Synthesis of C-9 oxidised N-acetylneuraminic acid derivatives as biological probes

Article abstract of DOI:10.1016/j.tetlet.2010.10.156

Sialic acids are 9-carbon acidic sugars involved in a number of important biological processes and human diseases. As part of our ongoing interest in the development of novel sialic acids as biological probes, we have developed an efficient and simple synthesis of C-9 oxidised sialic acid derivatives. The key oxidative step involves the use of TEMPO under carefully controlled aqueous pH conditions.

Full text of DOI:10.1016/j.tetlet.2010.10.156

Tetrahedron Letters 52 (2011) 98–100
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of C-9 oxidised N-acetylneuraminic acid derivatives
as biological probes
Milton J. Kiefel , Pradeep Chopra , Paul D. Madge, Alex Szyczew, 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:
Sialic acids are 9-carbon acidic sugars involved in a number of important biological processes and human
diseases. As part of our ongoing interest in the development of novel sialic acids as biological probes, we
have developed an efficient and simple synthesis of C-9 oxidised sialic acid derivatives. The key oxidative
step involves the use of TEMPO under carefully controlled aqueous pH conditions.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 18 August 2010
Revised 21 October 2010
Accepted 29 October 2010
Available online 4 November 2010
Keywords:
Sialic acid
Oxidation
Chain-extended
Biological probes
Carbohydrates
Sialic acids are typically found in nature at the non-reducing
terminus of a range of glycoproteins and glycolipids present on
the surface of cells.1–4 These 9-carbon acidic sugars are intimately
involved in a wide range of biological processes, ranging from cell–
cell interactions, cell differentiation, tumour metastasis and patho-
gen-host recognition phenomena.1,4–7 The most common member
of the sialic acid family in humans is N-acetylneuraminic acid
C-9 modified sialic acids, recent examples include the preparation
of hydrophobic chain-extended compounds such as 2 as probes for
myelin-associated glycoprotein.19 Several different types of sialic
acid derivatives have been used as probes for sialyltransferases,
including cytidine monophosphate (CMP)-activated phenyl substi-
tuted alkyl amide derivatives (e.g., 3)20 or glycerol side-chain mod-
ified compounds.21 Our own previous efforts towards C-9 modified
sialic acids were related to simple C-9 ether, ester and chain-ex-
tended compounds (e.g., 4) as probes for Neu5Ac aldolase.22 For
the majority of these C-9 modified sialic acids, the synthetic strat-
egy employed relies on the fact that the C-9 hydroxy group is
primary, and hence is more reactive and less sterically hindered
than the other hydroxy groups in the molecule.
(Neu5Ac, 1), which is usually found
a
(2,3)- or
a
(2,6)-linked to
either galactose or galactosamine residues,1,8 or
a(2,8)-linked to it-
self.9 The importance of sialic acid in pathogen-host recognition
events is of particular interest, since gaining a better understand-
ing of the way a particular pathogen utilizes sialic acid to either in-
fect a host or to evade detection by the host is necessary in order to
develop new drugs to combat such events.
We have had ongoing interest in the development of novel sialic
acid derivatives as probes for a number of important human patho-
gens, including the influenza virus,10,11 rotavirus,12,13 Vibrio chol-
erae14 and Plasmodium falciparum.15 As part of our investigations
H
H
H
OH
OH
R'
OH
CO2H
HO
N
O
O
CO2H
OBn
O
R
H
R
HO
HO
H
OH
OH
12,16
*
into the carbohydrate binding protein VP8 from rotavirus,
as
1 R = NHAc
2 R = NHAc, R' = alkyl or aryl
well as the link between sialic acid and the metastatic potential
of cancer cells, we required an efficient synthesis of sialic acids
modified at the C-9 position. There have been numerous reports
of the synthesis of modified sialic acid derivatives, and the reader
is directed to comprehensive reviews.4,17,18 For the synthesis of
O
H
H
OH
H
OH
OCMP
OH
N
Ph
R'O
n
O
O
CO2H
CO2H
R
H
R
HO
HO
H
OH
OH
4 R = NHAc, R' = alkyl, acyl
3 R = NHAc
⇑
Corresponding author. Tel.: +617 5552 7025; fax: +617 5552 9040.
E-mail address: m.vonitzstein@griffith.edu.au (M. von Itzstein).
Joint first authors.
In this current work, we were particularly interested in sialic
acid analogues wherein the C-9 hydroxy group was oxidized to a
0040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.10.156

M. J. Kiefel et al. / Tetrahedron Letters 52 (2011) 98–100
99
carboxylic acid. Such a modification would then allow subsequent
elaboration of the carboxyl group with amines to provide rapid ac-
cess to a series of amide-based compounds that would serve as
valuable probes for a series of sialic acid recognizing proteins.
the compound during the aqueous work-up. To test this hypothe-
sis, we repeated the TEMPO oxidation on compound 9, but used a
modified work-up procedure wherein the aqueous wash was re-
moved, and instead the crude reaction mixture was carefully acid-
ified (dil HCl), concentrated to dryness using a rotary evaporator,
and then subjected to acetylation conditions (pyridine/acetic anhy-
dride) overnight.30 In this way, the C-9 oxidised sialic acid deriva-
tive 10 was obtained in 66% yield after chromatographic
purification. Deprotection of the ester groups in 10 was achieved
efficiently and smoothly using aqueous lithium hydroxide solution,
Although 9-carboxy-Neu5Ac and 9-carboxy-Neu5Aca2Me have
been reported as probes for haemagglutination studies,23 to the
best of our knowledge the synthesis of these compounds has never
been described in detail, although Brossmer et al.24 reported that
they used ‘catalytic oxidation’. We therefore describe our synthesis
of both the a- and b-methyl glycosides of 9-carboxy-Neu5Ac.
Our approach towards C-9 oxidised sialic acids is summarized
in retrosynthetic terms in Scheme 1. Following directly from some
of our previous work,25 the desired 9-carboxy-derivative 5 should
be available by oxidation of the 9-hydroxy-Neu5Ac derivative 6,
which itself is obtained from the methyl sialoside 7 via a series
of simple and high yielding protecting group manipulations.
Our initial studies towards the preparation of 9-carboxy-
to give the bis-carboxylic acid
HPLC purification.
a-sialoside 11 in good yield after
Our synthesis of the b-methyl sialoside analogue of 11 followed
a similar route to that depicted in Scheme 2. Briefly, treatment of
Neu5Ac with acidic methanol according to the method of Kuhn
et al.31 gave the methyl ester b-methyl glycoside 12. Selective C-
9 silylation, followed by acetylation, gave the 9-O-silyl ether 13,
as previously described.25 Careful acid-promoted removal of the si-
lyl ether furnished the 9-hydroxy derivative 14 in modest yield
(Scheme 3). TEMPO-mediated oxidation of 14 gave the 9-carboxy
derivative 15. Given our initial water solubility problems in the
Neu5Ac centred on the
a-methyl glycoside series. Accordingly,
selective silylation of the C-9 hydroxy group in 826 followed by
acetylation and desilylation gave the pivotal 9-hydroxy derivative
9 in high yield (Scheme 2). Whilst there are numerous methods in
the literature for oxidation of a primary hydroxy to a carboxy
group,27,28 we opted to use a 2,2,6,6-tetramethylpiperidine-1-oxyl
(TEMPO) mediated approach, since this reagent is known to be
quite mild and the reaction can be conducted under relatively neu-
tral conditions.29 We reasoned that such mild conditions would be
needed with our substrate, especially because of the acid labile
a-series, we reasoned that an alternative option was to esterify
the newly formed acid functionality. Accordingly, the crude TEMPO
oxidation product 15 was exposed to methyl iodide in DMF, to give
16 in 41% yield from 14 after acetylation. De-esterification of 16
gave the bis-carboxylic acid b-sialoside 17 in good yield after HPLC
purification.
nature of the
TEMPO oxidation of the C-9 hydroxy group in
a
-methyl sialoside group. Our initial attempts at
During the aqueous acetic acid promoted removal of the 9-O-si-
lyl ether from 13, we found it extremely difficult to obtain pure 14.
As we have described in detail previously,25 this is due to acetate
group migration from the 8-position to the 9-position, resulting
in the 9-hydroxy product 14 being contaminated with the corre-
sponding 8-hydroxy-9-acetoxy analogue. In our hands, no amount
of manipulation of experimental conditions could overcome the
presence of small amounts of the 8-hydroxy-9-acetoxy analogue
in our purified 14. In an attempt to overcome problems associated
with minor contaminants, the TEMPO oxidation was repeated di-
rectly on the methyl ester b-methyl glycoside 12. To our delight,
after acetylation of the crude reaction product, the 9-carboxy b-
sialoside 15 was obtained directly from 12 in a very acceptable
56% yield (Scheme 4). Notably, a direct TEMPO oxidation of 8 affor-
9
produced
mixed results, with low (ꢀ20%) and variable yields of the desired
product 10.
Under TEMPO conditions, it is necessary to use aqueous base in
order for the reaction to proceed efficiently. We felt that the ace-
tate groups present in the sialoside 9 may be labile under such
reaction conditions and, in conjunction with the newly introduced
carboxy group at C-9, would result in a compound that would be
partially water soluble. Given that the work-up for a TEMPO oxida-
tion normally involves an aqueous wash, it is reasonable to con-
clude that our low and variable yields for 10 were due to loss of
ded the corresponding 9-carboxy
lated yield.
In summary, our synthesis of C-9 oxidised sialosides is efficient
(compound 10 in 43% overall yield from 8; compound 15 in 46%
overall yield from Neu5Ac), and provides ready access to com-
pounds (e.g., 10 and 14) that could serve as useful intermediates
a-methyl glycoside in a 40% iso-
H
H
OR'
OH
OMe
OMe
HO
HO
O
O
CO2H
CO2Me
R
H
R
O
HO
R'O
H
OH
OR'
5 R = NHAc
6 R = NHAc, R' = Ac
7 R = NHAc, R' = H
Scheme 1. Proposed approach towards C-9 oxidised sialic acids.
H
H
OH
OAc
R
OMe
OMe
HO
R'O
ii
i
O
O
CO2Me
CO2Me
R
H
H
OAc
R
OH
HO
AcO
CO2Me
OMe
CO2Me
OMe
H
H
OH
12 R = NHAc
OAc
HO
HO
i - iii
13 R = NHAc, R' = TBDMS
O
O
R
AcO
14 R = NHAc, R' = H
HO
H
H
OAc
OH
8 R = NHAc
9 R = NHAc
H
H
OH
OAc
R
OMe
OMe
R'O
iv
HO
H
OAc
R
v
H
iii
OH
CO2Me
CO2H
O
O
HO
CO2Me
CO2H
HO
v
iv
R
H
O
O
AcO
HO
O
OMe
O
OMe
H
OAc
15 R = NHAc, R' = H
16 R = NHAc, R' = Me
OH
R
O
O
AcO
HO
17 R = NHAc
H
OAc
10 R = NHAc
H
OH
11 R = NHAc
Scheme 2. Reagents and conditions: (i) TBDMSCl, pyridine, 3 h; (ii) Ac2O, pyridine,
16 h, 82% over two steps; (iii) 80% aq AcOH, 50 °C, 30 min, 79% based on recovered
starting material; (iv) TEMPO, CH2Cl2, satd aq NaHCO3 containing KBr and Bu4NBr,
aq NaOCl, 1 h, then aq HCl to pH ꢀ2, then Ac2O, pyridine, 16 h, 66%; (v) MeOH, LiOH
(2 M), 12 h, 62%.
Scheme 3. Reagents and conditions: (i) Ref. 25; (ii) 80% aq AcOH, 50 °C, 20 min, 69%
based on recovered starting material; (iii) TEMPO, CH2Cl2, satd aq NaHCO3
containing KBr and Bu4NBr, aq NaOCl, 1 h, then aq HCl to pH ꢀ2; (iv) DMF, MeI,
16 h, then Ac2O, pyridine, 16 h, 41% from 14; (v) NaOMe, MeOH, 3 h, then NaOH
(1 M), 4 h, 76%.

100
M. J. Kiefel et al. / Tetrahedron Letters 52 (2011) 98–100
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H
H
OH
OMe
OMe
HO
HO
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i
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O
O
CO2Me
CO2Me
R
R
H
O
HO
AcO
H
OH
12 R = NHAc
OAc
15 R = NHAc
Scheme 4. Reagents and conditions: (i) TEMPO, CH2Cl2, satd aq NaHCO3 containing
KBr and Bu4NBr, aq NaOCl, 1 h, then aq HCl to pH ꢀ2, then Ac2O, pyridine, 16 h, 56%.
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Acknowledgements
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We thank Ms. Faith Rose for assistance with HPLC purification
of final compounds and Mr. Sam Mallard for preparation of starting
materials and scale-up chemistry. The ARC (DP0774383) is
acknowledged for financial support. A GUPRS and financial support
from the Institute for Glycomics for P.C. is also acknowledged.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2010.10.156.
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30. Typical procedure for modified TEMPO oxidation is as follows: To a solution of
the alcohol 9 (1.10 g, 2.37 mmol) and TEMPO (5 mg, 0.03 mmol) in CH2Cl2
(15 mL) at 0 °C was added a solution of satd aq NaHCO3 (4.5 mL) containing
KBr (26 mg, 0.22 mmol) and Bu4NBr (38 mg, 0.12 mmol). The biphasic mixture
was stirred vigorously whilst a solution of NaOCl (10–15%, 5.5 mL) containing
satd aq NaHCO3 (2.6 mL) and satd aq NaCl (4.4 mL) was added over a 15 min
period. The mixture was allowed to warm to room temperature and stirred for
45 min before an additional portion of NaOCl (10–15%, 4.5 mL) was added.
After a further 15 min the pH was adjusted to ꢀ2 using dil. HCl (4 M) and the
entire mixture concentrated in vacuo. The residue was dissolved in pyridine
(15 mL) and Ac2O (8 mL), and stirred for 14 h at room temperature. The
mixture was concentrated to dryness, the residue dissolved in EtOAc (50 mL)
and washed with dil HCl (1 M, 30 mL), H2O (30 mL), dried (Na2SO4) and
concentrated. Flash chromatographic purification on silica gel (EtOAc/MeOH,
7:1) gave the desired 9-carboxy derivative 10 in 66% yield.
31. Kuhn, R.; Lutz, P.; MacDonald, D. L. Chem. Ber. 1966, 99, 611–617.