Transsialidase from Trypanosoma cruzi for regio- and stereoselective synthesis of N-acyl-modified sialylated oligosaccharides and measurement of transfer rates

Article abstract of DOI:10.1002/chem.200700439

Recombinant transsialidase from Trypanosoma cruzi (TcTS) was used for the sialylation with natural and non-natural derivatives of neuraminic acid. Neu5Ac-α(2→3)-Gal-β(1→6)-Glc-αOMe was prepared in 80% yield. Correspondingly, the modified trisaccharide derivatives Neu5Prop-α(2 →3)-Gal-β(1 →6)-GlcαOMe (32%) and Neu5Gc-α(2→3)-Gal-β(1→6)-Glc-αOMe (Prop = propanoyl, Gc = glycolyl) were obtained in 60 % yield, respectively.

Full text of DOI:10.1002/chem.200700439

DOI: 10.1002/chem.200700439
Transsialidase from Trypanosoma cruzi for Regio- and Stereoselective
Synthesis of N-Acyl-Modified Sialylated Oligosaccharides and Measurement
of Transfer Rates
Andreas Schroven,[a] Sebastian Meinke,[a] Patrick Ziegelmüller,[b] and Joachim Thiem*[a]
Abstract: Recombinant transsialidase from Trypanosoma cruzi (TcTS) was used
for the sialylation with natural and non-natural derivatives of neuraminic acid.
Keywords: enzyme
glycosylation · neuraminic acid ·
sialylation · transsialidase
catalysis
·
Neu5Ac-a
ingly, the modified trisaccharide derivatives Neu5Prop-a
aOMe (32%) and Neu5Gc-a
A
ACHTREUNG
E
ACHTREUNG
A
N
Gc=glycolyl) were obtained in 60% yield, respectively.
Introduction
In Chagas disease, transsialidase from Trypanosoma cruzi
(TcTS) causes the transfer of Neu5Ac from a human host
cell to the cell epitope of the pathogen. This unusual trans-
fer mechanism enables the pathogen to protect its own cell
surface against recognition by the human immune system.
Interestingly, this enzyme belongs to the superfamily of siali-
dases but shows merely transferase activity in the presence
of a suitable acceptor molecule. Thus, transsialidase allows
for transglycosylation of natural and non-natural donor sub-
strates, such as pNP-Neu5Ac to Galb-R acceptor structures,
leading to a large variety of complex and biologically active
oligosaccharides. Previously, a series of terminally sialylated
oligosaccharides could be obtained, and subsequently used
as building blocks for convenient syntheses of more complex
glycoconjugates in good yields.[2]
Neu5Gc, which is a foreign neuraminic acid derivative to
humans because of an inactivating mutation of the gene en-
coding the enzyme CMP-N-acetylneuraminic acid (CMP-
Neu5Ac) hydroxylase was found to be synthesized by many
cancerous tissues as a tumor-associated Hanganutziu–Dei-
cher antigen.[3] The mutation responsible for the absence of
Neu5Gc in humans occurred after our last common ancestor
with bonobos and chimpanzees, and before the origin of
present-day humans, prior to brain expansion.[4] Thus, it
could be of medical and even paleontological interest to
have the possibility to synthesize Neu5Gc-containing model
structures. In this case, the use TcTS offers a good solution,
utilising its stereo- and regioselectivity.
Neuraminic acid (Neu5Ac) plays an important role in
nature as a major constituent of a variety of glycoconjugates
(such as oligosaccharides, glycoproteins and gangliosides)
occurring in animals and several pathogens.[1] In most cases,
neuraminic acids are terminally a
R
G
to a galactose of the oligosaccharide cell epitope. Due to the
terminal position of Neu5Ac in these oligosaccharide scaf-
folds, the sialylation is associated with many processes, such
as cell-recognition and cell differentiation. Neu5Ac allows
recognition by a suitable receptor protein; on the other
hand, the presence of Neu5Ac is able to mask recognition
sites.
The application of the reversible nature of glycosidases in
the synthesis of various oligosaccharides presents a facile
glycosylation procedure. Sialidases from several sources
were exploited in transsialylations as their acceptance of ar-
tificial donor glycosides, such as pNP-aNeu5Ac (pNP=p-ni-
trophenyl) and MU-aNeu5Ac, has been recognized.
[a] Dipl.-Chem. A. Schroven, Dipl.-Chem S. Meinke, Prof. Dr. J. Thiem
University of Hamburg, Faculty of Science
Department of Chemistry, Institute of Organic Chemistry
Martin-Luther-King Platz 6, 20146
Hamburg (Germany)
Fax : (+49)40-42838-4325
E-mail: thiem@chemie.uni-hamburg.de
[b] Dr. P. Ziegelmüller
N-Acyl-modified sialic acids, such as Neu5Prop were pre-
viously reported to be obtained by an enzymatic approach
by Kayser et al. and other groups.[5–10] Herein, we report the
chemical synthesis of N-acyl-modified neuraminic acid
University of Hamburg, Faculty of Science
Department of Chemistry, Institute of Biochemistry
and Food Science, Martin-Luther-King Platz 6, 20146
Hamburg (Germany)
9012
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Chem. Eur. J. 2007, 13, 9012– 9021

FULL PAPER
donors by using the method of Komba et al.[11] and a highly
regio- and stereoselective enzymatic transglycosylation with
OH is not a suitable acceptor.[14] Six potential donor deriva-
tives were synthesised chemically. The most challenging
problem in the synthesis of compounds 3 to 6 is the selective
protection of the amino group. Further, care must be taken
TcTS to Gal-b(1!6)-Glc-aOMe.
A
Results and Discussion
Previously the transfer of side-
chain-shortened
neuraminic
acid derivatives to a variety of
different acceptor molecules
was reported with good
yields.[2,12] Figure 1 shows TcTS
in a complex with the excellent
donor-structure sialyllactose.[13]
Lactose is fixed by Tyr119 and
Trp312 and the carboxyl group
of Neu5Ac by Arg314. The C7–
C9 side chain of the substrate
is located outside the binding
pocket, and this may be the
reason for the high tolerance
of the enzyme towards changes
of this moiety. In contrast, the
acetyl group is located deep in
the pocket, fixed by Asp96. Thus, an acyl modification
should have stronger effects on the transfer process of the
protein, and enzymatic synthesis of sialylated oligosacchar-
ides with acyl modifications by using TcTS should be chal-
lenging. By use of molecular modelling with SYBYL we ar-
rived at the assumption that there should be space for elon-
gation of the acetyl substituent by at least one CH3 group.
to prevent neuraminic acid from formation of a five-mem-
bered ring between the amino and the keto group during de-
protection. Ring formation was avoided by protecting neu-
raminic acid from ring opening by formation of the thio-
phenyl glycoside which proved to be stable even under
strongly acidic conditions. Following the method of Rother-
mel et al., 7 was prepared in good yield.[15] Deprotection
with methanesulfonic acid and selective reprotection were
carried out in methanol following the method of Sugata
et al.[16] The use of alcohol as the solvent has the advantage
that no strict control of temperature and concentration are
needed, as the 5-amino function of neuraminic acid is nucle-
ophilic enough to withstand the solvent competition. In con-
trast, the hydroxyl groups were not acylated in the presence
of methanol. By using pyridine as the solvent, higher acylat-
ed byproducts were obtained.
After acidic deprotection of 7, Et3N and two equivalents
of the corresponding anhydride were added and stirred for
1 h at 08C. In contrast to the use of N-hydroxysuccinimide
esters,[16] the use of an anhydride was easier and therefore
preferred in the synthesis of glycoyl derivative 11.
The corresponding anhydride was easy to synthesise by
starting with the commercially available acetoxyacetyl chlo-
ride, following the method of Plusquellec et al.[17] The solu-
tion was evaporated and the residue was acetylated in pyri-
dine with acetic anhydride to give components 8–11. Hy-
drolysis of these thioglycosides by using NBS in acetone/
water at room temperature gave the hemiacetales 12–15
(Scheme 1).
Figure 1. Neu5Ac lactose in complex with TcTS; PDB-Code=1S0.
Synthesis of donor substrates: According to the donor spe-
cificity, pNP-Neu5Ac derivatives could be used as non-natu-
ral sialyl-donor structures, leading to a
A
ed oligosaccharides. In contrast to natural donor substrates,
the use of non-natural ones has the advantage that a strict
kinetic control to fix the equilibrium of sialyllactose and
product is not needed as the free phenolic compound pNP-
Despite the fact that protons at C-3 loose their character-
istic positions in 1H NMR spectra, the stereochemistry at
the anomeric position was easy to determine because of the
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J. Thiem et al.
nitrile) under neutral condi-
tions yielded compound 2 in
41% yield.
Transsialylation with transsiali-
dase TcTS: The activity of
TcTS is difficult to measure
and an assay would usually re-
quire radioactively labelled
material. To avoid this, the
quantity was determined by
the concentration of the pure
enzyme by using the same con-
centration for each test. This
allowed for a comparison of
the yields of different transsia-
lylations, following a procedure
established by R. Field et al.[19]
Potential donor substrates 1–6
were tested for transsialylation
with methyl a-allolactoside
(28), which proved to be an ex-
cellent acceptor substrate and
was therefore used to avoid
limiting effects due to an ac-
ceptor substrate. In addition,
this was, therefore, taken to
observe even marginal trans-
glycosylation activities with
non-natural donor substrates,
in the presence of TcTS for
17 h, giving yields from 32to
87% (Scheme 3). Purification
by Biogel (P2) chromatogra-
phy allowed an easy and fast
isolation of products.
Scheme 1. i) MsOH, MeOH, reflux; ii) Et3N, (RCO)2O, 0 8C; iii) Ac2O, pyridine; iv) acetone, NBS, H2O;
v) AcCl, MeOH; vi) pNP-OH, 1n NaOH, dichloromethane, 3 h; vii) MeOH, NaOMe; viii) 0.1n NaOH. NBS=
N-bromosuccinimide; Ms=methanesulfonyl.
Compound 2 could be used
coupling between H-3ax and the proton of the anomeric hy-
droxyl group. The subsequent formation of chlorides 16–19,
preparation of corresponding pNP glycosides 20–23 by
phase-transfer catalysis, ZemplØn deacetylation to com-
pounds 24–27 and hydrolysis to give 3–6 could be performed
partly in analogy to previous methods.[15] The synthesis of
the 9-O-acylated derivative 2 started with 1 by using a modi-
fied method of Ogura et al.[18] The reaction was carried out
in acetonitrile with trimethyl orthobutyrate in the presence
of catalytic amounts of p-toluenesulfonic acid followed by in
situ hydrolysis of the ortho ester (Scheme 2). Purification by
column chromatography with RP-18 silica gel (water/aceto-
for transsialylation comparable to compound 1. As expect-
ed, elongation of the N-acetyl group by one methylene
group led to a drastic decrease in yield from 87 to 32%.
Due to the relatively deep location of the acyl group in the
transition state (Figure 1), further modifications were chal-
lenging; however, potential donor substrates 4 and 5 were
not transferred. Despite the steric demand of an additional
hydroxyl group, the N-glycolyl derivative 6 turned out to be
a suitable donor and allowed the enzymatic transfer with
60% yield.
The stereochemistry of the linkage was assigned by a
characteristic shift of H-3eq and H-3ax of Neu5Acyl due to
the anisotropy of the carbonyl
group.[20] The regiochemistry
of the linkage could be deter-
mined by the chemical shift of
C-3. After sialylation, this
carbon showed a clear down-
field shift of approximately d=
Scheme 2. Selective 9-O-acylation with trimethyl orthobutyrate.
3.0 ppm compared to the non-
9014
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Chem. Eur. J. 2007, 13, 9012– 9021

Synthesis of N-Acyl-Modified Sialylated Oligosaccharides
FULL PAPER
91 nmolminꢀ1. By using pNP-
Neu5Gc as a substrate, KM in-
creased to 31 mm and Vmax de-
creased to 41 nmolminꢀ1. KM
and Vmax for pNP-Neu5Prop
were found to be 45 mm and
12nmolmin ꢀ1
,
respectively,
and for pNP-Neu5Ac9But
(But=butanoyl) 10 mm and
51 nmolminꢀ1, respectively.
Conclusion
Various neuraminic acid deriv-
atives were transferred enzy-
matically by employing trans-
Scheme 3. Transglycosylation of synthesised donor derivatives.
sialidase from Trypanosoma
cruzi. Changes of the substitu-
ent at nitrogen showed drastic
sialylated disaccharide.[2] The transfer rates of the novel
donor substrates were measured by recording 1H NMR
spectra and subsequent integration of the aromatic proton
signals of pNP-Neu5Ac derivatives and of the released pNP-
OH. The incubation of donor and acceptor followed the
procedure mentioned above by using D2O instead of H2O.
To determine the conversion at a given time, 100 mL of incu-
bated solution were transferred into ready NMR tubes con-
taining a mixture of 1:1 D2O/[D4]methanol to stop the reac-
tion by denaturing the enzyme. Conversion rates were calcu-
lated from the signals of the pNP moiety in the starting ma-
terial pNP-Neu5Ac and in the product pNP-OH (Figure 2).
By using the described procedure, maximum rates of con-
version (Vmax) and the Michaelis constants (KM) were ob-
tained by measuring reaction velocities in D2O at different
substrate concentrations. The resulting Lineweaver–Burk
plot is shown in Figure 3. Under the described conditions,
effects related to the transfer ratio. Nevertheless TcTS
turned out to be a useful tool for the synthesis of oligosac-
charides containing a2!3-linked glycolyl neuraminic acid.
Neu5Gc-a
G
G
60% yield. Propanoyl neuraminic acid was still obtained in
a yield of 30%. On the other hand, TcTS showed a high tol-
erance towards chemical changes of the glycerol side chain.
pNP-Neu5Ac showed
a KM of 10 mm and a Vmax of
Figure 3. Lineweaver–Burk plot of experimental data measured by
1H NMR spectroscopy.
Experimental Section
General remarks: Commercially available starting materials were used
without further purification. Solvents were dried according to standard
methods. Purification of the products was carried out by column chroma-
tography by using Merck silica gel 60 (230–400 mesh). The enzymatic re-
actions were incubated in a thermomixer Comfort (Merck) at 600 rpm.
The NMR spectra were recorded on a Bruker AMX-400 (100.62MHz
for 13C NMR spectra, 400.14 MHz for 1H NMR spectra) or DRX-500
Figure 2. Transsialylation of Neu5Acyl derivatives in D2O measured by
1H NMR spectroscopy.
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J. Thiem et al.
(125.77 MHz for 13C NMR spectra, 500.13 MHz for 1H NMR spectra)
spectrometer. All chemical shifts are quoted in ppm downfield from
TMS or referred to the characteristic signals of the used solvents CHCl3
in CDCl3 (7.26 ppm), [D5]C6H6 in [D6]C6H6 (7.16 ppm), [D3]MeOH in
[D4]MeOH (3.31 ppm) or HDO in D2O (4.79 ppm). Mass spectra were
recorded on a Bruker MALDI-Tof Biflex III.
aarom), 7.28 (d, J(Ha,Hb)arom =9.4, 2H; H-barom), 4.16 (dd, J5,6 =10.4, J6,7
1.5 Hz, 1H; H-6), 3.96 (dd, J4,5 =10.5, J5,6 =10.4 Hz, 1H; H-5), 3.86–3.81
(m, 3H; H-4/H-8/H-9a), 3.62(dd, 8,9b =6.6, J9a,9b =12.5 Hz, 1H; H-9b),
3.56 (dd, J6,7 =1.5, J7,8 =8.9 Hz, 1H; H-7), 2.84 (dd, J3ax,3eq =13.0, J3eq,4
4.8 Hz, 1H; H-3eq), 2.31 (ddd, JCH2,CH3 =7.6 Hz, 2H; CH2-Prop), 2.02 (dd,
=
J
=
J
3ax,3eq =13.0, J3ax,4 =11.7 Hz, 1H; H-3ax), 1.12ppm (dd, J(Ha,Hb)arom =
9.4 Hz, 3H; CH3); 13C NMR (101 MHz, D2O): d=125.88 (C-aarom), 120.46
Transsialylation with transsialidase from Trypanosoma cruzi CAHTRE(UNG method 1):
(C-barom), 102.77 (C-2), 74.12 (C-6), 71.79 (C-4), 68.66 (C-7), 67.97 (C-8),
A solution of donor (25 mmol) and acceptor (35 mmol) in degassed incu-
bation buffer (1.0 mL, 100 mm Tris/HCl, pH 7.5, 50 mg BSA, 0.02%
NaN3) was incubated with recombinant TcTS (80 mL, 1.3 mg/1 mL) at
238C for 24 h. The reaction was monitored by TLC (butanol/acetic acid/
water 5:2:2). After completion, the enzyme was denatured and centri-
fuged before the supernatant was lyophilized. The dry residue was dis-
solved in water and purified by biogel chromatography (P-2Biogel, 16ꢁ
900 mm) with water.
63.12(C-9), 51.92(C-5), 41.31 (C-3), 29.62(CH
2-Prop), 9.88 ppm (CH3-
Prop); MALDI-TOF: m/z: calcd for C18H24N2O11: 444.39; found: 466.7
[M+Na]+.
4-Nitrophenyl (5-N-butanoyl-3,5-dideoxy-a-d-glycero-d-galacto-2-nonu-
lopyranosylonic acid) (4): Compound 25 (99.0 mg, 210 mmol) was dis-
solved in aqueous NaOH (25 mL, 0.1m). After stirring for 0.5 h, the solu-
tion was neutralized with Amberlite IR 120 (H+) resin and lyophilized.
The dry residue was dissolved in water and purified by biogel chromatog-
raphy (P-2Biogel, 16ꢁ900 mm) with water. Compound 4 was obtained
as a colourless solid. Yield: 75.0 mg (78%); m.p. 858C; [a]25406 =+89 (c=1
in H2O); 1H NMR (400 MHz, D2O): d=8.23 (d, J(Ha,Hb)arom =9.4 Hz, 2H;
H-aarom), 7.30 (d, J(Ha,Hb)arom =9.4 Hz, 2H; H-barom), 4.17 (dd, J5,6 =10.2,
Deacetylation and selective N-acylation and O-acetylation of 7 (method
2): Compound 7 was dissolved in dry methanol (1 mL/100 mg) and meth-
anesulfonic acid (0.1 mL per 100 mg 7) was added. The solution was
heated under reflux for 24 h and was then neutralized with triethylamine
(Et3N). Further triethylamine (0.1 mL per 100 mg of 7) was added and
the solution was treated with the corresponding anhydride (1.5–
2.0 equiv) at 08C and stirred for 1 h. After this time, the solution was
evaporated and the residue dissolved in acetic anhydride. Dry pyridine
was added dropwise at 08C and stirred for 24 h. The solution was concen-
trated and the residue was taken-up in chloroform, washed with cold hy-
drochloric acid (1m) and water, dried (Na2SO4) and then concentrated.
The products were purified by column chromatography (ethyl acetate)
on silica gel.
J
6,7 =1.3 Hz, 1H; H-6), 3.98 (dd, J4,5 =10.2, J5,6 =10.2Hz, 1H; H-5), 3.88–
3.79 (m, 3H; H-4/H-8/H-9a), 3.63 (dd, J8,9b =7.0, J9a,9b =12.7 Hz, 1H; H-
9b), 3.59 (dd, J6,7 =1.3, J7,8 =8.9 Hz, 1H; H-7), 2.82 (dd, J3ax,3eq =13.0,
J
3eq,4 =4.6 Hz, 1H; H-3eq), 2.29 (dd, JCH2,CH2 =7.4 Hz, 2H; CH2-a-But),
2.03 (dd, J3ax,3eq =13.0, J3ax,4 =11.7 Hz, 1H; H-3ax), 1.63 (m, 2H; CH2-b-
But), 0.93 ppm (dd, JCH2,CH3 =7.4 Hz, 3H; CH3); 13C NMR (101 MHz,
D2O): d=125.93 (C-aarom), 120.30 (C-barom), 74.13 (C-6), 71.70 (C-4),
68.75 (C-7), 67.94 (C-8), 63.14 (C-9), 52.00 (C-5), 41.39 (C-3), 38.23 (C-2-
But), 19.39 (C-3-But), 13.21 ppm (CH3-But); MALDI-TOF: m/z: calcd
for C19H26N2O11: 458.42; found: 480.8 [M+Na]+.
Procedure for hydrolysis of neuraminic acid thioglycosides (method 3):
NBS (3 equiv) was added to a solution of the thioglycoside in acetone
(4 mL per 100 mg thioglycoside) and water (0.2mL per 100 mg thioglyco-
side) and the resulting mixture was stirred for 0.5 h at room temperature.
After this time, the solution was concentrated, diluted with CHCl3,
washed with saturated aqueous NaHCO3, dried (MgSO4) and evaporated.
The products were purified by column chromatography (ethyl acetate)
on silica gel.
4-Nitrophenyl (5-N-isobutanoyl-3,5-dideoxy-a-d-glycero-d-galacto-2-non-
ulopyranosylonic acid) (5): Compound 26 (50.0 mg, 106 mmol) was dis-
solved in aqueous NaOH (10 mL, 0.1m). After stirring for 0.5 h, the solu-
tion was neutralized with Amberlite IR 120 (H+) resin and lyophilized.
The dry residue was dissolved in water and purified by biogel chromatog-
raphy (P-2Biogel, 16ꢁ900 mm) with water. Compound 5 was obtained
as a colourless solid. Yield: 48.2mg (100%); m.p. 118 8C; [a]25406 =+11
(c=1 in H2O); 1H NMR (400 MHz, D2O): d=8.21 (d, J(Ha,Hb)arom =9.4,
4-Nitrophenyl (5-acetamido-9-O-butanoyl-3,5-dideoxy-a-d-glycero-d-gal-
acto-2-nonulopyranosylonic acid) (2): pNP-Neu5Ac (1, 180 mg, 418
mmol) was suspended in acetonitrile (4 mL). Trimethylorthobutyrate (260
mL, 1.67 mmol) and toluene-4-sulfonic acid (2mg) were added and stirred
for 1 h. Subsequently, the intermediate was hydrolysed by the addition of
H2O (1 mL). The solution was evaporated and the residue purified by
column chromatography (H2O/acetonitrile) on RP-18 silica gel. Com-
2H; H-aarom), 7.28 (d, J(Ha,Hb)arom =9.4 Hz, 2H; H-barom), 4.17 (dd, J5,6
=
10.4, J6,7 =1.5 Hz, 1H; H-6), 3.94 (dd, J4,5 =10.5, J5,6 =10.4 Hz, 1H; H-5),
3.86–3.80 (m, 3H; H-4/H-8/H-9a), 3.62(dd, J8,9b =6.6, J9a,9b =12.5 Hz, 1H;
H-9b), 3.53 (dd, J6,7 =1.5, J7,8 =8.9 Hz, 1H; H-7), 2.84 (dd, J3ax,3eq =12.8,
J
3eq,4 =4.6 Hz, 1H; H-3eq), 2.55 (h, J(CH,CH3a)But =7.1, J(CH,CH3b)But =7.1 Hz,
1H; CH-But), 2.02 (dd, J3ax,3eq =12.8, J3ax,4 =11.7 Hz, 1H; H-3ax), 1.12(d,
(CH,CH3a)But =7.1 Hz, 3H; CH3a-But), 1.11 ppm (d, J(CH,CH3b)But =7.1 Hz,
pound 2 was obtained as a colourless solid. Yield: 86 mg (41%); m.p.
20
1438C; [a] =+217 (c=1 in H2O); 1H NMR (400 MHz, D2O): d=8.17
546
J
(d, J(Ha,Hb)arom =9.2Hz, 2H; H-a arom), 7.23 (d, J(Ha,Hb)arom =9.2Hz, 2H; H-
barom), 4.33 (dd, J8,9a =2.3, J9a,9b =11.7 Hz, 1H; H-9a), 4.19 (dd, J5,6 =10.4,
3H; CH3b-But); 13C NMR (101 MHz, D2O): d=125.91 (C-aarom), 120.26
(C-barom), 102.68 (C-2), 74.14 (C-6), 71.73 (C-4), 68.70 (C-7), 67.78 (C-8),
63.10 (C-9), 51.81 (C-5), 41.35 (C-3), 35.58 (CH-But), 19.32(CH 3a-But),
18.79 ppm (CH3b-But); MALDI-TOF: m/z: calcd for C19H26N2O11:
458.42; found: 481.1 [M+Na]+.
J
6,7 =1.3 Hz, 1H; H-6), 4.17 (dd, J8,9b =5.6, J9a,9b =11.7 Hz, 1H; H-9b),
4.00 (ddd, J7,8 =9.2, J8,9a =2.3, J8,9b =5.6 Hz, 1H; H-8), 3.93 (dd, J4,5 =10.3,
5,6 =10.4 Hz, 1H; H-5), 3.78 (ddd, J3ax,4 =12.0, J3eq,4 =4.6, J4,5 =10.3 Hz,
1H; H-4), 3.59 (dd, J6,7 =1.3, J7,8 =9.2Hz, 1H; H-7), 2.79 (dd, J3ax,3eq
J
=
4-Nitrophenyl (5-N-glycolyl-3,5-dideoxy-a-d-glycero-d-galacto-2-nonulo-
pyranosylonic acid) (6): Compound 27 (18 mg, 39 mmol) was dissolved in
aqueous NaOH (5 mL, 0.1m). After stirring for 0.5 h, the solution was
neutralized with Amberlite IR 120 (H+) resin and lyophilized. The dry
residue was dissolved in water and purified by biogel chromatography
(P-2Biogel, 16ꢁ900 mm) with water. Compound 6 was obtained as a col-
ourless solid. Yield: 48.2mg (98%); m.p. 123 8C; [a]25406 =+83 (c=1 in
H2O); 1H NMR (400 MHz, D2O): d=8.24 (d, J(Ha,Hb)arom =9.3 Hz, 2H; H-
12.7, J3eq,4 =4.6 Hz, 1H; H-3eq), 2.36 (dd, JCH2,CH2 =7.1 Hz, 2H; CH2(2)-
But), 2.02 (s, 3H; COCH3), 1.99 (dd, J3ax,3eq =12.7, J3ax,4 =12.0 Hz, 1H; H-
3ax), 1.58 (sext,
J
CH2,CH2 =7.1,
JCH2,CH3 =7.4 Hz, 2H; CH2(3)-But),
0.88 ppm (dd, JCH2,CH3 =7.4 Hz, 3H; CH3(4)-but); 13C NMR (101 MHz,
D2O): d=177.27 (C-1), 160.36 (C-1-but), 125.89 (C-aarom), 120.16 (C-
barom), 102.58 (C-2), 74.04 (C-6), 69.38 (C-8), 68.76 (C-7), 67.94 (C-4),
66.07 (C-9), 52.09 (C-5), 41.26 (C-3), 36.12 (C-2-But), 22.40 (COCH3),
18.41 (C-3-But), 13.18 ppm (C-4-But); MALDI-TOF: m/z (%): calcd for
C21H28N2O12: 500.45; found: 523.0 [M+Na]+.
a
arom), 7.31 (d, J(Ha,Hb)arom =9.3 Hz, 2H; H-barom), 4.28 (dd, J5,6 =10.4, J6,7
=
=
1.3 Hz, 1H; H-6), 4.15 (s, 2H; CH2-Gc), 4.06 (dd, 4,5 =10.2, J5,6
J
4-Nitrophenyl (5-N-propanoyl-3,5-dideoxy-a-d-glycero-d-galacto-2-nonu-
lopyranosylonic acid) (3): Compound 24 (112mg, 244 mmol) was dis-
solved in aqueous NaOH (25 mL, 0.1m). After stirring for 0.5 h, the solu-
tion was neutralized with Amberlite IR 120 (H+) resin and lyophilized.
The dry residue was dissolved in water and purified by biogel chromatog-
raphy (P-2Biogel, 16ꢁ900 mm) with water. Compound 3 was obtained
as a colourless solid. Yield: 70.5 mg (70%); m.p. 988C; [a]25406 =+70 (c=1
in H2O); 1H NMR (400 MHz, D2O): d=8.21 (d, J(Ha,Hb)arom =9.4, 2H; H-
10.4 Hz, 1H; H-5), 3.93 (ddd, J3ax,4 =12.2, J3eq,4 =4.6, J4,5 =10.2Hz, 1H;
H-4), 3.89–3.84 (m, 2H; H-8/H-9a), 3.65 (dd, J8,9b =6.6, J9a,9b =12.5 Hz,
1H; H-9b), 3.60 (dd, J6,7 =1.3, J7,8 =8.9 Hz, 1H; H-7), 2.88 (dd, J3ax,3eq
=
12.7,
J3eq,4 =4.6 Hz, 1H; H-3eq), 2.05 ppm (dd, J3ax,3eq =12.7, J3ax,4
=
12.2 Hz, 1H; H-3ax); 13C NMR (101 MHz, D2O): d=125.93 (C-aarom),
120.32 (C-barom), 102.77 (C-2), 73.85 (C-6), 71.82 (C-4), 68.54 (C-7), 67.83
(C-8), 63.12(C-9), 61.36 (CH 2-Gc), 51.77 (C-5), 41.27 ppm (C-3);
9016
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 9012– 9021

Synthesis of N-Acyl-Modified Sialylated Oligosaccharides
FULL PAPER
MALDI-TOF: m/z: calcd for C17H22N2O12
[M+Na]+.
:
446.36; found: 469.3
20.29 (each COCH3), 19.60 (CH3a-But), 19.13 ppm (CH3b-But);
MALDI-TOF: m/z: calcd for C28H37NO12S: 611.66; found: 633.7
[M+Na]+, 649.6 [M+K]+.
Thiophenyl (methyl-5-N-propanoyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-
d-glycero-d-galacto-2-nonulopyranosylonate) (8): Compound 7 (500 mg,
857 mmol) was converted into the N-propanoyl derivative 8 by following
method 2with propionic anhydride (020 mL, 1.55 mmol). Compound 8
was obtained as an amorphous solid. Yield: 176 mg (34%); [a]25406 =+13
(c=0.5 in CHCl3); 1H NMR (400 MHz, C6D6): d=7.63 (dd, JH-m,H-o =8.4,
Thiophenyl (methyl-5-N-acetoxyacetyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
a-d-glycero-d-galacto-2-nonulopyranosylonate) (11): Compound
7
(500 mg, 857 mmol) was converted into the N-acetoxyacetyl derivative 11
by following method 2with an excess of acetoxyacetyl anhydride. Com-
pound 11 was obtained as an amorphous solid. Yield: 211 mg (38%);
[a] =+13 (c=0.1 in CHCl3); 1H NMR (400 MHz, C6D6): d=7.62(dd,
20
546
J
H-o,H-p =1.3 Hz, 2H; H-o), 7.16 (dd, JH-m,H-o =8.4, JH-m,H-p =7.3 Hz, 2H; H-
J
H-m,H-o =8.1, JH-o,H-p =1.3 Hz, 2H; H-o), 7.14 (dd, JH-m,H-o =8.1, JH-m,H-p
7.4 Hz, 2H; H-m), 7.06 (m, 1H; H-p), 5.65 (ddd, 7,8 =7.1, J8,9a =2.5,
8,9b =5.3 Hz, 1H; H-8), 5.58 (dd, J6,7 =2.0, J7,8 =7.1 Hz, 1H; H-7), 5.39
(d, 5,NH =9.9 Hz, 1H; NH), 5.12(ddd, 3ax,4 =12.0, 3eq,4 =3.6, J4,5
=
m), 7.08 (m, 1H; H-p), 5.64 (ddd, J7,8 =6.5, J8,9a =2.3, J8,9b =5.8 Hz, 1H;
H-8), 5.54 (dd, J6,7 =2.0, J7,8 =6.5 Hz, 1H; H-7), 4.90 (ddd, J3ax,4 =11.8,
J
J
J
3eq,4 =4.9, J4,5 =10.3 Hz, 1H; H-4), 4.75 (dd, J8,9a =2.3, J9a,9b =12.3 Hz,
J
J
J
=
1H; H-9a), 4.59 (d, J5,NH =10.3 Hz, 1H; NH), 4.47 (dd, J8,9b =5.8, J9a,9b
=
10.9 Hz, 1H; H-4), 4.73 (dd, J8,9a =2.5, J9a,9b =12.5 Hz, 1H; H-9a), 4.47
(dd, J8,9b =5.3, J9a,9b =12.5 Hz, 1H; H-9b), 4.47 (d, JCH2-Gc =15.0 Hz, 1H;
CH2a-Gc), 4.31 (ddd, J4,5 =10.9, J5,6 =10.7, J5,NH =9.9 Hz, 1H; H-5), 4.15
(d, JCH2-Gc =15.0 Hz, 1H; CH2b-Gc), 4.11 (dd, J5,6 =10.7, J6,7 =2.0 Hz, 1H;
H-6), 3.14 (s, 3H; COOCH3), 2.98 (dd, J3ax,3eq =12.7, J3eq,4 =3.6 Hz, 1H;
H-3eq), 2.04 (dd, J3ax,3eq =12.7, J3ax,4 =12.0 Hz, 1H; H-3ax), 1.93, 1.92, 1.79,
1.73, 1.69 ppm (eachs, 3H; COCH3); 13C NMR (101 MHz, C6D6): d=
130.16, 129.46, 128.59 (eachC-arom), 88.53 (C-2), 75.59 (C-6), 70.96 (C-
8), 69.21 (C-4), 68.41 (C-7), 63.16 (CH2-Gc), 62.49 (C-9), 52.55
(COOCH3), 50.14 (C-5), 39.24 (C-3), 21.15, 20.94, 20.80, 20.70, 20.39 ppm
(each COCH3); MALDI-TOF: m/z: calcd for C28H35NO14S: 641.64;
found: 664.1 [M+Na]+, 680.0 [M+K]+.
12.3 Hz, 1H; H-9b), 4.38 (ddd, J4,5 =10.3, J5,6 =10.3, J5,NH =10.3 Hz, 1H;
H-5), 4.01 (dd, J5,6 =10.3, J6,7 =2.0 Hz, 1H; H-6), 3.24 (s, 3H; COOCH3),
2.97 (dd, J3ax,3eq =12.8, J3eq,4 =4.9 Hz, 1H; H-3eq), 2.06 (dd, J3ax,3eq =12.8,
J
3ax,4 =11.8, 1H; H-3ax), 1.97, 1.96 (eachs, 3H; COCH3), 1.94–1.80 (m,
2H; CH2-Prop), 1.79, 1.61 (eachs, 3H; COCH3), 1.05 ppm (dd, JCH2,CH3
=
7.8, 3H; CH3-Prop); 13C NMR (101 MHz, C6D6): d=137.07 (C-ortho),
130.27 (C-meta), 129.58 (C-para), 88.63 (C-2), 75.99 (C-6), 71.35 (C-8),
70.27 (C-4), 68.39 (C-7), 62.88 (C-9), 52.67 (COOCH3), 49.40 (C-5), 39.29
(C-3), 30.09 (C-2-Prop), 21.32, 21.07, 20.93, 20.75 (each COCH3),
10.17 ppm (C-3-Prop); MALDI-TOF: m/z: calcd for C27H35NO12S:
597.63; found: 620.3 [M+Na]+.
Thiophenyl (methyl-5-N-butanoyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-d-
Methyl 5-N-propanoyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-b-d-glycero-d-
glycero-d-galacto-2-nonulopyranosylonate) (9): Compound
7 (430 mg,
galacto-2-nonulopyranosylonate (12): Thioglycoside
8
(290 mg,
734 mmol) was converted into the N-butanoyl derivative 9 by following
485 mmol) was hydrolysed by following method 3. Compound 12 was ob-
method 2with butanoic anhydride (420
mL, 1.47 mmol). Compound 9
tained as an amorphous solid. Yield: 160 mg (65%); [a]25406 =ꢀ5.1 (c=1
was obtained as an amorphous solid. Yield: 176 mg (39%); [a]25406 =+10
(c=0.5 in CHCl3); 1H NMR (400 MHz, C6D6): d=7.64 (dd, JH-m,H-o =8.4,
1
in CHCl3); H NMR (400 MHz, C6D6): d=5.72(dd, J6,7 =2.4, J7,8 =3.8 Hz,
1H; H-7), 5.62(ddd,
(ddd, J3ax,4 =10.4, J3eq,4 =6.4, J4,5 =10.4 Hz, 1H; H-4), 5.27 (d, J5,NH
10.4 Hz, 1H; NH), 5.08–5.05 (m, 2H; H-9a/OH), 4.60 (ddd, J4,5 =10.4,
5,6 =10.5, J5,NH =10.4 Hz, 1H; H-5), 4.40 (dd, J5,6 =10.5, J6,7 =2.4 Hz, 1H;
H-6), 4.25 (dd, 8,9b =8.1, 9a,9b =12.2 Hz, 1H; H-9b), 3.32 (s, 3H;
J8,9a =2.3, J8,9b =8.1, J9a,9b =12.2 Hz, 1H; H-8), 5.32
J
H-o,H-p =1.0 Hz, 2H; H-o); 7.16 (dd, JH-m,H-o =8.4, JH-m,H-p =7.6 Hz, 2H; H-
=
m), 7.07 (m, 1H; H-p), 5.66 (ddd, J7,8 =7.1, J8,9a =2.8, J8,9b =5.8 Hz, 1H;
H-8), 5.51 (dd, J6,7 =2.0, J7,8 =7.1 Hz, 1H; H-7), 4.88 (ddd, J3ax,4 =11.7,
J
J
3eq,4 =4.8, J4,5 =10.4 Hz, 1H; H-4), 4.76 (dd, J8,9a =2.8, J9a,9b =12.5 Hz,
1H; H-9a), 4.47 (dd, J8,9b =5.8, J9a,9b =12.5 Hz, 1H; H-9b), 4.35 (ddd,
4,5 =10.4, J5,6 =10.7, J5,NH =10.2Hz, 1H; H-5), 4.16 (d, 5,NH =10.2, 1H;
J
J
COOCH3), 2.27–2.21 (m, 2H; H-3ax/H-3eq), 2.01–1.84 (m, 2H; CH2-
Prop), 1.94, 1.88, 1.71, 1.62(eachs, 3H; COCH 3), 1.08 ppm (dd,
J
J
NH), 4.00 (dd, J5,6 =10.7, J6,7 =2.0 Hz, 1H; H-6), 3.21 (s, 3H; COOCH3),
2.99 (dd, J3ax,3eq =12.7, J3eq,4 =4.8 Hz, 1H; H-3eq), 2.04 (dd, J3ax,3eq =12.7,
J
(CH2,CH3)Prop =7.6, 3H; CH3-Prop); 13C NMR (101 MHz, C6D6): d=95.68
(C-2), 73.53 (C-8), 72.60 (C-6), 69.71 (C-4), 69.02 (C-7), 63.53 (C-9),
53.09 (COOCH3), 49.68 (C-5), 37.12 (C-3), 30.01 (C-2-Prop), 21.06, 20.91,
20.71, 20.69 (eachCOCH3), 10.07 ppm (C-3-Prop).
J
3ax,4 =11.7 Hz, 1H; H-3ax), 1.96, 1.93, 1.78, 1.58 (eachs, 3H; COCH3),
1.91–1.82(m, 1H; C HaHb-But), 1.74–1.67 (m, 1H; CHaHb-But), 1.63–1.51
(m, 1H; CH2-But), 0.83 ppm (dd, JCH2,CH3 =7.4, 3H; CH3-But); 13C NMR
(101 MHz, C6D6): d=172.63 (C-1), 137.06 (C-ortho), 130.25 (C-meta),
129.58 (C-para), 75.86 (C-6), 71.22 (C-8), 70.12 (C-4), 68.38 (C-7), 62.85
(C-9), 52.62 (COOCH3), 49.36 (C-5), 39.35 (C-3), 38.85 (C-2-But), 21.29,
21.04, 20.91, 20.75 (each COCH3), 19.39 (C-3-But), 14.72ppm (C-4-But);
MALDI-TOF: m/z: C28H37NO12S: 611.2; found: 634.4 [M+Na]+, 650.4
[M+K]+.
Methyl
5-N-butanoyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-b-d-glycero-d-
(100 mg, 164
galacto-2-nonulopyranosylonate (13): Thioglycoside
9
mmol) was hydrolysed by following method 3. Compound 13 was ob-
tained as an amorphous solid. Yield: 46 mg (54%); [a]25406 =ꢀ3 (c=0.5 in
CHCl3); 1H NMR (400 MHz, C6D6): d=5.66 (dd, J6,7 =2.3, J7,8 =4.3 Hz,
1H; H-7), 5.61 (ddd, J7,8 =4.3, J8,9a =2.0, J8,9b =7.9 Hz, 1H; H-8), 5.32(m,
1H; H-4), 5.03 (dd, J8,9a =2.0, J9a,9b =11.9 Hz, 1H; H-9a), 4.97 (d, J5,NH
10.2Hz, 1H; NH), 4.58 (ddd, J4,5 =10.2, J5,6 =10.6, J5,NH =10.2Hz, 1H; H-
5), 4.32(dd, 5,6 =10.6, 6,7 =2.3 Hz, 1H; H-6), 4.24 (dd, 8,9b =7.9,
J9a,9b11.9 Hz, 1H; H-9b), 3.29 (s, 3H; COOCH3), 2.25–2.20 (m, 2H; H-
3ax/3eq), 1.92, 1.87, 1.70, 1.63 (eachs, 3H; COCH3), 1.92–1.79 (m, 2H;
=
Thiophenyl (methyl-5-N-isobutanoyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-
d-glycero-d-galacto-2-nonulopyranosylonate) (10): Compound 7 (700 mg,
1.20 mmol) was converted into the N-isobutanoyl derivative 10 by follow-
ing method 2with isobutanoic anhydride (400 mL, 2.41 mmol). Com-
J
J
J
pound 10 was obtained as an amorphous solid. Yield: 401 mg (55%);
20
[a] =+2 1 c( =0.5 in CHCl3); 1H NMR (400 MHz, C6D6): d=7.64 (dd,
CH2a-But), 1.65–1.58 (m, 2H; CH2b-But), 0.83 ppm (dd, JCH2,CH3
7.6 Hz; 3HCH3-But); 13C NMR (101 MHz, C6D6): d=95.72(C-2), 73.41
=
546
J
H-m,H-o =7.6, JH-o,H-p =1.5 Hz, 2H; H-o), 7.15 (dd, JH-m,H-o =7.6, JH-m,H-p
7.6 Hz, 2H; H-m), 7.06 (m, 1H; H-p), 5.65 (ddd, 7,8 =7.1, J8,9a =2.8,
8,9b =5.9 Hz, 1H; H-8), 5.50 (dd, J6,7 =2.0, J7,8 =7.1 Hz, 1H; H-7), 4.90
(ddd, J3ax,4 =11.7, J3eq,4 =4.8, J4,5 =10.4 Hz, 1H; H-4), 4.75 (dd, J8,9a =2.8,
9a,9b =12.5 Hz, 1H; H-9a), 4.46 (dd, J8,9b =5.9, J9a,9b =12.5 Hz, 1H; H-9b),
4.37 (ddd, J4,5 =10.4, J5,6 =10.67, J5,NH =10.4 Hz, 1H; H-5), 4.25 (d, J5,NH
=
(C-6), 72.53 (C-8), 69.81 (C-4), 69.00 (C-7), 63.72 (C-9), 53.24
(COOCH3), 49.66 (C-5), 38.92 (C-3), 37.17 (C-2-But), 21.16, 21.04, 20.89,
20.83 (each COCH3), 19.45 (C-3-But), 14.29 ppm (CH3-But); MALDI-
TOF: m/z: calcd for C22H33NO13: 519.20; found: 542.2 [M+Na]+.
J
J
J
=
Methyl 5-N-isobutanoyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-b-d-glycero-d-
galacto-2-nonulopyranosylonate (14): Thioglycoside 10 (390 mg, 638
mmol) was hydrolysed by following method 3. Compound 14 was ob-
tained as an amorphous solid. Yield: 258 mg (78%); [a]25406 =ꢀ38 (c=1 in
CHCl3); 1H NMR (400 MHz, C6D6): d=5.64 (dd, J6,7 =2.3, J7,8 =4.0 Hz,
1H; H-7); 5.59 (ddd, J7,8 =4.0, J8,9a =2.3, J8,9b =8.0 Hz, 1H; H-8), 5.34
10.4 Hz, 1H; NH), 4.01 (dd, J5,6 =10.67, J6,7 =2.0 Hz, 1H; H-6), 3.20 (s,
3H; COOCH3), 2.97 (dd, J3ax,3eq =12.5, J3eq,4 =4.8 Hz, 1H; H-3eq), 2.06
(dd, J3ax,3eq =12.5, J3ax,4 =11.7 Hz, 1H; H-3ax), 1.95, 1.93 (eachs, 3H;
COCH3), 1.92–1.81 (m, 1H; CH-But), 1.78, 1.57 (eachs, 3H; COCH3),
1.14 (d,
J(CH,CH3a)But =6.9, 3H; CH3a-But), 0.98 ppm (d, J(CH,CH3b)But =
6.9 Hz, 3H; CH3b-But); 13C NMR (101 MHz, C6D6): d=170.00 (C-1),
136.66 (C-ortho), 129.82 (C-para), 129.68 (C-meta), 100.27 (C-2), 75.52
(C-6), 70.88 (C-8), 69.67 (C-4), 67.98 (C-7), 62.46 (C-9), 52.18
(COOCH3), 48.95 (C-5), 38.95 (C-3), 35.66 (CH-But), 20.84, 2.57, 20.46,
(ddd, J3ax,4 =11.3, J3eq,4 =5.3, J4,5 =10.3 Hz, 1H; H-4), 5.03 (d, J5,NH
10.3 Hz, 1H; NH), 5.02(dd, J8,9a =2.3, J9a,9b =12.3 Hz, 1H; H-9a), 4.90 (d,
3ax,OH =1.5 Hz, 1H, OH), 4.57 (ddd, J4,5 =10.3, J5,6 =10.5, J5,NH =10.3 Hz,
1H; H-5), 4.33 (dd, J5,6 =10.5, J6,7 =2.3 Hz, 1H; H-6), 4.23 (dd, J8,9b =8.0,
=
J
Chem. Eur. J. 2007, 13, 9012 – 9021
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9017

J. Thiem et al.
J
9a,9b =12.3 Hz, 1H; H-9b), 3.30 (s, 3H; COOCH3), 2.26 (ddd, J3ax,3eq
12.8, 3ax,4 =11.3, 3ax,OH =1.5 Hz, 1H; H-3ax), 2.19 (dd, 3ax,3eq =12.8,
3eq,4 =5.3 Hz, 1H; H-3eq), 2.05–1.99 (m, 1H; CH-But), 1.92, 1.87, 1.70,
=
Methyl 5-N-isobutanoyl-4,7,8,9-tetra-O-acetyl-2-chloro-3,5-dideoxy-b-d-
glycero-d-galacto-2-nonulopyranosylonate (18): Compound 14 (253 mg,
487 mmol) was dissolved in acetyl chloride (30 mL). Methanol (0.6 mL)
was added dropwise at 08C. The solution was stirred for 17 h and subse-
J
J
J
J
1.63 (eachs, 3H; COCH3), 1.17 (d, J(CH,CH3a)But =6.9, 3H; CH3a-But),
1.03 ppm (d, J(CH,CH3b)But =6.9 Hz, 3H; CH3b-But); 13C NMR (101 MHz,
C6D6): d=95.33 (C-2), 73.00 (C-8), 72.15 (C-6), 69.32 (C-4), 68.54 (C-7),
63.31 (C-9), 52.81 (COOCH3), 49.27 (C-5), 36.79 (C-3), 35.72 (CH-But),
20.71, 20.57, 20.42, 20.38 (each COCH3), 19.69 (CH3a-But), 19.18 ppm
(CH3b-But); MALDI-TOF: m/z: calcd for C22H33NO13: 519.50; found:
542.0 [M+Na]+.
quently evaporated. Compound 18 was obtained as an amorphous solid.
20
Yield: 261 mg (100%); [a] =+8 (c=1 in CHCl3); 1H NMR (400 MHz,
546
C6D6): d=5.56–5.51 (m, 2H; H-7/H-8), 5.29 (ddd, J3ax,4 =11.2, J3eq,4 =4.8,
J
4,5 =10.7 Hz, 1H; H-4), 4.81 (dd, J8,9a =2.0, J9a,9b =12.5 Hz, 1H; H-9a),
4.47 (ddd, J4,5 =10.7, J5,6 =10.7, J5,NH =10.7 Hz, 1H; H-5), 4.34 (d, J5,NH
=
10.7 Hz, 1H; NH), 4.28 (dd, J8,9b =5.8, J9a,9b =12.5 Hz, 1H; H-9b), 4.23
(dd, J5,6 =10.7, J6,7 =2.0 Hz, 1H; H-6), 3.35 (s, 3H; COOCH3), 2.72 (dd,
Methyl 5-N-acetoxyacetyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-b-d-glycero-
d-galacto-2-nonulopyranosylonate (15): Thioglycoside 11 (200 mg, 312
mmol) was hydrolysed by following method 3. Compound 15 was ob-
tained as an amorphous solid. Yield: 111 mg (65%); [a]25406 =ꢀ2 0 (c=1 in
CHCl3); 1H NMR (400 MHz, C6D6): d=5.83 (d, J5,NH =9.9, 1H; NH),
5.64 (dd, J6,7 =2.0, J7,8 =4.8 Hz; 1HH-7), 5.59 (ddd, J7,8 =4.8, J8,9a =2.0,
J
3ax,3eq =13.0, J3eq,4 =4.8 Hz, 1H; H-3eq), 2.01 (dd, J3ax,3eq =13.0, J3ax,4
11.2Hz, 1H; H-3 ax), 1.96–1.91 (m, 1H; CH-But), 1.90, 1.89, 1.74, 1.60
(eachs, 3H; COCH3), 1.16 (d, (CH,CH3a)But =6.8 Hz, 3H; CH3a-But),
=
J
1.01 ppm (d, J(CH,CH3b)But =7.1 Hz, 3H; CH3b-But); 13C NMR (101 MHz,
C6D6): d=98.03 (C-2), 72.11 (C-6), 71.35 (C-8), 68.75 (C-4), 67.46 (C-7),
62.90 (C-9), 53.48 (COOCH3), 48.57 (C-5), 41.47 (C-3), 35.90 (CH-But),
21.02, 20.83, 20.82, 20.68 (each COCH3), 19.39 (CH3a-But), 19.36 ppm
(CH3b-But); MALDI-TOF: m/z: calcd for C22H32ClNO12: 537.94; found:
559.9 [M+Na]+.
J
J
8,9b =7.6, J9a,9b =12.2 Hz, 1H; H-8), 5.45 (ddd, J3ax,4 =11.7, J3eq,4 =5.1,
4,5 =10.4 Hz; 1HH-4), 4.96 (dd, J8,9a =2.0, J9a,9b =12.2 Hz, 1H; H-9a), 4.90
(d, J3ax,OH =2.0 Hz, 1H; OH), 4.56 (ddd, J4,5 =10.4, J5,6 =10.4, J5,NH
9.9 Hz, 1H; H-5), 4.53 (d, JCH2-Gc =15.0 Hz, 1H; CH2a-Gc), 4.29 (dd,
5,6 =10.4, J6,7 =2.0 Hz, 1H; H-6), 4.23 (d, JCH2-Gc =15.0 Hz, 1H; CH2b-
Gc), 4.20 (dd, 8,9b =7.6, 9a,9b =12.2 Hz, 1H; H-9b), 3.29 (s, 3H;
=
Methyl 5-N-acetoxyacetyl-4,7,8,9-tetra-O-acetyl-2-chloro-3,5-dideoxy-b-
J
d-glycero-d-galacto-2-nonulopyranosylonate
(19):
Compound
15
J
J
(104 mg, 189 mmol) was dissolved in acetyl chloride (30 mL). Methanol
(0.6 mL) was added dropwise at 08C. The solution was stirred for 17 h
and subsequently evaporated. Compound 19 was obtained as an amor-
phous solid. Yield: 102mg (95%); [ a]25406 =ꢀ41 (c=1 in CHCl3);
1H NMR (400 MHz, C6D6): d=5.60 (dd, J6,7 =2.3, J7,8 =6.4 Hz, 1H; H-7),
COOCH3), 2.26 (ddd, J3ax,3eq =12.7, J3ax,4 =11.7, J3ax,OH =2.0 Hz, 1H; H-
3ax), 2.15 (dd, J3ax,3eq =12.7, J3eq,4 =5.1 Hz, 1H; H-3eq), 1.90, 1.84, 1.79,
1.75, 1.69 ppm (eachs, 3H; COCH3); 13C NMR (101 MHz, C6D6): d=
95.01 (C-2), 72.13 (C-8), 71.46 (C-6), 68.44 (C-4), 68.24 (C-7), 62.95 (C-
9), 62.65 (CH2-Gc), 52.53 (COOCH3), 49.68 (C-5), 36.50 (C-3), 20.39,
20.32, 20.23, 20.09, 19.88 ppm (each COCH3); MALDI-TOF: m/z: calcd
for C22H31NO15: 549.48; found: 571.9 [M+Na]+.
5.53 (ddd, J7,8 =6.4, J8,9a =2.8, J8,9b =6.1 Hz, 1H; H-8), 5.38 (ddd, J3ax,4
=
11.2, J3eq,4 =4.8, J4,5 =10.4 Hz, 1H, H-4), 5.24 (d, J5,NH =10.4, 1H, NH);
4.79 (dd, J8,9a =2.8, J9a,9b =12.5 Hz, 1H; H-9a), 4.48 (d, JCH2-Gc =14.8, 1H;
CH2a-Gc), 4.45 (ddd, J4,5 =10.4, J5,6 =10.7, J5,NH =10.4 Hz, 1H; H-5), 4.26
Methyl
5-N-propanoyl-4,7,8,9-tetra-O-acetyl-2-chloro-3,5-dideoxy-b-d-
glycero-d-galacto-2-nonulopyranosylonate (16): Compound 12 (150 mg,
297 mmol) was dissolved in acetyl chloride (35 mL). Methanol (0.6 mL)
was added dropwise at 08C. The solution was stirred for 17 h and subse-
(dd,
J8,9b =6.1, J9a,9b =12.5 Hz, 1H; H-9b), 4.21 (dd, J5,6 =10.7, J6,7 =
2.3 Hz, 1H; H-6), 4.16 (d, JCH2-Gc =14.8 Hz, 1H; CH2b-Gc), 3.34 (s, 3H;
COOCH3), 2.64 (dd, J3ax,3eq =14.0, J3eq,4 =4.8 Hz, 1H; H-3eq), 1.97 (dd,
quently evaporated. Compound 16 was obtained as an amorphous solid.
J
3ax,3eq =14.0, J3ax,4 =11.2Hz, 1H; H-3 ax), 1.91, 1.88, 1.78, 1.76, 1.74 ppm
20
Yield: 154 mg (99%); [a] =+55 (c=1 in CHCl3); 1H NMR (400 MHz,
(eachs, 3H; COCH3); 13C NMR (101 MHz, C6D6): d=74.92(C-6), 71.15
(C-8), 68.07 (C-4), 67.56 (C-7), 63.16 (C-9), 62.73 (CH2-Gc), 53.46
(COOCH3), 49.30 (C-5), 41.48 (C-3), 21.02, 20.90, 20.68, 20.67, 20.43 ppm
(each COCH3); MALDI-TOF: m/z: calcd for C22H30ClNO14: 567.92;
found: 589.9 [M+Na]+.
546
C6D6): d=5.58 (dd, J6,7 =2.2, J7,8 =6.3 Hz, 1H; H-7), 5.53 (ddd, J7,8 =6.3,
J
8,9a =2.5, J8,9b =6.0 Hz, 1H; H-8), 5.29 (ddd, J3ax,4 =10.0, J3eq,4 =4.5, J4,5
10.0 Hz, 1H; H-4), 4.81 (dd, J8,9a =2.5, J9a,9b =12.6 Hz, 1H; H-9a), 4.54 (d,
5,NH =10.0 Hz, 1H; NH), 4.48 (ddd, J4,5 =10.0, J5,6 =10.0, J5,NH =10.0 Hz,
=
J
1H; H-5), 4.28 (dd, J8,9b =6.0, J9a,9b =12.6 Hz, 1H; H-9b), 4.25 (dd, J5,6
10.0, J6,7 =2.2 Hz, 1H; H-6), 3.36 (s, 3H; COOCH3), 2.73 (dd, J3ax,3eq
=
=
4-Nitrophenyl (methyl-5-N-propanoyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
a-d-glycero-d-galacto-2-nonulopyranosylonate) (20): Compound 16
(160 mg, 305 mmol), tetrabutylammonium hydrogen sulfate (120 mg) and
4-nitrophenol (75.0 mg, 539 mmol) were dissolved in dichloromethane
(4.0 mL) and stirred vigorously with aqueous sodium hydroxide (4.0 mL,
1m) for 3 h at room temperature. After this time, the organic layer was
washed with saturated aqueous sodium hydrogencarbonate, dried
(MgSO4) and evaporated. The residue was purified by column chroma-
14.0, J3eq,4 =4.5 Hz, 1H; H-3eq), 2.02 (dd, J3ax,3eq =14.0, J3ax,4 =10.0 Hz,
1H; H-3ax), 1.92, 1.91 (eachs, 3H; COCH3), 1.90–1.76 (m, 2H; CH2-
Prop), 1.75, 1.62(eachs, 3H; COCH 3), 1.07 ppm (dd, JCH2,CH3 =7.7, 3H;
CH3-Prop); 13C NMR (101 MHz, C6D6): d=97.75 (C-2), 74.80 (C-6),
71.08 (C-8), 68.66 (C-4), 67.24 (C-7), 62.60 (C-9), 53.18 (COOCH3), 48.46
(C-5), 41.15 (C-3), 29.62 (C-2-Prop), 20.74, 20.54, 20.36, 20.26 (each
COCH3), 9.65 ppm (C-3-Prop); MALDI-TOF: m/z: calcd for
C21H30ClNO12: 523.92; found: 545.9 [M+Na]+.
tography (ethyl acetate) on silica gel. Compound 20 was obtained as an
20
546
amorphous solid. Yield: 80.0 mg (42%); [a] =+2 0 c(=1 in CHCl3);
1H NMR (400 MHz, C6D6): d=8.09 (d, J(Ha,Hb)arom =9.2Hz, 2H; H-a arom),
7.04 (d, J(Ha,Hb)arom =9.2Hz, 2H; H-b arom), 5.71 (ddd, J7,8 =8.6, J8,9a =2.3,
Methyl
5-N-butanoyl-4,7,8,9-tetra-O-acetyl-2-chloro-3,5-dideoxy-b-d-
glycero-d-galacto-2-nonulopyranosylonate (17): Compound 13 (251 mg,
484 mmol) was dissolved in acetyl chloride (65 mL). Methanol (1.2mL)
was added dropwise at 08C. The solution was stirred for 17 h and subse-
quently evaporated. Compound 17 was obtained as an amorphous solid.
Yield: 260 mg (100%); [a]25406 =+45 (c=1 in CHCl3); 1H NMR
J
8,9b =5.6 Hz, 1H; H-8), 5.49 (dd, J6,7 =1.8, J7,8 =8.6 Hz, 1H; H-7), 4.94
(ddd, J3ax,4 =12.4, J3eq,4 =4.6, J4,5 =10.4 Hz, 1H; H-4), 4.67 (dd, J5,6 =10.7,
6,7 =1.8 Hz, 1H; H-6), 4.44 (ddd, J4,5 =10.4, J5,6 =10.7, J5,NH =9.5 Hz, 1H;
H-5), 4.44 (dd, J8,9a =2.3, J9a,9b =12.7 Hz, 1H; H-9a), 4.23 (dd, J8,9b =5.6,
9a,9b =12.7 Hz, 1H; H-9b), 4.21 (d, J5,NH =9.5 Hz, 1H; NH), 3.09 (s, 3H;
COOCH3), 2.74 (dd, J3ax,3eq =13.0, J3eq,4 =4.6 Hz, 1H; H-3eq), 2.23 (dd,
3ax,3eq =13.0, J3ax,4 =12.4 Hz, 1H; H-3ax), 2.06, 1.86 (eachs, 3H; COCH3),
J
J
(400 MHz, CDCl3): d=5.35–5.28 (m, 2H; H-4/H-7), 5.20 (d, J5,NH
9.7 Hz, 1H, NH), 5.06 (ddd, J7,8 =6.6, J8,9a =2.8, J8,9b =5.8 Hz, 1H; H-8),
4.31 (dd, J8,9a =2.8, J9a,9b =12.4 Hz, 1H; H-9a), 4.25 (dd, J5,6 =10.9, J6,7
1.8 Hz, 1H; H-6), 4.12(ddd, 4,5 =10.0, J5,6 =10.9, J5,NH =9.7 Hz, 1H; H-
=
J
=
1.84–1.68 (m, 2H; CH2-Prop), 1.77, 1.59 (eachs, 3H; COCH3), 1.03 ppm
(dd, JCH2,CH3 =7.4 Hz, 3H; CH3-Prop); 13C NMR (101 MHz, C6D6): d=
126.23 (C-aarom), 119.20 (C-barom), 100.26 (C-2), 74.85 (C-6), 69.87 (C-8),
68.73 (C-4), 67.73 (C-7), 62.87 (C-9), 53.10 (COOCH3), 49.43 (C-5), 39.65
(C-3), 30.04 (C-2-Prop), 21.23, 20.86, 20.76, 20.61 (eachCOCH3),
J
5), 3.95 (dd, J8,9b =5.8, J9a,9b =12.4 Hz, 1H; H-9b), 3.77 (s, 3H; COOCH3),
2.68 (dd, J3ax,3eq =13.9, J3eq,4 =4.7 Hz, 1H; H-3eq), 2.17 (dd, J3ax,3eq =13.9,
J
3ax,4 =11.2Hz, 1H; H-3 ax), 2.02, 1.97, 1.95, 1.93 (eachs, 3H; COCH3),
1.99–1.91 (m, 2H; CH2a-But), 1.51–1.44 (m, 2H; CH2b-But), 0.82ppm
(dd, JCH2,CH3 =7.4 Hz, 3H; CH3-But); 13C NMR (101 MHz, CDCl3): d=
97.00 (C-2), 74.37 (C-6), 70.35 (C-8), 69.05 (C-4), 67.28 (C-7), 62.48 (C-
9), 54.19 (COOCH3), 48.97 (C-5), 41.07 (C-3), 39.00 (C-2-But), 21.34,
21.26, 21.17, 21.18 (each COCH3), 19.18 (C-3-But), 14.10 ppm (C-4-But).
10.11 ppm (CH3-Prop); MALDI-TOF: m/z: calcd for C27H34NO15
626.56; found: 649.0 [M+Na]+, 665.0 [M+K]+.
:
4-Nitrophenyl (methyl-5-N-butanoyl-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-
d-glycero-d-galacto-2-nonulopyranosylonate) (21): Compound 17
9018
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 9012– 9021

Synthesis of N-Acyl-Modified Sialylated Oligosaccharides
FULL PAPER
(260 mg, 483 mmol), tetrabutylammonium hydrogen sulfate (200 mg) and
4-nitrophenol (105 mg, 755 mmol) were dissolved in dichloromethane
(2.5 mL) and stirred vigorously with aqueous sodium hydroxide (2.0 mL,
1m) for 3 h at room temperature. After this time, the organic layer was
washed with saturated aqueous sodium hydrogencarbonate, dried
(MgSO4) and evaporated. The residue was purified by column chroma-
1.79, 1.78, 1.69 ppm (each s, 3H; COCH3); 13C NMR (101 MHz, C6D6):
d=125.79 (C-aarom), 118.85 (C-barom), 74.07 (C-6), 69.24 (C-8), 67.63 (C-
7), 67.40 (C-4), 62.83 (CH2-Gc), 62.21 (C-9), 52.70 (COOCH3), 49.96 (C-
5), 39.33 (C-3), 20.78, 20.45, 20.35, 20.03 ppm (eachCOCH3); MALDI-
TOF: m/z: calcd for C28H34N2O17: 670.57; found: 693.0 [M+Na]+, 709.0
[M+K]+.
tography (ethyl acetate) on silica gel. Compound 25 was obtained as an
4-Nitrophenyl (methyl-5-N-propanoyl-3,5-dideoxy-a-d-glycero-d-galacto-
2-nonulopyranosylonate) (24): Compound 20 (80 mg, 128 mmol) was dis-
solved in methanolic sodium methoxide solution (15 mL, 0.1m) and
stirred for 1.5 h at room temperature. After this time, the solution was
neutralized with Amberlite IR 120 (H+) resin and lyophilized. The resi-
due was purified by column chromatography (ethyl acetate/methanol) on
silica gel. Compound 24 was obtained as a colourless solid. Yield: 40 mg
(68%); m.p. 1038C; [a]52406 =+33 (c=1 in MeOH); 1H NMR (400 MHz,
20
amorphous solid. Yield: 177 mg (57%); [a] =+16 (c=0.5 in CHCl3);
546
1H NMR (400 MHz, C6D6): d=8.09 (d, J(Ha,Hb)arom =9.4 Hz, 2H; H-aarom),
7.05 (d, J(Ha,Hb)arom =9.4 Hz, 2H; H-barom), 5.73 (ddd, J7,8 =8.6, J8,9a =2.7,
J
8,9b =5.6 Hz, 1H, H-8), 5.50 (dd, J6,7 =2.0, J7,8 =8.6 Hz, 1H; H-7), 4.97
(ddd, J3ax,4 =12.7, J3eq,4 =4.6, J4,5 =10.9 Hz, 1H; H-4), 4.72(dd, 5,6 =10.6,
6,7 =2.0 Hz, 1H; H-6), 4.49–4.42 (m, 2H; H-5/H-9a), 4.24 (dd, J8,9b =5.6,
9a,9b =12.5 Hz, 1H; H-9b), 4.20 (d, J5,NH =9.7 Hz, 1H; NH), 3.08 (s, 3H;
J
J
J
COOCH3), 2.72 (dd, J3ax,3eq =13.2, J3eq,4 =4.6 Hz, 1H; H-3eq), 2.23 (dd,
3ax,3eq =13.2, J3ax,4 =12.7 Hz, 1H; H-3ax), 2.07, 1.85, 1.77, 1.61 (eachs, 3H;
MeOD): d=8.20 (d,
J(Ha,Hb)arom =9.4 Hz, 2H; H-aarom), 7.34 (d,
J
J
(Ha,Hb)arom =9.4, 2H; H-barom), 4.17 (dd, J5,6 =10.4, J6,7 =1.5 Hz, 1H, H-6),
COCH3), 1.90–1.49 (m, 4H; 2ꢁCH2), 0.84 ppm (dd, JCH2,CH3 =7.2Hz, 3H;
CH3); 13C NMR (101 MHz, C6D6): d=126.23 (C-aarom), 119.20 (C-barom),
74.86 (C-6), 69.89 (C-8), 68.72(C-4), 67.83 (C-7), 6.292(C-9), 53.11
(COOCH3), 49.37 (C-5), 39.69 (C-3), 38.79 (C-2-But), 21.24, 20.88, 20.76,
20.69 (eachCOCH3), 19.40 (C-3-But), 14.24 ppm (CH3-But); MALDI-
TOF: m/z: calcd for C28H36N2O15: 640.60; found: 663.3 [M+Na]+, 679.4
[M+K]+.
3.90 (dd, J4,5 =10.4, J5,6 =10.4 Hz, 1H; H-5), 3.83–3.74 (m, 3H; H-4/H-8/
H-9a), 3.72(s, 3H; COOCH ), 3.62(dd, J8,9b =5.9, J9a,9b =11.7 Hz, 1H; H-
3
9b), 3.51 (dd, J6,7 =1.5, J7,8 =8.9 Hz, 1H; H-7), 2.78 (dd, J3ax,3eq =12.7,
J
3eq,4 =4.3 Hz, 1H; H-3eq), 2.29 (ddd, J(CH2,CH3) =7.6 Hz, 2H; CH2-Prop),
2.04 (dd, 3ax,3eq =12.7, 3ax,4 =13.0 Hz, 1H; H-3ax), 1.15 ppm (dd,
(CH2,CH3) =7.6 Hz, 3H, CH3); 13C NMR (101 MHz, MeOD): d=126.71
J
J
J
(C-aarom), 121.67 (C-barom), 102.34 (C-2), 76.70 (C-6), 72.33 (C-8), 70.56
(C-7), 68.23 (C-4), 65.54 (C-9), 53.91 (COOCH3), 53.57 (C-5), 42.65 (C-
3), 30.59 (C-2-Prop), 10.71 ppm (CH3-Prop); MALDI-TOF: m/z: calcd
for C19H26N2O11: 458.42; found: 481.3 [M+K]+.
4-Nitrophenyl
(methyl-5-N-isobutanoyl-4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-a-d-glycero-d-galacto-2-nonulopyranosylonate) (22): Compound
18 (255 mg, 474 mmol), tetrabutylammonium hydrogen sulfate (200 mg)
and 4-nitrophenol (105 mg, 755 mmol) were dissolved in dichloromethane
(2.8 mL) and stirred vigorously with aqueous sodium hydroxide (2.0 mL,
1m) for 3 h at room temperature. The organic layer was washed with sa-
turated aqueous sodium hydrogencarbonate, dried (MgSO4) and evapo-
rated. The residue was purified by column chromatography (ethyl ace-
4-Nitrophenyl (methyl-5-N-butanoyl-3,5-dideoxy-a-d-glycero-d-galacto-
2-nonulopyranosylonate) (25): Compound 21 (170 mg, 265 mmol) was dis-
solved in methanolic sodium methoxide solution (15 mL, 0.1m) and
stirred for 1.5 h at room temperature. After this time, the solution was
neutralized with Amberlite IR 120 (H+) resin and lyophilized. The resi-
due was purified by column chromatography (ethyl acetate/methanol) on
silica gel. Compound 25 was obtained as a colourless solid. Yield: 103 mg
(82%), m.p. 95 8C; [a]52406 =+60 (c=1 in MeOH); 1H NMR (400 MHz,
tate) on silica gel. Compound 22 was obtained as an amorphous solid.
Yield: 133 mg (44%); [a] =+16 (c=1 in CHCl3); 1H NMR (400 MHz,
20
546
C6D6): d=8.01 (d, J(Ha,Hb)arom =9.2Hz, 2H; H-a arom), 7.04 (d, J(Ha,Hb)arom
9.2Hz, 2H; H-b arom), 5.72(ddd, 7,8 =8.7, J8,9a =2.8, J8,9b =5.9 Hz, 1H; H-
8), 5.49 (dd, J6,7 =2.0, J7,8 =8.7 Hz, 1H; H-7), 4.98 (ddd, J3ax,4 =12.7,
3eq,4 =4.6, J4,5 =10.2Hz, 1H; H-4), 4.72 (dd, 5,6 =10.2, J6,7 =2.0 Hz, 1H;
H-6), 4.46 (ddd, J4,5 =10.2, J5,6 =10.2, J5,NH =10.9 Hz, 1H; H-5), 4.43 (dd,
8,9a =2.8, J9a,9b =12.5 Hz, 1H; H-9a), 4.25 (d, J5,NH =10.9 Hz, 1H; NH),
=
J
MeOD): d=8.21 (d,
J(Ha,Hb)arom =9.2Hz, 2H; H-a arom), 7.35 (d,
J
J
J
(Ha,Hb)arom =9.2Hz, 2H; H-b arom), 4.17 (dd, J5,6 =10.4, J6,7 =1.3 Hz, 1H; H-
6), 3.92(dd, J4,5 =10.4, J5,6 =10.4 Hz, 1H; H-5), 3.84–3.77 (m, 3H; H-4/H-
8/H-9a), 3.74 (s, 3H; COOCH3), 3.63 (dd, J8,9b =5.6, J9a,9b =11.5 Hz, 1H;
H-9b), 3.54 (dd, J6,7 =1.3, J7,8 =8.5 Hz, 1H; H-7), 2.79 (dd, J3ax,3eq =12.5,
J
4.23 (dd, J8,9b =5.9, J9a,9b =12.5 Hz, 1H; H-9b), 3.08 (s, 3H; COOCH3),
2.73 (dd, J3ax,3eq =12.7, J3eq,4 =4.6 Hz, 1H; H-3eq), 2.24 (dd, J3ax,3eq =12.7,
J
3eq,4 =4.5 Hz, 1H; H-3eq), 2.28–2.20 (m, 2H; CH2-a-But), 2.05 (dd,
2J3ax,3eq =12.5 Hz, J3ax,4 =12.2 Hz, 1H; H-3ax), 1.72–1.62 (m, 2H; CH2b-
But), 0.98 ppm (dd, JCH2,CH3 =7.4 Hz, 3H; CH3); 13C NMR (101 MHz,
MeOD): d=125.18 (C-aarom), 120.17 (C-barom), 75.19 (C-6), 70.81 (C-8),
69.12(C-7), 66.70 (C-4), 64.07 (C-9), 52.38 (COO CH3), 52.31 (C-5), 41.19
(C-3), 37.93 (C-2-But), 19.18 (C-3-But), 12.96 ppm (CH3-But); MALDI-
TOF: m/z: calcd for C20H28N2O11: 472.44; found: 693.0 [M+Na]+, 709.0
[M+K]+.
J
3ax,4 =12.7 Hz, 1H; H-3ax), 2.06 (s, 3H; COCH3), 1.95–1.91 (m, 1H; CH-
But), 1.85, 1.77, 1.60 (eachs, 3H; COCH3), 1.13 (d, J(CH,CH3a)But =6.9 Hz,
3H; CH3a-But), 0.97 ppm (d,
J(CH,CH3b)But =6.9 Hz, 3H; CH3b-But);
13C NMR (101 MHz, C6D6): d=118.88 (C-aarom), 115.37 (C-barom), 74.48
(C-6), 69.52 (C-8), 68.25 (C-4), 67.41 (C-7), 62.52 (C-9), 52.67
(COOCH3), 48.95 (C-5), 39.26 (C-3), 38.79 (CH-But), 20.80, 20.41, 20.31,
20.23 (eachCOCH3), 19.62(CH a-But), 19.08 ppm (CH3b-But); MALDI-
3
TOF: m/z: calcd for C28H36N2O15: 640.59; found: 663.0 [M+Na]+.
4-Nitrophenyl (methyl-5-N-isobutanoyl-3,5-dideoxy-a-d-glycero-d-galac-
to-2-nonulopyranosylonate) (26): Compound 22 (118 mg, 184 mmol) was
dissolved in methanolic sodium methoxide solution (8 mL, 0.1m) and
stirred for 2h at room temperature. After this time, the solution was neu-
tralized with Amberlite IR 120 (H+) resin and lyophilized. The residue
was purified by column chromatography (ethyl acetate/methanol) on
silica gel. Compound 26 was obtained as a colourless solid. Yield:
56.3 mg (65%); m.p. 1158C; [a]25406 =+32( c=1 in MeOH); 1H NMR
(400 MHz, D2O): d=8.21 (d, J(Ha,Hb)arom =9.4 Hz, 2H; H-aarom), 7.28 (d,
4-Nitrophenyl
(methyl-5-N-acetoxyacetyl-4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-a-d-glycero-d-galacto-2-nonulopyranosylonate) (23): Compound
19 (92mg, 192 mmol), tetrabutylammonium hydrogen sulfate (69 mg) and
4-nitrophenol (38.0 mg, 273 mmol) were dissolved in dichloromethane
(1.0 mL) and stirred vigorously with aqueous sodium hydroxide (0.7 mL,
1m) for 3 h at room temperature. After this time, the organic layer was
washed with saturated aqueous sodium hydrogencarbonate, dried
(MgSO4) and evaporated. The residue was purified by column chroma-
tography (ethyl acetate) on silica gel. Compound 23 was obtained as an
20
546
J
(Ha,Hb)arom =9.4 Hz, 2H; H-barom), 4.17 (dd, J5,6 =10.4, J6,7 =1.5 Hz, 1H; H-
amorphous solid. Yield: 61 mg (47%); [a] =+2 1 c(=1 in CHCl3);
1H NMR (400 MHz, C6D6): d=8.09 (d, J(Ha,Hb)arom =9.4 Hz, 2H; H-aarom),
6), 3.94 (dd, J4,5 =10.5, J5,6 =10.4 Hz, 1H; H-5), 3.86–3.80 (m, 3H; H-4/H-
8/H-9a), 3.62(dd, J8,9b =6.6, J9a,9b =12.5 Hz, 1H; H-9b), 3.53 (dd, J6,7 =1.5,
7.04 (d, J(Ha,Hb)arom =9.4 Hz, 2H; H-barom), 5.72(ddd,
J
7,8 =8.6, J8,9a =3.0,
J
7,8 =8.9 Hz, 1H; H-7), 2.84 (dd, J3ax,3eq =12.8, J3ax,3eq =12.8 Hz, 1H; H-
eq), 2.55 (m, 1H; CH-But), 2.02 (dd, J3ax,4 =11.7, J3ax,3eq =12.8 Hz, 1H;
H-3ax), 1.12(d, (CH,CH3a)But =7.1 Hz, 3H; CH3a-But), 1.11 ppm (d,
(CH,CH3b)But =7.1 Hz, 3H; CH3b-But); 13C NMR (101 MHz, D2O): d=
J
8,9b =5.3 Hz, 1H; H-8), 5.55 (dd, J6,7 =1.8, J7,8 =8.6 Hz, 1H; H-7), 5.37
3
(d, J5,NH =10.2Hz, 1H; NH), 5.24 (ddd,
J3ax,4 =12.5, J3eq,4 =4.6, J4,5 =
J
10.7 Hz, 1H; H-4), 4.84 (dd, J5,6 =10.7, J6,7 =1.8 Hz, 1H; H-6), 4.42(d,
CH2-Gc =14.7 Hz, 1H; CH2a-Gc), 4.42(dd, 8,9a =3.0, J9a,9b =12.5 Hz, 1H;
H-9a), 4.34 (ddd, J4,5 =10.7, J5,6 =10.7, J5,NH =10.2Hz, 1H; H-5), 4.24 (dd,
8,9b =5.3, 9a,9b =12.5 Hz, 1H; H-9b), 4.11 (d, CH2-Gc =14.7 Hz, 1H;
J
J
J
125.91 (C-aarom), 120.26 (C-barom), 102.68 (C-2), 74.14 (C-6), 71.73 (C-4),
68.70 (C-7), 67.78 (C-8), 63.10 (C-9), 51.81 (C-5), 41.35 (C-3), 35.58 (CH-
But), 19.32(CH 3a-But), 18.79 ppm (CH3b-But); MALDI-TOF: m/z:
calcd for C20H28N2O11: 472.44; found: 495.2 [M+Na]+, 511.1 [M+K]+.
J
J
J
CH2b-Gc), 3.04 (s, 3H; COOCH3), 2.75 (dd, J3ax,3eq =13.0, J3eq,4 =4.6 Hz,
1H; H-3eq), 2.22 (dd, J3ax,3eq =13.0, J3ax,4 =12.5 Hz, 1H; H-3ax), 2.02, 1.83,
Chem. Eur. J. 2007, 13, 9012 – 9021
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9019

J. Thiem et al.
4-Nitrophenyl (methyl-5-N-glycolyl-3,5-dideoxy-a-d-glycero-d-galacto-2-
nonulopyranosylonate) (27): Compound 23 (55 mg, 82 mmol) was dis-
solved in a methanolic sodium methoxide solution (5 mL, 0.1m) and
stirred for 2h at room temperature. After this time, the solution was neu-
tralized with Amberlite IR 120 (H+) resin and lyophilized. The residue
was purified by column chromatography (ethyl acetate/methanol) on
silica gel. Compound 27 was obtained as a colourless solid. Yield: 28 mg
(74%); m.p. 1148C; [a]52406 =+46 (c=1 in MeOH); 1H NMR (400 MHz,
1H; H-1’), 4.20 (dd, J5,6a =2.0, J6a,6b =9.8 Hz, 1H; H-6a), 4.12(dd, J2’,3’ =
11.8, J3’,4’ =3.3 Hz, 1H; H-3’), 3.97–3.53 (m, 17H; H-2/H-2’/H-3/H-4/H-4’/
H-4’’/H-5/H-5’/H-5’’/H-6b/H-6’a/H-6’b/H-6’’/H-7’’/H-8’’/H-9’’a/H-9’’b), 3.45
(s, 3H; OCH3), 2.79 (dd, J3’’ax,3’’eq =12.3, J3’’eq,4’’ =4.5 Hz, 1H; H-3’’eq), 2.32
(ddd, JCH2,CH3 =7.8 Hz, 2H; CH2-Prop), 1.82(dd,
J3’’ax,4’’ =12.1, J3’’ax,3’’eq =
12.5 Hz, 1H; H-3’’ax), 1.13 ppm (dd, JCH2,CH3 =7.8 Hz, 3H; CH3-Prop);
13C NMR (101 MHz, D2O): d=103.38 (C-1’), 99.75 (C-1), 76.16 (C-3’),
75.28 (C-7’’), 73.24 (C-5’), 73.24 (C-6’’), 73.12(C-5), 71.48, 70.93, 70.63
(C-2/C-2’/C-8’’), 69.52, 69.45 (C-4/C-3), 68.55 (C-6), 68.43 (C-4’’), 67.85
(C-4’), 62.89 (C-9’’), 61.34 (C-6’), 55.62(OCH 3), 51.88 (C-5’’), 40.13 (C-
3’’), 29.59 (CH2-Prop), 9.85 ppm (CH3-Prop); MALDI-TOF: m/z: calcd
for C25H43NO19: 661.60; found: 684.1 [M+Na]+.
MeOD): d=8.20 (d,
(Ha,Hb)arom =9.3 Hz, 2H; H-barom), 4.24 (dd, J5,6 =10.2, J6,7 =1.5 Hz, 1H; H-
6), 4.05 (s, 2H; CH2-Gc), 3.97 (dd, J4,5 =10.2, J5,6 =10.2Hz, 1H; H-5),
3.90 (ddd, J3ax,4 =11.5, J3eq,4 =4.6, J4,5 =10.2Hz, 1H; H-4), 3.82(dd, J8,9a
2.8, 9a,9b =11.2Hz, 1H; H-9a), 3.78 (ddd, 7,8 =9.2, 8,9a =2.8, J8,9b
5.3 Hz, 1H; H-8), 3.73 (s, 3H; COOCH3), 3.62(dd, 8,9b =5.3, J9a,9b
11.2Hz, 1H; H-9b), 3.54 (dd,
J(Ha,Hb)arom =9.3 Hz, 2H; H-aarom), 7.35 (d,
J
=
J
J
J
J
=
=
Methyl (5-N-glycolyl-3,5-dideoxy-a-d-glycero-d-galacto-2-nonulopyrano-
sylonic acid)-(2!3)-(b-d-galactopyranosyl)-(1!6)-a-d-glucopyranoside
(34): Following method 1, donor 6 was incubated with the allolactoside
28 and TcTS for 17 h, yielding compound 34 as a colourless solid. Yield:
10 mg (60%); m.p. 1398C; [a]25406 =+31 (c=1 in H2O); 1H NMR
J
6,7 =1.5, J7,8 =9.2Hz, 1H; H-7), 2.81 (dd,
J
3ax,3eq =13.0, J3eq,4 =4.6 Hz, 1H; H-3eq), 2.05 ppm (dd, J3ax,3eq =13.0, J3ax,4 =
11.5 Hz, 1H; H-3ax); 13C NMR (101 MHz, MeOD): d=125.14 (C-aarom),
120.33 (C-barom), 74.79 (C-6), 70.95 (C-8), 68.94 (C-7), 66.47 (C-4), 63.97
(CH2-Gc), 61.51 (C-9), 52.42 (COOCH3), 52.15 (C-5), 41.06 ppm (C-3);
(400 MHz, D2O): d=4.74 (d, J1,2 =3.8 Hz, 1H; H-1), 4.44 (d, J1’,2’
=
7.9 Hz, 1H; H-1’), 4.12(dd, 5,6a =1.8, J6a,6b =11.4 Hz, 1H; H-6a), 4.07–
J
3.44 (m, 18H; H-2/H-2’/H-3/H-3’/H-4/H-4’/H-4’’/H-5/H-5’/H-5’’/H-6b/H-
6’a/H-6’b/H-6’’/H-7’’/H-8’’/H-9’’a/H-9’’b), 3.36 (s, 3H; OCH3), 2.72 (dd,
MALDI-TOF: m/z: calcd for C18H24N2O12
[M+Na]+, 499.9.0 [M+K]+.
: 460.39; found: 484.0
J
J
3’’ax,3’’eq =12.5, J3’’eq,4’’ =4.6 Hz, 1H; H-3’’eq), 1.74 ppm (dd, J3’’ax,4’’ =12.1,
Methyl (5-acetamido-3,5-dideoxy-a-d-glycero-d-galacto-2-nonulopyrano-
sylonic acid)-(2!3)-(b-d-galactopyranosyl)-(1!6)-a-d-glucopyranoside
(29): Following method 1, donor 1 was incubated with the allolactoside
28 and TcTS for 17 h, yielding compound 29 as a colourless solid. Yield:
14 mg (87%), m.p. 1428C; [a]25406 =+33 (c=1 in H2O); 1H NMR
3’’ax,3’’eq =12.5 Hz, 1H; H-3’’ax); 13C NMR (101 MHz, D2O): d=103.41
(C-1’), 100.16 (C-2’’), 99.77 (C-1), 76.19 (C-3’), 75.32(C-7 ’’), 73.27 (C-5’),
72.96 (C-6’’), 72.42 (C-5), 71.51, 70.96, 69.55 (C-2/C-2’/C-8’’), 69.50 (C-4),
68.58 (C-6), 68.45 (C-3), 68.34 (C-4’’), 67.86 (C-4’), 62.89 (C-9’’), 61.38
(CH2-Gc), 61.37 (C-6’), 55.65 (OCH3), 51.76 (C-5’’), 40.18 ppm (C-3’’);
MALDI-TOF: m/z: calcd for C24H41NO20: 663.58; found: 686.4 [M+Na]+
.
(400 MHz, D2O): d=4.85 (d, J1,2 =1.8 Hz, 1H; H-1); 4.54 (d, J1’,2’
=
8.0 Hz, 1H; H-1’), 4.22 (dd, J5,6a =2.0; J6a,6b =11.8 Hz, 1H; H-6a), 4.14
(dd, J2’,3’ =9.8, J3’,4’ =3.3 Hz, 1H; H-3’), 4.00 (m, 17H; H-2/H-2’/H-3/H-4/
H-4’/H-4’’/H-5/H-5’/H-5’’/H-6b/H-6’a/H-6’b/H-6’’/H-7’’/H-8’’/H-9’’a/H-
9’’b), 3.47 (s, 3H; OCH3), 2.81 (dd, J3’’ax,3’’eq =12.3, J3’’eq,4’’ =4.5 Hz, 1H; H-
3’’eq), 2.08 (s, 3H; COCH3), 1.84 ppm (dd,
J3’’ax,4’’ =12.1, J3’’ax,3’’eq =
12.3 Hz, 1H; H-3’’ax); 13C NMR (101 MHz, D2O): d=103.39 (C-1’),
100.10 (C-2’’), 99.76 (C-1), 76.17 (C-3’), 75.29 (C-7’’), 73.25, 73.23 (C-5’/C-
6’’), 72.15 (C-5), 71.49, 70.94, 69.52 (C-2/C-2’/C-8’’), 69.45 (C-4), 68.69 (C-
3), 68.56 (C-6), 68.41 (C-4’’), 67.86 (C-4’), 62.91 (C-6’), 61.36 (C-9’’), 55.63
(OCH3), 52.03 (C-5’’), 40.09 (C-3’’), 22.40 ppm (COCH3); MALDI-TOF:
Acknowledgement
Support of this work by the Deutsche Forschungsgemeinschaft (SFB 470,
A5) and the Fonds der Chemischen Industrie is gratefully acknowledged.
m/z: calcd for C24H41NO19
[M+K]+.
:
647.58; found: 670.4 [M+Na]+, 686.3
[1] R. Schauer, J. P. Kamerling, New Comp. Biochem. 1997, 29b, 243–
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[2] B. Neubacher, D. Schmidt, P. Ziegelmüller, J. Thiem, Org. Biomol.
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382, 233–240.
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ChemBioChem 2006, 7, 896–899.
Methyl (5-acetamido-9-O-butanoyl-3,5-dideoxy-a-d-glycero-d-galacto-2-
nonulopyranosylonic acid)-(2!3)-(b-d-galactopyranosyl)-(1!6)-a-d-glu-
copyranoside (30): Following method 1, donor 2 was incubated with the
allolactoside 28 and TcTS for 17 h, yielding compound 30 as a colourless
20
546
solid. Yield: 14 mg (78%); m.p. 1128C; [a] =+11 (c=1 in H2O);
1H NMR (400 MHz, D2O): d=4.80 (d, J1,2 =3.6, 1H; H-1), 4.49 (d, J1’,2’
=
8.1 Hz, 1H; H-1’), 4.39 (dd, J8’’,9’’a =2.3, J9’’a,9’’b =11.7 Hz, 1H; H-9’’a), 4.25
(dd, J8’’,9’’b =5.6, J9’’a,9’’b =11.7 Hz, 1H; H-9’’b), 4.18 (dd, J5,6a =2.0, J6a,6b
=
11.4 Hz, 1H; H-6a), 4.09 (dd, J2’,3’ =9.9, J3’,4’ =3.1 Hz, 1H; H-3’), 4.05
(ddd, J7’’,8’’ =5.6, J8’’,9’’a =2.3, J8’’,9’’b =5.6 Hz, 1H; H-8’’), 3.94–3.63 (m, 11H;
H-3/H-4’/H-4’’/H-5/H-5’/H-5’’/H-6b/H-6’a/H-6’b/H-6’’/H-7’’), 3.60–3.55 (m,
2H; H-2/H-2 ’’), 3.52(dd,
J3,4 =9.4, J4,5 =9.4 Hz, 1H; H-4), 3.42(s, 3H;
OCH3), 2.76 (dd, J3’’ax,3’’eq =12.5, J3’’eq,4’’ =4.6 Hz, 1H; H-3’’eq), 2.41 (dd,
J
CH2,CH2 =7.4, 2H; CH2(2)-But), 2.03 (s, 3H; COCH3), 1.79 (dd, J3’’ax,3’’eq
12.5, J3’’ax,4’’ =12.2 Hz, 1H; H-3’’ax), 1.69 (sext, JCH2,CH2 =7.4, JCH2,CH3
7.6 Hz, 2H; CH2(3)-But), 0.92ppm (dd,
=
=
J
CH2,CH3 =7.6 Hz, 3H; CH3(4)-
But); 13C NMR (101 MHz, D2O): d=103.41 (C-1’), 100.09 (C-2’’), 99.76
(C-1), 76.18 (C-3’), 75.32(C-7 ’’), 73.25 (C-5’), 73.01 (C-6’’), 71.52(C-5),
70.97, 69.78, 69.78 (C-2/C-2’/C-8’’), 69.51 (C-4), 68.67 (C-3), 68.60 (C-6),
68.37 (C-4’’), 67.80 (C-4’), 65.70 (C-9’’), 61.38 (C-6’), 55.64 (OCH3), 52.04
(C-5’’), 40.16 (C-3’’), 36.11 (C-2-But), 22.39 (COCH3), 18.73 (C-3-But),
13.21 ppm (C-4-But); MALDI-TOF: m/z: calcd for C28H47NO20: 717.67;
found: 740.2[ M+Na]+.
Methyl (5-N-propanoyl-3,5-dideoxy-a-d-glycero-d-galacto-2-nonulopyra-
nosylonic acid)-(2!3)-(b-d-galactopyranosyl)-(1!6)-a-d-glucopyrano-
side (31): Following method 1, donor 3 was incubated with the allolacto-
side 28 and TcTS for 17 h, yielding compound 31 as a colourless solid.
[13] M. F. Amaya, A. G. Watts, I. Damager, A. Wehenkel, T. Nguyen, A.
Buschiazzo, G. Paris, A. C. Frasch, S. G. Withers, P. M. Alzari, Struc-
ture 2004, 12, 775–784.
20
Yield: 7.4 mg (32%); m.p. 103 8C; [a] =+16 (c=1 in H2O); 1H NMR
546
(400 MHz, D2O): d=4.82(d,
J1,2 =3.8, 1H; H-1), 4.52(d, J1’,2’ =8.0 Hz,
9020
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 9012– 9021

Synthesis of N-Acyl-Modified Sialylated Oligosaccharides
FULL PAPER
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Received: March 20, 2007
Published online: August 6, 2007
Chem. Eur. J. 2007, 13, 9012 – 9021
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
9021