-9A), 3.77–3.84 (7H, m, H-5, H-7,
5
J
R. Chimie 2011, 14, 224–234.
1
3
1
1
1
5. (a) Nüchter, M.; Ondruschka, B.; Lautenschläger, W. Synth. Commun. 2001, 31,
H-8, H-9B, CO
[NC(O)Me], 39.0 (C-3), 50.87 (OMe), 51.5 (C-5), 53.4 (CO
(C-4), 67.8, 69.6 and 70.4 (C-6, C-7, C-8), 99.0 (C-2), 170.3 (C-1), 174.6
2
Me), 3.94–4.01 (1H, m, H-4);
C
NMR (CD
3
OD): d 21.9
1
3
277–1283; (b) Bornaghi, L. F.; Poulsen, S.-A. Tetrahedron Lett. 2005, 46, 3485–
488; (c) Roy, D. K.; Bordoloi, M. J. Carbohydr. Chem. 2008, 27, 300–307.
2
Me), 63.2 (C-9), 66.3
+
6. (a) Šardzík, R.; Noble, G. T.; Weissenborn, M. J.; Martin, A.; Webb, S. J.; Flitsch, S.
L. Beilstein J. Org. Chem. 2010, 6, 699–703; (b) Patane, J.; Trapani, V.; Villavert, J.;
McReynolds, K. D. Carbohydr. Res. 2009, 344, 820–824.
[NC(O)Me]; LRMS: C13
Compound 3: R = 0.50 (EtOAc–MeOH–H
(1H, dd, J3ax, 3eq 12.9 Hz, J3ax, 4 11.4 Hz, H-3ax), 2.00 (3H, s, NAc), 2.17 (1H, dd,
3eq, 3ax 12.9 Hz, J3eq, 5.1 Hz, H-3eq), 3.45 (1H, dd, J7,6 1.5 Hz, J7,8 9.3 Hz, H-7),
3.57 (1H, dd, J9A,9B 11.1 Hz, J9A,8 5.7 Hz, H-9A), 3.66 (1H, m, H-8), 3.76 (3H, s,
CO Me), 3.79–3.83 (2H, m, H-5, H-9B), 3.96 (1H, dd, J6,7 1.5 Hz, J6,5 10.5 Hz, H-
6), 4.02 (1H, m, H-4); C NMR (CD
(CO Me), 54.3 (C-5), 64.8 (C-9), 67.8 (C-4), 70.1, 71.6 and 72.6 (C-6, C-7, C-8),
H23NO
9
m/z 360.3 ([M+Na] , 100%).
O 7:2:1); 1H NMR (CD
f
2
3
OD): d 1.84
7. A CEM DiscoverÒ SP Explorer Hybrid-12 microwave system with single mode
cavity was used in this study. The microwave can perform reactions with
volumes of 0.2–75 mL and run open vessel or pressurised vessel reactions. The
system embodies volume-independent infrared temperature or fibre optic
temperature measurement, automated power control, air-cooling for
simultaneous cooling (PowerMax) and reaction quenching and ActiVent self-
venting technology for pressure relief during or after reaction.
J
4
2
13
3
OD): d 22.6 [NC(O)Me], 40.7 (C-3), 53.1
2
96.6 (C-2), 171.7 (C-1), 175.1 [NC(O)Me]; LRMS:
([M+Na] , 100%).
12 9
C H21NO m/z 345.8
+
1
8. General procedure for the synthesis of Neu5Acb1,2Me
2
(2) and Neu5Ac1Me (3)
Synthesis of Neu5Ac1Me (3): The crude syrupy product was dried under high
vacuum to provide an amorphous solid. The amorphous solid was washed with
EtOAc (3 Â 10 mL) and dried under high vacuum to provide 2 (0.99 g, 94%).
Characterisation is as given above.
under microwave irradiation conditions: In a Teflon (septum)-sealed 10 mL
pressure tube, a mixture of N-acetylneuraminic acid (1) (0.05 g, 0.16 mmol)
and dry DowexÒ 50x8 (H ) resin (0.05 g) in anhydrous MeOH (2 mL) was
+
irradiated for a specific time at a specific temperature. After completion of the
21. General procedure for the conventional synthesis of Neu5Acb1,2Me (2): A
2
1
Ò
holding time, an aliquot of the reaction mixture was analysed by TLC and
NMR spectroscopy. 1H NMR spectra were recorded in D
of the anomeric OMe which is overlapped by residual MeOH when the spectra
are run in CD OD.
H
mixture of N-acetylneuraminic acid (1) (5.0 g, 16.18 mmol) and dry Dowex
+
2
O to allow observation
50x8 (H , 12.5 g) in anhydrous MeOH (250 mL) was refluxed for 48 h, then
cooled to room temperature and the resin removed by filtration. The filtrate
was concentrated under reduced pressure to give a yellow syrup. The syrup
was then dissolved in a small volume of EtOAc–MeOH (3:1, v/v). On standing at
ꢀ4 °C, crystals were deposited which were further washed with ethyl acetate
3
It should be noted, we observed that the age and quality of the resin used
influenced reaction outcomes. In the reported experiments the resin was
Ò
+
prepared according to the following procedure: A slurry of Dowex 50x8 (H )
resin (50 g) in H O (400 mL) was gently agitated, allowed to settle, and the H
f
to obtain crystalline 2 (3.4 g, 62%). R = 0.2 (EtOAc–MeOH, 5:1).
2
2
O