Synthesis and evaluation of C-9 modified N-acetylneuraminic acid derivatives as substrates for N-acetylneuraminic acid aldolase

Article abstract of DOI:10.1016/S0968-0896(99)00325-9

Several C-9 modified N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetylneuraminic acid aldolase. Simple C-9 acyl or ether modified derivatives of N-acetylneuraminic acid were found to be accepted as substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. ¹H NMR spectroscopy was used to evaluate the extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00325-9

Bioorganic & Medicinal Chemistry 8 (2000) 657±664
Synthesis and Evaluation of C-9 Modi®ed N-Acetylneuraminic
Acid Derivatives as Substrates for N-Acetylneuraminic Acid
Aldolase
Milton J. Kiefely, Jennifer C. Wilsony, Simon Bennett, Matt Gredley
and Mark von Itzstein*,y
Department of Medicinal Chemistry, Monash University (Parkville Campus), 381 Royal Parade, Parkville, Victoria 3052, Australia
Received 29 October 1999; accepted 10 December 1999
AbstractÐSeveral C-9 modi®ed N-acetylneuraminic acid derivatives have been synthesised and evaluated as substrates of N-acetyl-
neuraminic acid aldolase. Simple C-9 acyl or ether modi®ed derivatives of N-acetylneuraminic acid were found to be accepted as
substrates by the enzyme, albeit being transformed more slowly than Neu5Ac itself. 1H NMR spectroscopy was used to evaluate the
extent of the enzyme catalysed transformation of these compounds. Interestingly, the chain-extended Neu5Ac derivative 16 is not a
substrate for N-acetylneuraminate lyase and behaves as an inhibitor of the enzyme. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
Several workers have attempted to elucidate the cataly-
tic mechanism of Neu5Ac aldolase by exposing the
enzyme to various structurally modi®ed ManNAc11±16
and Neu5Ac17±22 derivatives. A vast number of sub-
strate speci®city studies for the aldol condensation (i.e.
ManNAc+pyruvate to give Neu5Ac) have shown that
only pyruvate is accepted as the donor,23 whilst a wide
variety of hexoses, pentoses, d- and l-sugars, are
accepted as substrates.11±15 In contrast, less attention
has been paid to the retro-aldol condensation with
structurally modi®ed sialic acids. From the few investi-
gations conducted to date it is evident, and perhaps not
surprising, that C-4 modi®ed Neu5Ac derivatives are
not cleaved by the enzyme and in some instances act as
inhibitors.18,20,21 Studies into the retro-aldol condensa-
tion catalysed by Neu5Ac aldolase have shown that 9-
deoxy-Neu5Ac is a relatively good substrate.20,21 Simi-
larly, C-5 modi®cations are accepted by Neu5Ac aldo-
lase,21 with the exception of 5-epi-derivatives.21 Glycerol
sidechain modi®cations other than at C-9 of Neu5Ac,
involving either stereochemical changes or deoxygena-
tion at C-7 and C-8, typically result in compounds
which are cleaved more slowly by the enzyme than the
naturally occuring substrate Neu5Ac.19±21
In the forward direction, N-acetylneuraminic acid aldo-
lase (Neu5Ac aldolase, EC 4.1.3.3) catalyses a retro-
aldol condensation of N-acetylneuraminic acid (Neu5Ac
(1)) to produce pyruvate and N-acetyl-d-mannosamine
(ManNAc) (Scheme 1).1,2 The synthetically more useful
reverse aldol condensation provides 1 from ManNAc,
with the equilibrium of the two reactions being modu-
lated by the concentration of pyruvate.1±3 Neu5Ac
aldolase is found in several species of bacteria1,2,4,5 as
well as in mammals,1,6 and plays an important role in
the recycling of Neu5Ac (1) in nature.1,2 The X-ray
crystal structure of the active site of Neu5Ac aldolase
from Escherichia coli has been resolved to 2.2 A.7 From
the crystallographic data and related studies it has been
elucidated for the forward reaction that the enzyme
forms a Schi€ base between an active site lysine residue
and the acetal carbonyl of the open chain form of the
substrate (Scheme 1), followed by proton abstraction
ultimately resulting in cleavage of the C3±C4 bond of
Neu5Ac.5,8,9 Unfortunately, little more is known
regarding the precise mechanism of this transformation,
although recently it has been suggested that it is the
carboxylate group of the substrate which acts as the
base in the proton abstaction step.10
As part of our continued interest in the chemistry and
biochemistry of structurally modi®ed sialic acid deriva-
tives,24±26 we thought it of value to prepare a range of
C-9 modi®ed Neu5Ac derivatives as probes for N-
acetylneuraminic acid aldolase. Importantly, our target
compounds were designed to explore the steric and
structural constraints of the active site of Neu5Ac
*Corresponding author. Tel.: +617-5594-8232; fax: +617-5594-8908;
e-mail: m.vonitzstein@mailbox.gu.edu.au
yPresent address: Centre for Biomolecular Science and Drug Dis-
covery, Grith University (Gold Coast Campus), PMB50 Gold Coast
Mail Centre, Queensland 9726, Australia.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00325-9

658
M. J. Kiefel et al. / Bioorg. Med. Chem. 8 (2000) 657±664
Scheme 1. Neu5Ac aldolase catalysed transformation of Neu5Ac to ManNAc via a Schi€-base.
aldolase, as well as examining the e€ects of di€erent
functionalities such as acyl, ether and chain-extended
derivatives on the reaction of the modi®ed sialic acid
with the enzyme. In addition, we envisaged that many
of our simple target compounds would be accessible
from Neu5Ac with a minimum of protecting group
chemistry.
Our preliminary e€orts towards the preparation of C-9
ether substituted Neu5Ac derivatives utilised the 9-O-
methanesulfonyl derivative 6 of Neu5Ac1,a2Me2.29
Sequential exposure of the mesylate 6 to NaOMe in
MeOH followed by acidi®cation a€orded the 9-O-
methyl ethyl 7 in 38% yield after puri®cation. Despite
the successful introduction of the 9-O-methyl sub-
stituent, the moderate chemical yield and the fact that
the ether 7 still required both deglycosidation and dees-
teri®cation before use as a probe for Neu5Ac aldolase
prompted further investigation of this transformation.
In an attempt to overcome the need for deprotection
steps, we aimed to prepare the 9-O-methyl ether 8
directly from Neu5Ac itself. Disappointingly, the direct
transformation of Neu5Ac to Neu5Ac9Me (8) proved
fruitless, with several attempts using standard methyla-
tion conditions (such as NaH and MeI using various
solvents and temperatures)28 failing to furnish any of
the desired compound, instead resulting in complex
reaction mixtures.
Results and Discussion
Synthesis of C-9 modi®ed Neu5Ac derivatives
It was anticipated that C-9 acylated derivatives of
Neu5Ac would be directly available from Neu5Ac itself,
without the need for any laborious protecting group
chemistry. Accordingly, the known 9-O-acetyl Neu5Ac
derivative 2 was prepared by treatment of Neu5Ac with
trimethylorthoacetate in DMSO containing p-TsOH.27
In this way, Neu5,9Ac2 (2) was obtained in 78% yield
after puri®cation, which is comparable to the results
obtained by Ogura et al.27 Unfortunately, initial
attempts at forming the sterically more demanding 9-O-
benzoyl derivative 3 by treatment of Neu5Ac under
typical benzoylation conditions (BzCl in pyridine)28
failed to furnish any of the desired product. After some
experimentation it was established that the 9-O-benzoyl
group could be successfully introduced if the methyl
ester 4 was used as substrate instead of Neu5Ac. In this
way the 9-O-benzoyl derivative 5 of Neu5Ac was
obtained in high yield (71% after chromatography).
In an alternative approach towards limiting the need
for deprotecting steps, we attempted to employ
Neu5Ac1Me (4) in an analogous sequence to that suc-
cessfully used in the preparation of 7. Accordingly,
treatment of 4 under tosylation conditions (TsCl/pyri-
dine) gave the desired 9-O-tosyl derivative 9 in 55%
yield (Scheme 2). Exposure of the tosylate 9 to NaOMe
in MeOH followed by acidi®cation (Amberlite IR-
120H+ resin) furnished the requisite 9-O-methyl ether
10 in moderate yield (Scheme 2). Interestingly, reaction
of the 9-O-mesylate 11 with NaOMe in MeOH followed
by acidi®cation (as described above) resulted in an inse-

M. J. Kiefel et al. / Bioorg. Med. Chem. 8 (2000) 657±664
659
Scheme 2. Conditions: (a) TsCl, pyridine, 5 ꢁC, 16 h, 55%; (b) NaOMe, MeOH, rt, 0.5 h; (c) Amberlite IR-120H+ resin (pH=2), MeOH, rt, 2 h, 53%.
parable mixture of Neu5Ac1,9Me2 (10) and unreacted
mesylate 11.
In addition to the compounds described above, we have
previously described31 the synthesis of the C11 chain-
extended analogue 14 of Neu5Ac. The additional struc-
tural features of 14 may provide valuable information
regarding the steric and electronic speci®city of Neu5Ac
aldolase. Interestingly however, attempted NaOMe cat-
alysed deacetylation of 14 resulted in the instantaneous
deep red colouration of the reaction mixture (even at
10 ꢁC) and partial decomposition (to a highly polar
and unidenti®able yellow material) of the chain-extended
Neu5Ac derivative. Whilst yields of up to 60% of the
deacetylated material 15 could be obtained under
carefully controlled conditions,32 it was felt that an
alternative deprotection strategy was appropriate, par-
ticularly since methyl glycoside and methyl ester func-
tionalities are still present in 15. Towards this end,
exposure of the chain-extended compound 14 to acidic
Dowex1 resin (50WÂ8) in water at 80 ꢁC gave the
fully deprotected compound 16 in 68% yield after
chromatography.
An alternative C-9 ether derivative of Neu5Ac that
could be useful for exploring the substrate speci®city of
Neu5Ac aldolase was the 9-O-methoxymethyl ether 12,
since it was envisaged that the extra oxygen atom may
participate in a range of additional interactions including
hydrogen-bonding interactions. Accordingly, exposure of
a solution of 4 in DMF containing Hunigs base to
chloromethyl methylether furnished the 9-O-methoxy-
methyl ether 13 in 81% yield after chromatography.
Of the C-9 modi®ed Neu5Ac derivatives prepared, all
except Neu5,9Ac2 (2) required deprotection of the
methyl ester before exposure to Neu5Ac aldolase. For
the 9-O-methyl derivative 10 and the 9-O-methoxy-
methyl derivative 13, saponi®cation was achieved by
exposure to dilute NaOH in MeOH, to a€ord
Neu5Ac9Me (8) and Neu5Ac9MOM (12), respectively,
in high yield. The base sensitive nature of the 9-O-ben-
zoyl group in 5 precluded the use of NaOH in the
saponi®cation of the methyl ester group, as exempli®ed
by the attempted careful saponi®cation (at pH 10.5) of 5
which showed (by TLC analysis) that the 9-O-benzoyl
group was being hydrolysed more rapidly than the
methyl ester. Fortunately, treatment of the 9-O-benzoyl
derivative 5 with 25% aqueous Et3N30 furnished
Neu5Ac9Bz (3) in 72% yield after chromatography.
Only a small amount (ꢀ10%) of Neu5Ac was isolated
from this reaction. Unfortunately, all attempts to
hydrolyse the methyl ester group in the 9-O-tosyl deri-
vative 9, using either dilute NaOH in MeOH or aqueous
Et3N, failed to yield any of the desired product.
Evaluation of C-9 modi®ed Neu5Ac derivatives as
substrates for Neu5Ac aldolase
Each of the C-9 modi®ed Neu5Ac derivatives (100 mg)
were exposed to Neu5Ac aldolase (from E. coli, 1.4 units)
using membrane-enclosed enzyme catalysis (MEEC)33

660
M. J. Kiefel et al. / Bioorg. Med. Chem. 8 (2000) 657±664
at pH 7.4 and 37 ꢁC over a period of 4 days (see
Experimental for full details). After this time the solu-
tion was concentrated and examined by 1H NMR
spectroscopy to determine the ratio of Neu5Ac deriva-
tive versus ManNAc derivative. The mixture of Neu5Ac
and ManNAc derivatives from each reaction were then
separated using chromatography. The results in Table 1
show that, with the exception of the C11 chain-extended
derivative 16, all other C-9 modi®ed Neu5Ac derivatives
were recognised as substrates by Neu5Ac aldolase.
There are clear trends in the relative rates of cleavage of
these compounds, which correlate, in part, with the
steric demand of the functionality at C-9. For example,
the sterically more demanding 9-O-MOM ether (12) is
transformed more slowly than the 9-O-methyl ether (6)
by Neu5Ac aldolase. Similarly, the 9-O-benzoyl deriva-
tive 3 is cleaved signi®cantly more slowly than the 9-O-
acetyl derivative 2. Whilst these observations may sug-
gest that a group as large as a benzoyl moiety could be
approaching the limit of the steric tolerance of Neu5Ac
aldolase it is worthwhile remembering that a number of
9-O-acyl Neu5Ac derivatives, including a sterically
demanding BOC-glycyl derivative,34 can be easily pre-
pared by the reverse enzyme reaction.14,35 As the
enzyme substrate is bound in the acyclic form,5,9,36 at
this stage it is dicult to rationalise the apparent dif-
ferences in steric tolerance between the forward and
reverse Neu5Ac aldolase catalysed transformations.
Studies directed towards a better understanding of the
steric constraints of Neu5Ac aldolase are continuing.
The progress of the transformation of Neu5Ac to
ManNAc could then easily be determined by measuring
the relative integrals of Neu5Ac H3e and ManNAc H1
(full details included in Experimental). In an analogous
experiment, the C11 chain-extended Neu5Ac derivative
16 (6 mg) was added to a mixture containing Neu5Ac
(4 mg) and Neu5Ac aldolase (0.1 units) and the mixture
1
monitored by H NMR spectroscopy. The results from
these studies are shown in Figure 1, and demonstrate
quite clearly that the rate of hydrolysis of Neu5Ac is
signi®cantly reduced in the presence of 16.
It is not clear at this stage why the chain-extended
compound 16 is an inhibitor of Neu5Ac aldolase. There
are a number of plausible explanations of how this
compound might inhibit the catalytic activity of
Neu5Ac aldolase, including the possibility that the
additional carboxylate group may be interacting with
the Schi€-base forming lysine. Additional investigations
Since the C11 chain-extended Neu5Ac derivative 16 was
not recognised as a substrate by Neu5Ac aldolase it was
investigated for its inhibitory action against this
1
enzyme. H NMR spectroscopy was conveniently used
to monitor the rate of transformation of Neu5Ac to
ManNAc by Neu5Ac aldolase,37 since the H3e proton
of Neu5Ac (dꢀ2.3) and the H1 proton of ManNAc
(dꢀ5.1/5.2 for a/b) are not obscured by other resonances.
Figure 1. Rate of hydrolysis of 1 by Neu5Ac aldolase in the presence
(Ð&Ð) and absence (- -*- -) of 16.
Table 1. Survey of C-9 modi®ed Neu5Ac derivatives as substrates for Neu5Ac aldolase
Ratio from 1H NMR analysisa
Ratio from isolated materialb
Relative cleavage
rates (%)
Compound
R0
8-R0-Neu5Ac
5-R0-ManNac
8-R0-Neu5Ac
5-R0-ManNAc
1
8
12
2
3
16
CH2OH
CH2OMe
CH2OMOM
CH2OAc
CH2OBz
10.0
10.0
10.0
10.0
10.0
10.0
20.5
11.0
7.4
8.1
2.8
0.0c
nd
nd
9.5
7.1
7.5
2.5
ndd
100
54
36
39
14
ndd
10.0
10.0
10.0
10.0
10.0
CHˆCHCO2H
aRatio refers to the average result from at least two concordant experiments and is determined by the analysis of the integral from the 1H NMR
spectrum recorded on the entire enzyme reaction product.
bRatio refers to the yield of material after chromatography.
cIn addition to no transformation, this compound repeatedly caused precipitation of Neu5Ac aldolase.
dnd=not determined.

M. J. Kiefel et al. / Bioorg. Med. Chem. 8 (2000) 657±664
661
are required in order to understand more fully the nature
of the interactions of compounds like 16 within the cat-
alytic site of Neu5Ac aldolase.
(1.4 units, 5.6 mg per unit) and BSA (3.0 mg) were then
dissolved in ca. 200 mL of milli-Q H2O and added to a
dialysis bag.33 The dialysis bag was then placed in the
test-tube and the reaction stirred for four days at 37 ꢁC.
The water was removed from the reaction vessel, and
H2O (2Â3 mL) stood with the dialysis bag for 30 min.
The combined aqueous fractions were concentrated and
In conclusion, this investigation has demonstrated that
the retro-aldol condensation catalysed by Neu5Ac
aldolase is susceptible to structural modi®cation at C-9
of Neu5Ac. In particular, we have shown by a direct
NMR spectroscopic method that large ester (e.g. 3) or
large ether (e.g. 12) groups at C-9 result in a signi®cant
reduction in the rate of Neu5Ac aldolase catalysed
transformation to the corresponding ManNAc deriva-
tive. In addition, we have demonstrated three di€erent
methods for the ecient deprotection of the methyl
ester group at C-1 in derivatives of Neu5Ac, which adds
an additional degree of ¯exibility to synthetic manip-
ulations with sialic acid derivatives.
1
the total product examined by H NMR spectroscopy
to establish the ratio of Neu5Ac compound versus
ManNAc compound present (see Table 1). Column
chromatography then provided the pure components.
General procedure for performing NMR tube reactions
with Neu5Ac aldolase
Neu5Ac (1) (4 mg) was dissolved in 0.6 mL of 50 mM
deuterated PBS (pH 7.4) and a 1H NMR spectrum
recorded (600 MHz) at 37 ꢁC representing time=0. To
the NMR tube was added Neu5Ac aldolase (0.4 mg, 0.1
1
unit), and H NMR spectra recorded every ꢀ15±20 min
Experimental
over 4 h. The relative integrals of H3e of Neu5Ac
(dꢀ2.3) and H1 of ManNAc (dꢀ5.1/5.2 for a/b) were
then measured for each time interval.
General procedures
1H and 13C spectra were recorded using a Bruker DRX-
300 spectrometer unless indicated otherwise. Chemical
shifts are given in ppm relative to the solvent used
(CDCl3: 7.26 for 1H; 77.0 for 13C; CD3OD: 3.31 for 1H;
49.0 for 13C) or relative to external Me4Si for D2O
spectra. Two-dimensional DQF-COSY and HMQC
experiments were recorded in order to assist with spec-
tral assignment. Typically, the following parameters
were used: DQF-COSYÐ16 scans, 512 slices, relaxation
delay 2.0 s; 1H±13C HMQCÐ48 scans, 256 slices,
relaxation delay 2.5 s. FAB mass spectra were obtained
using a Jeol JMS-DX 300 mass spectrometer. Infrared
spectra were recorded as KBr discs using a Hitachi 270-
30 spectrophotometer. Optical rotations were measured
using a Jasco DIP-370 digital polarimeter. Reactions
The identical experiment was then carried out, with the
exception that the C11 chain-extended derivative 16
(6 mg) was added with the Neu5Ac at the beginning of
the experiment (time=0). In each experiment, 1H NMR
spectra were recorded using a Bruker 600MHz DRX
spectrometer operating at 37 ꢁC. Spectra were acquired
with 32 scans and 16 k data points, over a spectral width
of 7800 Hz.
Synthesis of C-9 modi®ed Neu5Ac derivatives
5-Acetamido-9-O-acetyl-3,5-dideoxy-D-glycero-ꢀ-D-
galacto-2-nonulopyranosonic acid (2). To a solution
of Neu5Ac (0.50 g, 1.6 mmol) in DMSO (5 mL) under
N2 at rt was added trimethylorthoacetate (2.0 mL,
were monitored by TLC (Merck silica gel plates GF254
,
.
cat. No. 1.05554) and products were generally puri®ed
by ¯ash chromatography using Merck silica gel 60
(0.040±0.063 mm, cat. No. 1.09385) or by HPLC using
reverse phase C18 Waters mBondapakTM columns (ana-
lytical: 8Â100 mm, part No. WAT085721; preparative:
25Â100 mm, part No. WAT038505), detecting at
220 nM with a Waters 484 tunable absorbance detector.
All solvents were distilled prior to use or were of analy-
tical grade. Microanalyses were performed at the
Department of Chemistry, University of Queensland,
Australia. N-Acetylneuraminic acid aldolase (EC
4.1.3.3) from E. coli was generously provided by Dr.
Yoji Tsukada (Kyoto Research Laboratories, Japan).
16 mmol) and p-TsOH H2O (20 mg). After being stirred
at rt for 1.5 h, the mixture was applied directly to a col-
umn (8Â1 cm) of Dowex1 1-Â4 (HCO2 ) ion exchange
resin and the column eluted with H2O (100 mL) and
then formic acid (1 N, 200 mL). The formic acid eluant
was concentrated under reduced pressure and then pur-
i®ed by HPLC (reverse phase, 3% CH3CN in H2O) to
a€ord Neu5,9Ac2 (2) (0.44 g, 78%) as an amorphous
white solid: [a]d 15.3ꢁ (c 1.03, H2O); nmax 3440(br),
1
1730, 1634, 1554, 1376, 1264 and 1036 cm 1; H NMR
(D2O) d 1.80 (1H, dd, J3a,3e 12.9, J3a,4 11.9 Hz, H-3a),
1.98 (3H, s, AcN), 2.04 (3H, s, AcO), 2.23 (1H, dd, J3e,3a
12.9, J3e,4 4.5 Hz, H-3e), 3.53 (1H, d, J7,8 9.2 Hz, H-7),
3.78±3.89 (2H, m, H-5/H-8), 3.94±4.03 (2H, m, H-4/H-
0
6), 4.10 (1H, dd, J9,9 11.7, J9,8 5.4 Hz, H-9), 4.29 (1H,
General procedure for performing reactions with Neu5Ac
aldolase
dd, J9 ,9 11.7, J9 ,8 2.1 Hz, H-90), assignments con®rmed
by DQF-COSY; 13C NMR (D2O) d 22.9 (OC(O)Me),
24.7 (NC(O)Me), 41.4 (C-3), 54.7 (C-5), 68.9 (C-9),
69.3 (C-4*), 70.3 (C-7*), 70.8 (C-8*), 72.9 (C-6*), 97.8
(C-2), 175.6 (C-1), 177.0, 177.4 (NC(O)Me/OC(O)Me),
*assignments interchangeable; FABMS: 352 [(M+1)+,
64%], 334 (16), 316 (21), 201 (52), 185 (100). Anal. calcd
0
0
The Neu5Ac derivative (ꢀ100 mg) to be treated with
Neu5Ac aldolase was weighed accurately into a quick®t
test-tube (12Â1 cm) and then dissolved in 2.5 mL milli-
Q H2O. The pH of the solution was then adjusted to pH
ca. 7.4 with dilute NaOH solution (0.5%, typically ca.
2 mL) and then the total volume of the solution made
up to ca. 6 mL with milli-Q H2O. Neu5Ac aldolase
.
for C13H21NO10 0.5H2O: C, 43.2; H, 6.3; N, 3.6. Found:
C, 43.3; H, 6.15; N, 3.9.

662
M. J. Kiefel et al. / Bioorg. Med. Chem. 8 (2000) 657±664
Methyl 5-acetamido-9-O-benzoyl-3,5-dideoxy-D-glycero-
ꢀ-D-galacto-2-nonulopyranosylonate (5). To a solution
of Neu5Ac1Me (4) (0.50 g, 1.55 mmol) in pyridine
(5 mL) under N2 at 0 ꢁC was added benzoyl chloride
(200 mL, 1.7 mmol). The mixture was allowed to warm
to rt and stirred for 15 h before MeOH (ca. 0.5 mL) was
added and the solution concentrated. Column chroma-
tography (Silica, EtOAc:MeOH, 10:1; Rf 0.3) a€orded
Neu5Ac9Bz1Me (5) (0.47 g, 71%) as an amorphous
2.10 (1H, dd, J3e,3a 12.0, J3e,4 4.9 Hz, H-3e), 2.32 (3H, s,
ArMe), 3.34 (1H, d, J7,8 9.2 Hz, H-7), 3.65 (3H, s,
CO2Me), 3.71±3.98 (5H, m, H-4/H-5/H-7/H-8/H-9),
4.18 (1H, dd, J9 ,9 9.9, J9 ,8 1.8 Hz, H-90), 7.32±7.67 (4H,
m, ArH); 13C NMR (d4-MeOH) d 24.6 (NC(O)Me),
25.7 (ArMe), 43.6 (C-3), 55.2 (C-5), 56.2 (CO2Me), 69.6
(C-4*), 71.2 (C-6*), 71.9 (C-7*), 73.4 (C-8*), 75.7 (C-9),
98.5 (C-2), 131.0, 133.0, (2ÂArH), 136.0 (ArC-4), 148.3
(ArC-1), 173.6 (C-1), 177.1 (NC(O)Me), *assignments
interchangeable; FABMS: 478 [(M+1)+, 80%; Found:
478.13677 C19H28NO11S requires 478.13831]. Anal.
0
0
white solid: [a]d 2.7ꢁ (c 1.03, MeOH); nmax 3415(br),
1
1735, 1720, 1636, 1552, 1450, 1276, 1122 and 1066 cm
;
1H NMR (D2O) d 1.84 (1H, dd, J3a,3e 12.8, J3a,4 11.8 Hz,
H-3a), 1.93 (3H, s, AcN), 2.23 (1H, dd, J3e,3a 12.8, J3e,4
4.7 Hz, H-3e), 3.64 (1H, d, J7,8 9.3 Hz, H-7), 3.74 (3H, s,
CO2Me), 3.85 (1H, dd, J5,6=J5,4=10.1 Hz, H-5), 3.96±
calcd for C19H27NO11S H2O: C, 46.1; H, 5.9; N, 2.8.
Found: C, 46.5; H, 5.8; N, 2.4.
.
Methyl 5-acetamido-9-O-methyl-3,5-dideoxy-D-glycero-
ꢀ-D-galacto-2-nonulopyranosylonate (10). To a stirred
solution of the tosylate 9 (0.63 g, 1.32 mmol) in MeOH
(5 mL) under N2 at rt was added 2 mL of a freshly pre-
pared NaOMe solution (from 0.15 g of sodium dis-
solved in 10 mL of MeOH). After 20 min a further 2 mL
of the NaOMe solution was added and the mixture
stirred for 30 min. The mixture was acidi®ed (to pH 2)
with Amberlite IR-120H+ resin and stirred for a further
2 h, before being ®ltered and concentrated. Chromato-
graphy (Silica, EtOAc:MeOH, 4:1; Rf 0.4) gave 10 con-
taminated with p-TsOH. Ion exchange chromatography
(AG-1Â4, 100±200 mesh, HCO2 form, eluted with
H2O) removed the p-TsOH to give Neu5Ac1,9Me2 (10)
(0.24 g, 53%) as a colourless solid: [a]d 10.4ꢁ (c 0.97,
H2O); nmax 3448(br), 2940, 1744, 1600, 1440, 1376 and
0
4.04 (3H, m, H-4/H-6/H-8), 4.36 (1H, dd, J9,9 11.8, J9,8
4.9 Hz, H-9), 4.50 (1H, dd, J9 ,9 11.8, J9 ,8 1.3 Hz, H-90),
7.40±7.45 (2H, m, Ph), 7.52±7.58 (1H, m, Ph), 7.86±7.91
(2H, m, Ph); 13C NMR (D2O) d 24.6 (NC(O)Me), 41.3
(C-3), 54.8 (C-5), 56.1 (CO2Me), 69.1 (C-8*), 69.3 (C-9),
70.6 (C-4*), 70.8 (C-7*), 72.9 (C-6*), 97.9 (C-2), 131.2,
131.9, 136.3 (all Ph), 131.7 (ipso Ph), 170.9 (PhC(O)O),
173.9 (C-1), 177.3 (NC(O)Me), *assignments inter-
changeable; FABMS: 450 [(M+Na)+, 100%], 428
[(M+1)+, 90]. Anal. calcd for C19H25 NO10: C, 53.4; H,
5.9; N, 3.3. Found: C, 53.4; H, 6.1; N, 2.9.
0
0
Methyl (methyl 5-acetamido-9-O-methyl-3,5-dideoxy-D-
glycero-ꢁ-D-galacto-2-nonulopyranosid)onate (7). To a
stirred solution of the 9-O-mesylate 629 (0.78 g,
1.89 mmol) in MeOH (15 mL) under N2 at rt was added
2 mL of a freshly prepared NaOMe solution (from
0.15 g of sodium dissolved in 10 mL of MeOH). After
60 min the mixture was acidi®ed (to pH 2) with Dowex1
50WÂ8 H+ resin and stirred for a further 2 h, before
being ®ltered and concentrated. Chromatography
(Silica, EtOAc:MeOH, 10:1; Rf 0.3) gave 7 (0.23 g, 38%)
as a white foam: nmax 3410(br), 1735, 1660, 1172, and
1
1124 cm 1; H NMR (D2O) d 1.89 (1H, dd, J3a,3e 13.1,
J3a,4 12.1 Hz, H-3a), 2.03 (3H, s, AcN), 2.29 (1H, dd,
J3e,3a 13.1, J3e,4 4.6 Hz, H-3e), 3.36 (3H, s, OMe), 3.49±
3.54 (2H, m, H-7/H-9), 3.68 (1H, d, J9 ,9 10.7 Hz, H-90),
0
3.82 (3H, s, CO2Me), 3.89 (1H, dd, J5,6=J5,4=10.2 Hz,
H-5), 4.00±4.10 (2H, m, H-6/H-4); 13C NMR (D2O) d
24.8 (NC(O)Me), 43.0 (C-3), 56.3 (C-5), 57.8 (CO2Me),
62.8 (OMe), 71.0 (C-4*), 72.6 (C-6*), 72.8 (C-7*),
74.6 (C-8), 78.1 (C-9), 99.6 (C-2), 173.6 (C-1), 177.8
(NC(O)Me), *assignments interchangeable; FABMS:
360 [(M+Na)+, 100%], 338 [(M+1)+, 92; Found:
338.14576 C13H24NO9 requires 338.14511]. Anal. calcd
1
1034 cm 1; H NMR (d4-MeOH) d 1.76 (1H, dd, J3a,3e
12.7, J3a,4 11.7 Hz, H-3a), 1.92 (3H, s, AcN), 2.57 (1H,
dd, J3e,3a 12.7, J3e,4 4.5 Hz, H-3e), 3.26 (3H, s, 9-OMe),
3.30 (3H, s, 2-OMe), 3.34±3.58 (5H, m, H-4/H-6/H-7/H-
9/H-90), 3.64 (3H, s, CO2Me), 3.66 (1H, ddd,
J5,6=J5,4=J5,NH=10.1 Hz, H-5), 3.87 (1H, ddd, J8,7
.
for C13H23NO9 H2O: C, 43.9; H, 7.1; N, 3.9. Found: C,
43.7; H, 7.1; N, 4.0%.
8.6, J8,9 5.5, J8,9 2.2 Hz, H-8); 13C NMR (d4-MeOH) d
0
22.7 (NC(O)Me), 41.4 (C-3), 51.9 (2-OMe), 53.4 (C-5*),
53.9 (CO2Me*), 59.4 (9-OMe), 68.6 (C-9), 70.2 (C-4*),
71.1 (C-6*), 74.7 (C-7*), 75.3 (C-8*), 100.3 (C-2), 170.8
(C-1), 175.1 (NC(O)Me), *assignments interchangeable;
FABMS: 374 [(M+Na)+, 70%], 352 [(M+1)+, 90].
Methyl 5-acetamido-9-O-methanesulfonyl-3,5-dideoxy-D-
glycero-ꢀ-D-galacto-2-nonulopyranosylonate (11). To a
stirred solution of Neu5Ac1Me (4) (0.50 g, 1.55 mmol)
in pyridine (7 mL) at 0 ꢁC under N2 was added metha-
nesulphonyl chloride (209 mL, 2.71 mmol) and the mix-
ture stirred for 3 h. MeOH (3 mL) was added and the
mixture concentrated to a yellow syrup. Chromato-
graphy (Silica, EtOAc:MeOH, 4:1; Rf 0.7) gave the
desired 9-O-mesylate 11 (0.38 g, 61%): [a]d 16.6ꢁ (c
0.9, MeOH); nmax 3388(br), 1736, 1664, 1528, 1356,
1278, 1170, 1132 and 1032 cm 1; 1H NMR (D2O) d 1.85
(1H, dd, J3a,3e=J3a,4=12.5 Hz, H-3a), 1.99 (3H, s,
AcN), 2.26 (1H, dd, J3e,3a 12.5, J3e,4 4.6 Hz, H-3e), 3.16
(3H, s, SO3Me), 3.56 (1H, d, J7,8 9.2 Hz, H-7), 3.77 (3H,
s, CO2Me), 3.81±4.02 (4H, m, H-4/H-5/H-6/H-8), 4.37
Methyl 5-acetamido-9-O-tosyl-3,5-dideoxy-D-glycero-ꢀ-
D-galacto-2-nonulopyranosylonate (9). To a stirred solu-
tion of Neu5Ac1Me (4) (1.05 g, 3.25 mmol) in pyridine
(5 mL) at 0 ꢁC under N2 was added p-toluenesulfonyl
chloride (0.74 g, 3.90 mmol) and the mixture stirred at
5 ꢁC for 16 h. MeOH (3 mL) was added to the mixture
which was then concentrated to a yellow syrup. Chro-
matography (Silica, EtOAc:MeOH, 4:1; Rf 0.7) gave the
tosylate 9 (0.87 g, 55%) as a white foam: [a]d 16.3ꢁ (c
1.28, MeOH); nmax 3396(br), 1744, 1640, 1554, 1172,
0
0
(1H, dd, J9,9 10.6, J9,8 4.9 Hz, H-9), 4.47 (1H, dd, J9 ,9
0
1132 and 1034 cm 1; H NMR (d4-MeOH) d 1.76 (1H,
10.6, J9 ,8 1.6 Hz, H-90); 13C NMR (D2O) d 24.7
(NC(O)Me), 39.0 (SO3Me), 41.3 (C-3), 54.7 (C-5), 54.7
1
dd, J3a,3e=J3a,4=12.0 Hz, H-3a), 1.90 (3H, s, AcN),

M. J. Kiefel et al. / Bioorg. Med. Chem. 8 (2000) 657±664
663
(CO2Me), 69.2 (C-4*), 70.0 (C-6*), 70.3 (C-7*), 70.5 (C-
8*), 72.9 (C-6), 75.0 (C-9), 97.9 (C-2), 173.9 (C-1), 175.5
(NC(O)Me), *assignments interchangeable; FABMS:
424 [(M+Na)+, 4%], 402 [(M+1)+, 100; Found:
402.10868 C13H24NO11S requires 402.10699].
amorphous mass: nmax 3400(br), 1710, 1632, 1550, 1375,
1
1120 and 1032 cm 1; H NMR (D2O) d 1.82 (1H, dd,
J3a,3e 12.7, J3a,4 11.6 Hz, H-3a), 1.98 (3H, s, AcN), 2.19
(1H, dd, J3e,3a 12.7, J3e,4 4.7 Hz, H-3e), 3.34 (3H, s,
OCH2OMe), 3.51 (1H, d, J7,8 9.2 Hz, H-7), 3.64 (1H,
0
dd, J9,9 12.0, J9,8 6.5 Hz, H-9), 3.76±3.82 (2H, m, H-8/
Methyl 5-acetamido-3,5-dideoxy-9-O-methoxymethyl-D-
glycero-ꢀ-D-galacto-2-nonulopyranosylonate (13). To a
solution of Neu5Ac1Me (4) (0.41 g, 1.27 mmol) in DMF
(4 mL) under N2 at 0 ꢁC was added diisopropylethyl
amine (775 mL, 4.45 mmol) and then chloromethyl
methylether (115 mL, 1.52 mmol). The mixture was
allowed to warm to rt and stirred for 48 h before MeOH
(ca. 0.4 mL) was added and the solution concentrated.
Column chromatography (Silica, EtOAc:MeOH, 4:1)
furnished recovered Neu5Ac1Me (0.10 g) together with
the title compound 13 [Rf 0.3, 0.29 g, 62% (81% based
on recovered Neu5Ac1Me)] as an amorphous white
solid: [a]d 19.5ꢁ (c 1.05, H2O); nmax 3380(br), 1746,
H-90), 3.84 (1H, dd, J5,6=J5,4=10.1 Hz, H-5), 3.91±4.02
(2H, m, H-4/H-6), 4.64 (2H, s, OCH2OMe), assign-
ments con®rmed by DQF-COSY; 13C NMR (D2O)
d 24.7 (NC(O)Me), 41.5 (C-3), 54.7 (C-5), 57.6
(OCH2OMe), 69.3 (C-4), 70.8 (C-7), 71.5 (C-8), 72.1
(C-9), 73.0 (C-6), 98.0 (C-2), 98.9 (OCH2OMe), 175.5
(C-1), 177.5 (NC(O)Me), assignments con®rmed by
HMQC; FABMS: 398 [(M+Na)+, 8%], 376 [(M+1)+,
13], 354 (100).
5-Acetamido-9-O-benzoyl-3,5-dideoxy-D-glycero-ꢀ-D-
galacto-2-nonulopyranosonic acid (3). To a solution of
Neu5Ac9Bz1Me (5) (0.15 g, 0.35 mmol) in H2O (1 mL)
was added Et3N (0.25 mL)30 and the mixture stirred at
rt for 1.5 h. Concentration of the mixture under reduced
pressure and column chromatography (Silica, EtOAc:-
MeOH:H2O, 3:2:0.1) a€orded Neu5Ac (Rf 0.2, 18 mg,
17%) together with Neu5Ac9Bz (3) (Rf 0.3, 104 mg,
72%) as an amorphous white solid: nmax 3380(br), 1708,
1
1642, 1552, 1440, 1122 and 1032 cm 1; H NMR (D2O)
d 1.86 (1H, dd, J3a,3e 12.9, J3a,4 11.8 Hz, H-3a), 1.99
(3H, s, AcN), 2.25 (1H, dd, J3e,3a 12.9, J3e,4 4.8 Hz, H-
3e), 3.34 (3H, s, OCH2OMe), 3.54 (1H, d, J7,8 8.7 Hz,
0
H-7), 3.63 (1H, dd, J9,9 11.0, J9,8 6.1 Hz, H-9), 3.76±
3.83 (2H, m, H-8/H-90), 3.78 (3H, s, CO2Me), 3.85 (1H,
dd, J5,6=J5,4=10 Hz, H-5), 3.94±4.05 (2H, m, H-4/H-
6), 4.65 (2H, s, OCH2OMe), assignments con®rmed by
DQF-COSY; 13C NMR (D2O) d 24.6 (NC(O)Me), 41.3
(C-3), 54.7 (C-5), 56.1 (CO2Me), 57.6 (OCH2OMe),
69.1 (C-4), 70.7 (C-7), 71.4 (C-8), 72.0 (C-9), 72.9
(C-6), 97.9 (C-2), 98.9 (OCH2OMe), 173.9 (C-1), 177.4
(NC(O)Me), *assignments con®rmed by HMQC;
FABMS: 390 [(M+Na)+, 97%], 368 [(M+1)+, 95].
Anal. calcd for C14H25NO10: C, 45.8; H, 6.9; N, 3.8.
Found: C, 45.6; H, 7.1; N, 3.1.
1
1630, 1376, 1314, 1278, 1122 and 1032 cm 1; H NMR
(D2O) d 1.76 (1H, dd, J3a,3e 13.0, J3a,4 11.6 Hz, H-3a),
1.92 (3H, s, AcN), 2.17 (1H, dd, J3e,3a 13.0, J3e,4 4.5 Hz,
H-3e), 3.69 (1H, d, J7,8 8.6 Hz, H-7), 3.87 (1H, d, J6,5
9.6 Hz, H-6), 3.90±4.10 (3H, m, H-4/H-5/H-8), 4.36
0
0
(1H, dd, J9,9 11.7, J9,8 5.0 Hz, H-9), 4.47 (1H, dd, J9 ,9
0
11.7, J9 ,8 2.0 Hz, H-90), 7.42±7.47 (2H, m, Ph), 7.57±
7.62 (1H, m, Ph), 7.91±7.97 (2H, m, Ph); 13C NMR
(D2O) d 24.7 (NC(O)Me), 42.0 (C-3), 54.9 (C-5), 69.1
(C-9), 69.6 (C-8*), 70.6 (C-4*), 70.9 (C-7*), 72.8 (C-6*),
99.4 (C-2), 131.4, 132.0, 136.5 (all Ph), 131.8 (ipso
Ph), 171.1 (C-1), 177.3 (NC(O)Me), 179.2 (PhC(O)O),
*assignments interchangeable; FABMS: 436 [(M+Na)+,
59%], 414 [(M+1)+, 33]. Anal. calcd for C18H23
5-Acetamido-9-O-methyl-3,5-dideoxy-D-glycero-ꢀ-D-
galacto-2-nonulopyranosylonic acid (8). Neu5Ac1,9Me2
(10) (0.18 g, 0.54 mmol) was dissolved in MeOH (2 mL)
and 2 N NaOH added to pH 13. The mixture was stir-
red for 4 h at rt and then Amberlite IR 120H+ resin was
added to pH 5. The resin was removed by ®ltration,
washed with aq MeOH (3Â10 mL) and the combined
®ltrate concentrated under reduced pressure. HPLC
puri®cation (reverse phase, 5% CH3CN in H2O) gave
Neu5Ac9Me (8) (0.11 g, 62%) as an amorphous mass:
nmax 3456(br), 2932, 1742, 1632, 1376, 1120 and
.
NO10 1.5H2O: C, 49.1; H, 5.9; N, 3.2. Found: C, 49.1;
H, 5.5; N, 3.0.
5-Acetamido-9,10-didehydro-3,5,9,10-tetradeoxy-10-car-
acid
boxy-D-glycero-ꢀ-D-galacto-2-deculopyranosonic
(16). A solution of the C11 chain-extended Neu5Ac
derivative 1431 (200 mg, 0.38 mmol) in H2O (10 mL)
containing Dowex1 50WÂ8 acidic resin (500 mg) was
heated at 80 ꢁC for 48 h. After cooling to rt the mixture
was ®ltered, the resin washed with H2O and the solution
concentrated. HPLC chromatography (reverse phase,
H2O) gave 16 (92 mg, 68%) as an amorphous white
solid: nmax 3350(br), 1710, 1642, 1554, 1376, 1272, 1124
1
1030 cm 1; H NMR (D2O) d 1.82 (1H, dd, J3a,3e 13.0,
J3a,4 12.1 Hz, H-3a), 1.98 (3H, s, AcN), 2.24 (1H, dd,
J3e,3a 13.0, J3e,4 3.8 Hz, H-3e), 3.32 (3H, s, OMe), 3.48±
3.51 (2H, m, H-7/H-9), 3.64, (1H, d, J9 ,9 10.6 Hz, H-90),
0
3.78 (1H, dd, J8,7 8.8, J8,9 6.6 Hz, H-8), 3.88 (1H, dd,
J5,6=J5,4=10.1 Hz, H-5), 3.97±4.01 (2H, m, H-6/H-4);
13C NMR (D2O) d 24.6 (NC(O)Me), 41.3 (C-3), 54.6
(C-5), 61.0 (OMe), 69.2 (C-4*), 70.8 (C-6*), 71.1 (C-7*),
72.9 (C-8*), 76.4 (C-9), 98.2 (C-2), 175.6 (C-1), 177.3
(NC(O)Me), *assignments interchangeable.
1
and 1036 cm 1; H NMR (D2O) d 1.83 (1H, dd, J3a,3e
12.5, J3a,4 11.7 Hz, H-3a), 1.99 (3H, s, AcN), 2.26 (1H,
dd, J3e,3a 12.5, J3e,4 4.5 Hz, H-3e), 3.51 (1H, d, J7,8
8.3 Hz, H-7), 3.88 (1H, dd, J5,6=J5,4=10.1 Hz, H-5),
3.98±4.07 (3H, m, H-4/H-6), 4.35 (1H, m, H-8), 6.05
(1H, d, J10,9 15.7 Hz, H-10), 7.08 (1H, dd, J9,10 15.7, J9,8
4.9 Hz, H-9), assignments con®rmed by DQF-COSY;
13C NMR (D2O) d 24.7 (NC(O)Me), 41.5 (C-3), 54.7
(C-5), 69.3 (C-4), 72.4 (C-8), 72.8 (C-6), 73.1 (C-7),
98.2 (C-2), 124.1 (C-10), 152.1 (C-9), 172.6, 175.8
(2ÂCO2H), 177.4 (NC(O)Me), assignments con®rmed
5-Acetamido-9-O-methoxymethyl-3,5-dideoxy-D-glycero-
ꢀ-D-galacto-2-nonulopyranosylonic acid (12). Prepared
from the 9-O-MOM ether 13 using the saponi®cation
procedure detailed above to give 12 in 72% yield after
HPLC (reverse phase, 3% CH3CN in H2O) as an

664
M. J. Kiefel et al. / Bioorg. Med. Chem. 8 (2000) 657±664
by HMQC; FABMS: 372 [(M+Na)+, 18%], 350
[(M+1)+, 44].
16. Lin, C.-C.; Lin, C.-H.; Wong, C.-H. Tetrahedron Lett.
1997, 38, 2649.
17. Baumann, W.; Freidenreich, J.; Weisshaar, G.; Brossmer,
R.; Freibolin, H. Biol. Chem. Hoppe-Seyler 1989, 370, 141.
18. Grob, H. J.; Brossmer, R. FEBS Lett. 1988, 232, 145.
19. Suttajit, M.; Urban, C.; McLean, R. L. J. Biol. Chem.
1971, 246, 810.
Acknowledgements
The authors thank Glaxo Wellcome (UK) for ®nancial
support, and Dr. Yoji Tsukada (Kyoto Research
Laboratories, Japan) for the generous gift of Neu5Ac
aldolase.
20. Schauer, R.; Stoll, S.; Zbiral, E.; Schreiner, E.; Brand-
stetter, H. H.; Vasella, A.; Baumberger, F. Glycoconjugate J.
1987, 4, 361.
21. Zbiral, E.; Kleineidam, R. G.; Schreiner, E.; Hartmann,
M.; Christian, R.; Schauer, R. Biochem J. 1992, 282, 511.
22. Shukla, A. K.; Schauer, R. Anal. Biochem. 1986, 158, 158.
23. Kim, M.-J.; Hennen, W. J.; Sweers, H. M.; Wong, C.-H. J.
Am. Chem. Soc. 1988, 110, 6481.
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