Synthesis of α-(2 → 5)Neu5Gc oligomers

Article abstract of DOI:10.1002/chem.200390101

A facile synthesis of the sialic acid oligomers α-(2 → 5)NeuSGc (1) is presented. Monosaccharides 2-4 with suitable functionality were used as the building blocks. After selective removal of the paired carboxyl and amine protecting groups, the fully prote

Full text of DOI:10.1002/chem.200390101

FULL PAPER
Synthesis of a-(2 ! 5)Neu5Gc Oligomers
Chien-Tai Ren,[a] Chien-Sheng Chen,[b] Yi-Ping Yu,[c] Yow-Fu Tsai,[a] Ping-Yu Lin,[d]
Yu-Ju Chen,[d] Wei Zou,[e] and Shih-Hsiung Wu*[a, c, f]
Abstract: A facile synthesis of the sialic
acid oligomers a-(2 ! 5)Neu5Gc (1) is
presented. Monosaccharides 2 4 with
suitable functionality were used as the
building blocks. After selective removal
of the paired carboxyl and amine pro-
tecting groups, the fully protected
oligomers were assembled through con-
secutive coupling of the building blocks
by well established peptide coupling
techniques. By this approach, fully pro-
tected oligomers as large as an octasac-
charide were synthesized. Deprotection
of these fully protected oligomers was
conducted in two steps (LiCl in refluxing
pyridine and 0.1n NaOH) to afford the
desired products in high yield. Enzymat-
ic degradation of the octamer with
neuraminidase, monitored by capillary
electrophoresis (CE), was also accom-
plished. The stepwise exo-cleavage ad-
ducts were all well separated and iden-
tified in the CE spectrum. The strategy
described here for solution-phase syn-
thesis also provides the basis for future
solid-phase synthesis of poly-a-(2 ! 5)-
Neu5Gc.
Keywords: carbohydrates ¥ electro-
phoresis
acids
¥ neuraminidase ¥ sialic
the acetyl moiety of Neu5Ac by a hydroxy group, is one of
these Neu5Ac derivatives. Neu5Gc has been found as a
component of all types of glycoconjugates in animals of the
deuterostomate lineage.[2] The formation of CMP-Neu5Gc in
vivo is through the introduction–by the action of a CMP-
Neu5Ac hydroxylase (monooxygnease), a cytosolic enzyme–
of a single oxygen atom to the N-acetyl group of CMP-
Neu5Ac.[3] According to previous reports,[3, 4] this is the only
biosynthetic pathway for the production of Neu5Gc in
biological systems. A mutation resulting in the inactivation
of this enzyme in the human genome can explain the almost
Introduction
Sialic acids comprise a family of more than 40 acidic sugars. N-
Acetylneuraminic acid (Neu5Ac) is the most common sialic
acid and seems to be the precursor for all other neuraminic
acid derivatives.[1] N-Glycolylneuraminic acid (Neu5Gc),
formed by the substitution of one of the hydrogen atoms in
[a] Prof. Dr. S.-H. Wu, Dr. C.-T. Ren, Dr. Y.-F. Tsai
Institute of Biological Chemistry
Academia Sinica, Taipei (Taiwan)
Fax : (‡886)2-2653-9142
[5]
complete lackof Neu5Gc in human tissues.
E-mail: shwu@gate.sinica.edu.tw
In the marine deuterostomes, including sea urchins, starfish,
and sea cucumbers, Neu5Gc is often the main sialic acid and is
widely distributed in glycoproteins and gangliosides, suggest-
ing that it is involved in a number of important biological
functions in these marine invertebrates.[6] The N-glycolyl
group of Neu5Gc can serve as an acceptor site for O-
acetylation, O-methylation, and glycosylation to form some
unusual compounds.[7, 8] Besides the a-(2 ! 8)- or a-(2 ! 9)-
linked Neu5Ac polymers found in microbial and vertebrate
neural cells, a polysialic acid (PSA) chain with Neu5Gc
residues ketosidically linked to the glycolyl group of NeuGc
[(!5-OglycolylNeu5Gca2 ! )n, called poly-a-(2 ! 5)-N-glyco-
lylneuraminic acid (NeuGc)] (1), has been isolated from the
jelly coat of sea urchin egg and found to play an important
role in the fertilization of eggs.[8] In order to investigate the
biosynthesis of this kind of PSA and the mechanism of
fertilization involving the PSA, a-(2 ! 5)Neu5Gc oligomers
were synthesized as described below.
[b] C.-S. Chen
Department of Chemistry
National Taiwan University
Taipei (Taiwan)
[c] Prof. Dr. S.-H. Wu, Dr. Y.-P. Yu
Graduate Institute of Life Science
National Defense Medical Center
Taipei (Taiwan)
[d] P.-Y. Lin, Prof. Dr. Y.-J. Chen
Institute of Chemistry
Academia Sinica
Taipei (Taiwan)
[e] Dr. W. Zou
Institute for Biological Sciences
National Research Council of Canada
Ottawa (Canada)
[f] Prof. Dr. S.-H. Wu
Graduate Institute of Biochemical Science
National Taiwan University
Taipei (Taiwan)
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S.-H. Wu et al.
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Results and Discussion
Because of its linear structure,
oligo-a-(2 ! 5)Neu5Gc (com-
pound 1) should be accessible
from three suitable monosac-
charides: namely, the non-re-
ducing end building block 2,
the internal building block 3,
and the reducing end unit 4[9a]
(Scheme 1). Compounds 2
4
can be regarded as amino acid
equivalents. By this synthetic
strategy the stereoselective sia-
lylation[9b] should be performed
at an early stage to avoid the
use of two sialic acid intermedi-
ates (donor and acceptor) and
the potential difficulty in sepa-
ration of anomeric oligomers.
More importantly, our ap-
proach essentially translates a
normally problematic oligosac-
charide synthesis into a peptide
synthesis by assembling these
building blocks through amide
bond linkages by use of well
established peptide coupling
techniques.[10]
Scheme 1. Retrosynthesis of a-(2 ! 5)Neu5Gc oligomers 1.
As it was to be employed for
sequential chain elongation, the
internal building block 3 was
designed with a pair of carboxyl
and amine protecting groups
that could be selectively re-
moved under mild conditions.
The N-(2,2,2-trichloroethoxy)-
carbonyl (N-Troc) group was
chosen to protect amino moi-
eties because N-Troc is compat-
ible with both sialylation and
amide condensation and can be
easily removed by zinc powder
under acidic conditions. On the
other hand, the benzyl ester in 2
and 3 can be converted into the
corresponding carboxylic acid
by catalytic hydrogenation.
Preparation of monosaccharide
Scheme 2. Synthesis of the amino building blocks.
building blocks: The protected
building blocks (2 4) were all
prepared from thiosialoside 5[9a] (Scheme 2), which had
previously been prepared from 6.[11] Treatment of 5 with
benzyl glycolate in the presence of N-iodosuccinimide (NIS)/
TfOH[12] at À258C afforded, after chromatographic separa-
tion, the desired a-anomer 3 as a major product (67%),
together with the b-anomer 3b (19%). Similarly, 4 was
obtained by treatment of 5 with benzyl alcohol in 66% yield,
together with 14% of the b-anomer 4b. One of the major
difficulties encountered in the previous synthesis had been in
obtaining the desired sialosides with high anomeric purity.
The N-Troc[14] group introduced here appears to facilitate the
separation of two sialoside anomers by silica gel chromatog-
raphy, which allowed us to obtain pure 3 and 4 on multigram
scales. In order to prepare building block 2, the N-Troc group
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a-(2 ! 5)Neu5Gc Oligomers
1085 1095
in 3 was first removed with Zn/
HOAc to give the amine 7
(76%), which was in turn treat-
ed with acetoxyacetyl chloride
and Hunig×s base in CH2Cl2 at
08C to afford 2 in 96% yield.
However, we prepared 2 more
practically from 3 in 80% yield
by direct acetoxyacetylation of
the crude 7 (Scheme 3), to min-
imize the O N acetyl migra-
tion observed in the process
after the removal of N-Troc.
Although the expected 7 was
indeed the major product
(76%) from 3, we also isolated
N-acetylated 8 in 8% yield, an
intramolecular O N acetyl mi-
gration apparently having tak-
en place. The same migration
was also observed in the re-
moval of N-Troc from 4, which
led to the major product 9
Scheme 3. Synthesis of the carboxy building blocks.
(84%) and a by-product 10 (5%) as shown in Scheme 2. It
was speculated that the regioselective migration is caused by
the proximity of the 8-OAc group and the 5-NH2 group in
compounds 7 and 9. Furthermore, it is noteworthy that
spontaneous O N acetyl migrations were also observed in
the pure compounds 7 and 9[15, 16] even during storage at
À208C, resulting in about 50% formation of 8 and 10,
respectively, within two months. Protected building blocks 2
and 3, after catalytic hydrogenation (Pd/C, H2) in ethyl
acetate, were converted in
the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodi-
imide (EDC; 1.5 equiv) and activating reagent HOBt[17]
(0.2 equiv), which led to the formation of an amide bond
between the 5-NH2 group of 9 and the aglycon carboxylic
group of 12 and afforded the fully protected disaccharide 13 in
54% yield based on 4 (Scheme 4). For further elongation, the
N-Troc moiety in 13 was removed (Zn/HOAc) to generate
amine 14, which was in turn again coupled with 12 under same
conditions to give the corresponding trisaccharide 15 in 58%
quantitative yield into 11 and
12, respectively, each with a
free carboxylic group for amide
coupling.
With building blocks 7, 9, 11,
and 12 to hand we have two
coupling routes towards a-(2 !
5)Neu5Gc oligomers, either by
construction from the non-re-
ducing end, by use of 7 and 11,
or by starting from the reducing
end, with 9 and 12. We decided
to try both routes for the syn-
theses of two tetramers (17 and
27) and to perform a final [4‡4]
coupling to provide an octasac-
charide 28 (see Scheme 4 and
Scheme 5). In the process, we
would be able to evaluate the
overall efficiency of both pro-
cedures.
Assembly of the oligosacchar-
ides from the reducing end:
Condensation of amine 9 and
Scheme 4. Assembly of the oligomers from the reducing end.
12 was performed in CH3CN in
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S.-H. Wu et al.
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yield. After repetition of the above procedure (i.e., N-Troc
removal and amide coupling), tetrasaccharide 17 was ob-
tained from 15 (56%) in two steps. To terminate the
elongation cycle, the unstable amines (14, 16, and 18) derived
from removal of the N-Troc group were converted into
acetoxyacetamides 19 21 in 72 74% yields by use of
acetoxyacetyl chloride and Hunig×s base. As previously
discussed with compounds 7 and 9, there was also significant
able to perform a convergent coupling. Thus, the N-Troc
group of 17 was removed to produce 18, which was condensed
with 27 to provide octamer 28 in 49% yield (two steps). The
major difficulty encountered was the purification of these
oligomers, more specifically the amines, due to O-N acetyl
migration.[18] Fortunately, the fully protected oligomers could
be isolated in high purity by crystallization. On comparison of
the two procedures it is clear that the construction of a-(2 !
5)-linked Neu5Gc oligomers from the non-reducing end gives
a higher overall coupling yield. Such difference is very
probably the result of the lower yield in the N-Troc removal
step because of the acetyl migration. In addition, the use of
amine 7 as a building blockinstead of the generation of amine
groups on oligomers for each elongation step also simplified
the purification.
O
N acetyl migration associated with these oligomer inter-
mediates, which not only made their purification difficult but
also effectively prevented further elongation. To minimize
these adverse effects these amine compounds were used
immediately in the crude state in the coupling reactions,
without chromatographic purification.
Assembly of the oligosaccharides from the non-reducing end:
The alternative route for elongation, with carboxylic acid 11
and amine 7, was also examined (Scheme 5). The coupling of 7
and 11 (EDC/HOBt) gave the desired disaccharide 22 (79%).
The benzyl ester protecting group was removed by catalytic
hydrogenolysis (Pd-C/H2) to afford the carboxylic acid 23 for
further coupling with 7. By repetition of this deprotection and
coupling cycle, trimer 24 and tetramer 26 were obtained in
yields of about 75% from 23 and 25, respectively. For the
introduction of a reducing end monosaccharide unit, the
carboxylic acid 25, derived from 24, was coupled with 9 to give
the fully protected tetramer 21 (59%).
Deprotection of the fully protected oligo-a-(2 ! 5)Neu5Gc
compounds: To complete the synthesis, an efficient depro-
tection method for these protected oligomers was required. A
model study on a protected disaccharide 29 showed that the
Zemplen removal of acetyl groups resulted in two positional
isomers: the desired product 31 and the amide migration
product 32 in a ratio of 1:4 (Scheme 6). Both structures were
characterized by various NMR techniques. By HMBC, the
heteronuclear cross-signal pairs 2J(C,H) A-NH/B-C1 and
3J(C,H) B-CH3/B-C22 were observed for 32 (Figure 1). This
compound was probably formed through a cyclic imide
intermediate 30 (Scheme 6), followed by a nucleophilic attack
on the carbonyl group of the glyocolyl amide. Because the
glycolyl amide is sterically more
With two tetrasaccharide fragments to hand, one carboxylic
acid 27 and the other N-Troc-protected amine 17, we were
accessible than the C1 amide
carbonyl group, competitive ring-
opening by NaOMe gave the
undesired 32 as the major product.
As an alternative (Scheme 7),
we first removed the methyl ester
groups in 19, 21, and 28 with LiCl
in refluxing pyridine,[19] to obtain
the corresponding lithium salts
33 35 (86 89%), which effec-
tively prevented cyclization and
subsequent amide migration. Ba-
sic hydrolysis of 33 35 with 0.1n
NaOH followed, to remove the
O-acetyl groups and to afford
sodium salts 36 38 in 87 89%
yield after purification on Sepha-
dex LH-20.
Characterization of synthetic a-
(2 ! 5)Neu5Gc oligomers with
neuraminidase: As one of the
exo-glycosidases, neuraminidase
recognizes the carboxyl group of
sialic acid and specifically releas-
es the a-linked sialic acid from
the non-reducing terminal.[20] We
should therefore be able to char-
acterize the synthetic oligomers
Scheme 5. Assembly of the fully protected oligomers from the non-reducing end.
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a-(2 ! 5)Neu5Gc Oligomers
1085 1095
the dimer and tetramer derived
from neuraminidase degrada-
tion. Within 24 h, octamer 38
was completely converted to
Neu5Gc (peaki) by neuramini-
dase. This stepwise digestive
process clearly confirms both
the purity and the a configura-
tion of octamer 38.
In summary, we have effi-
ciently synthesized a-(2 ! 5)-
Neu5Gc oligomers of up to
eight repeating units, by a proc-
ess in which the amide linkage,
rather than the glycosidic bond,
was assembled by peptide cou-
pling techniques. The method
should be applicable to the
construction of higher oligom-
ers. Access to these oligosac-
charides should allow us to
study further the biological
roles played by poly-a-(2 ! 5)-
Neu5Gc and to identify the
sialyltransferase involved in
the biosynthesis of Neu5Gc
PSA.
Scheme 6. Acyl migration during basic deprotection.
Experimental Section
General: NMR spectra were recorded
with Bruker AM 400 (400 MHz) and
Bruker DMX 500 (500 MHz) spec-
trometers. Assignment of 1H NMR
spectra was achieved by 2D methods
(COSY, NOESY, HMQC, HMBC).
Chemical shifts are expressed in ppm
with residual CHCl3 or CHD2OD as
reference. High-resolution FAB-MS
were recorded with a JEOL SX-120
mass spectrometer. Matrix-assisted la-
ser desorption ionization time-of-
flight (MALDI-TOF) spectra were
obtained on a Voyager-DETM mass
spectrometer. Reactions were moni-
tored by TLC on aluminaor glass
plates coated with silica gel (60 F254
,
Merck) and visualized either by use of
UV light or by charring with a molyb-
date solution (a 0.02m solution of
Figure 1. Partial HMBC spectra (500 MHz) of 32 in [D6]DMSO.
ammonium cerium sulfate dihydrate
and ammonium molybdate tetrahy-
drate in aqueous 10% H2SO4). Column chromatography was performed
on silica gel 60 (Merck70 230 mesh). Methanol was dried by heating
under reflux with magnesium methoxide and distilled immediately before
use. Dioxane, acetonitrile, and pyridine were freshly distilled under N2 over
CaH2. Molecular sieves (4 ä) were activated in vacuo at 3008C (16 h) and
stored under N2. Neuraminidase from Anthrobacter ureafaciens was
purchased from Nacalai Tesque (Kyoto, Japan).
such as 38 by use of neuraminidase and thus verify the a
configuration. High-performance capillary electrophoresis
(HPCE)[21] should allow the determination of the number of
repeating units. As shown in Figure 2, neuraminidase degrad-
ed a-(2 ! 5)Neu5Gc octamer 38 (peaka) into heptamer
(peakb), hexamer (peakc), pentamer (peakd), tetramer 37
(peake), trimer (peakf), dimer 36 (peakg), and monomer-
OBn (peakh). Each peak was compared with authentic
a-(2-8)Neu5Ac oligomers by coinjection, and synthetic
a-(2 ! 5)Neu5Gc oligomers 36 and 37 were used to identify
General procedure for the glycosidation reactions of thioglycoside 5 with
benzyl glycolate and benzyl alcohol–preparation of the building blocks 3
and 4:[9a]
A mixture of thioglycoside 5 (6.484 g, 9.04 mmol), benzyl
glycolate (2.252 g, 13.56 mmol), and molecular sieves (4 ä, 9.0 g) in
acetonitrile (40 mL) was stirred under N2 for 1 h. After the solution had
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S.-H. Wu et al.
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filtrate was washed with saturated aqueous Na2S2O3 (15 mL) and saturated
aqueous NaHCO3 (20 mL). The organic layer was dried over MgSO4,
filtered, and concentrated under reduced pressure. The residue was
purified by flash silica gel chromatography (hexane/CH2Cl2/acetone
10:10:1) to afford the a anomer 3 (4.682 g, 67%) and the b anomer 3b
(1.327 g, 19%) as white foams.
Compound 3: 1H NMR (400 MHz, CDCl3): d ˆ 7.36 7.28 (m, 5H), 5.34 (s,
2H), 5.15 (dd, J ˆ 12.3, 23.6 Hz, 2H), 5.04 (ddd, J ˆ 12.4, 10.4, 4.6 Hz, 1H),
4.90 (d, J ˆ 10.6 Hz, 1H), 4.87 (d, J ˆ 12.0 Hz, 1H), 4.45 (d, J ˆ 12.0 Hz,
1H), 4.34 (d, J ˆ 16.3 Hz, 1H), 4.23 (d, J ˆ 16.3 Hz, 1H), 4.22 (d, J ˆ
12.4 Hz, 1H), 4.08 (dt, J ˆ 12.4, 2.2 Hz, 1H), 4.02 (d, J ˆ 10.4 Hz, 1H),
3.72 (s, 3H), 3.60 (q, 10.4 Hz, 1H), 2.75 (dd, J ˆ 12.4, 4.6 Hz, 1H), 2.11 (s,
3H), 2.09(s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.93 (t, J ˆ 12.4 Hz, 1H) ppm;
13C NMR (100 MHz, CDCl3): d ˆ 170.63 (C), 170.37 (C), 170.10 (C), 169.88
(C), 169.08 (C), 167.39 (C), 154.04 (C), 135.41 (C), 128.53 (CH), 128.35
(CH), 98.05 (C), 95.36 (C), 74.50 (CH2), 71.93 (CH), 68.30 (CH), 67.98
(CH), 67.20 (CH), 66.64 (CH2), 62.08 (CH2), 61.90 (CH2), 52.98 (CH3), 51.56
(CH), 37.69 (CH2), 21.03 (CH3), 20.82 (CH3), 20.78 (CH3), 20.71
‡
(CH3) ppm; LRMS (FAB): m/z (%): 796 (2) [M‡Na‡2] , 794 (2)
‡
‡
‡
[M‡Na] , 774 (1) [M‡H‡2] , 772 (1) [M‡H] , 714 (6), 712 (6), 607 (1),
548 (28), 546 (28), 488 (5), 486 (5), 368 (7), 366 (7), 330 (8), 328 (8), 154 (12),
‡
137 (16), 91 (100); HRMS (FAB, [M‡H] ): found 772.1174; C30H37Cl3O16N
calcd 772.1178.
Compound 3b: 1H NMR (400 MHz, CDCl3): d ˆ 7.38 7.30 (m, 5H), 5.29
(dd, J ˆ 4.4, 1.8 Hz, 1H), 5.24 (ddd, J ˆ 11.8, 10.6, 4.9 Hz, 1H), 5.20 (ddd,
J ˆ 7.5, 4.4, 2.4 Hz, 1H), 5.16 (dd, J ˆ 25.8, 12.0 Hz, 2H), 4.86 (d, J ˆ
12.0 Hz, 1H), 4.72 (d, J ˆ 9.8 Hz, 1H), 4.66 (dd, J ˆ 12.4, 2.4 Hz, 2H),
4.45 (d, J ˆ 12.0 Hz, 1H), 4.26 (s, 2H), 4.14 (dd, J ˆ 10.6, 1.8 Hz, 1H), 3.98
(dd, J ˆ 12.4, 7.5 Hz, 1H), 3.72 (q, J ˆ 10.6 Hz, 1H), 3.72 (s, 3H), 2.52 (dd,
J ˆ 13.0, 4.9 Hz, 1H), 2.10 (s, 3H), 2.01 (s, 3H), 1.97 (s, 3H), 1.96 (s, 3H),
1.90 (dd, J ˆ 13.0, 11.8 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ
170.60 (C), 170.37 (C), 170.25 (C), 169.89 (C), 169.04 (C), 166.64 (C), 154.29
(C), 135.21 (C), 128.78 (CH), 128.72 (CH), 98.42 (C), 95.39 (C), 74.58
(CH2), 71.86 (CH), 71.25 (CH), 68.42 (CH), 68.30 (CH), 67.10 (CH2), 62.39
(CH2), 61.61 (CH2), 52.89 (CH3), 51.34 (CH), 36.96 (CH2), 20.88 (CH3),
20.81 (CH3), 20.70 (CH3) ppm; LRMS (FAB): m/z (%): 796 (1)
Scheme 7. Deprotection of the fully protected oligomers.
‡
‡
‡
‡
[M‡Na‡2] , 794 (1) [M‡Na] , 774 (1) [M‡H‡2] , 772 (1) [M‡H] ,
714 (2), 712 (2), 607 (1), 548 (30), 546 (30), 488 (5), 486 (5), 368 (8), 366 (8),
‡
330 (9), 328 (9), 137 (35), 91 (100); HRMS (FAB, [M‡H] ): found
772.1166; C30H37Cl3O16N calcd 772.1178.
Under the same glycosidation conditions,[9a] compound
5 (5.201 g,
7.25 mmol) and benzyl alcohol (1.10 mL, 10.87 mmol) afforded the a-
anomer 4 (3.407 g, 66%) and the b-anomer 4b (0.702 g, 14%) as white
foams after purification by flash silica gel chromatography (10 30%
gradient EtOAc in hexane).
General procedure for N-Troc removal–preparation of free amino
compounds 7 and 9: Freshly activated zinc dust (1.51 g) was added to a
solution of compound 3 (0.254 g, 0.33 mmol) in glacial acetic acid (3.0 mL).
The mixture was stirred at room temperature for 1.5 h, diluted with ethyl
acetate (100 mL), and filtered. The filtrate was washed with saturated
aqueous NaHCO3 solution, dried over Na2SO4, filtered, and concentrated.
The residue was purified by flash silica gel chromatography (50 100%
gradient EtOAc in hexane) to afford 7 (150 mg, 76%) and 8 (16 mg, 8%) as
white foams.
Compound 7: 1H NMR (400 MHz, CDCl3): d ˆ 7.33 7.25 (m, 6H), 5.50
(dd, J ˆ 3.5, 2.0 Hz, 1H), 5.44 (dd, J ˆ 9.4, 1.0 Hz, 1H), 5.38 (ddd, J ˆ 9.4,
3.0, 2.2 Hz, 1H), 5.15 (dd, J ˆ 22.2, 12.3 Hz, 1H), 4.66 (ddd, J ˆ 12.2, 9.9,
4.6 Hz, 1H), 4.32 (d, J ˆ 16.3 Hz, 1H), 4.25 (dd, J ˆ 12.7, 2.2 Hz, 1H), 4.20
(dd, J ˆ 12.7, 3.0 Hz, 1H), 4.20 (d, J ˆ 16.3 Hz, 1H), 3.70 (s, 3H), 3.62 (dd,
J ˆ 9.9, 1.0 Hz, 1H), 2.77 (dd, J ˆ 12.3, 4.6 Hz, 1H), 2.57 (t, J ˆ 9.9 Hz, 1H),
2.13 (s, 3H), 2.11 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 1.76 (t, J ˆ 12.3 Hz,
1H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 170.71 (C), 170.62 (C), 170.32
(C), 169.91 (C), 169.16 (C), 167.46 (C), 128.50 (CH), 128.30 (CH), 128.28
(C), 98.02 (C), 74.90 (CH), 71.80(CH), 67.66 (CH), 66.54 (CH2), 61.89
(CH2), 61.67 (CH2), 52.77 (CH3), 50.98 (CH), 37.12 (CH2), 21.04 (CH3),
21.00 (CH3), 20.72 (CH3), 20.67 (CH3) ppm; MS (MALDI-TOF): m/z:
Figure 2. The products obtained from the hydrolysis of a-(2 ! 5)Neu5Gc
octamer 38 by neuraminidase were analyzed by CE at different times
intervals (I, 0 min; II, 1 h; III, 90 mins; IV, 3 h; V, 4 h; VI, 6 h; VII, 1 day).
Peaks a h were assigned to octamer, heptamer, hexamer, pentamer,
tetramer, trimer, dimer (2 ! 5)Neu5Gc-2-OBn, and monomer Neu5Gc-2-
OBn respectively, peaki was monomer Neu5Gc-2-OH.
‡
found 598.2129; [C27H36O14N] calcd 598.2136.
been cooled to À408C, NIS (3.051 g, 13.56 mmol) and TfOH (0.15 mL,
1.71 mmol) were added successively. The reaction mixture was stirred at
À258C for 1 h, diluted with CH2Cl2 (200 mL), and filtered through a pad of
celite. The celite was washed with CH2Cl2 (3 Â 50 mL), and the combined
Compound 8: 1H NMR (400 MHz, CDCl3): d ˆ 7.40 7.25 (m, 6H), 5.33 (d,
J ˆ 9.7 Hz, 1H), 5.14 (s, 2H), 5.07 (dd, J ˆ 8.6, 2.1 Hz, 1H), 4.89 (ddd, J ˆ
11.8, 10.3, 4.8 Hz, 1H), 4.30 (s, 2H), 4.15 4.00 (m, 4H), 3.70 (dd, J ˆ 10.6,
1090
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/03/0905-1090 $ 20.00+.50/0
Chem. Eur. J. 2003, 9, No. 5

a-(2 ! 5)Neu5Gc Oligomers
1085 1095
2.1 Hz, 1H), 3.70 (s, 3H), 2.80 (dd, J ˆ 13.0, 4.9 Hz, 1H), 2.07 (s, 3H), 2.05
(s, 3H), 2.02 1.98 (m, 4H), 1.84 (s, 3H) PPM; 13C NMR (100 MHz,
CDCl3): d ˆ 170.88 (C), 170.28 (C), 168.86 (C), 135.07 (C), 128.64 (CH),
128.57 (CH), 128.42 (CH), 98.26 (C), 72.86 (CH), 69.23(CH), 68.82 (CH),
68.00 (CH), 66.95 (CH2), 64.71 (CH2), 61.54 (CH2), 53.76 (CH3), 49.02
(CH), 36.94 (CH2), 23.06 (CH3), 20.83 (CH3); MS (MALDI-TOF): m/z:
atmosphere for 6 20 h until TLC analysis indicated that the reaction had
gone to completion. The catalyst was removed by filtration through a pad
of celite, and the cake was washed with EtOAc (3 Â 20 mL). The filtrate
was concentrated under reduced pressure to give 11 as a crystalline solid
(0.394 g, 100%).
By the same procedure, 3 (0.461 g, 0.58 mmol) afforded 12 as a crystalline
solid (0.412 g, 100%).
‡
Found 598.2135; [C27H36O14N] calcd 598.2136.
Under the same deprotection conditions, compound 4 (235 mg, 0.33 mmol)
in glacial acetic acid (5.0 mL) afforded 9 (151 mg, 84%) and 10 (9 mg, 5%)
as white foams after purification by flash silica gel chromatography (50
100% gradient EtOAc in hexane).
1
Compound 11: H NMR (400 MHz, CDCl3): d ˆ 5.93 (d, J ˆ 9.7 Hz, 1H),
5.36 (ddd, J ˆ 8.4, 5.8, 2.7 Hz, 2H), 5.21 (dd, J ˆ 8.4, 2.0 Hz, 1H), 5.04 (ddd,
J ˆ 11.6, 10.2, 4.6 Hz, 1H), 4.58 (d, J ˆ 15.3 Hz, 1H), 4.35 (d, J ˆ 16.6 Hz,
1H), 4.29 (d, J ˆ 15.3 Hz, 1H), 4.27 (dd, J ˆ 12.4, 2.7 Hz, 1H), 4.24 (d, J ˆ
16.6 Hz, 1H), 4.11 (dd, J ˆ 10.7, 2.0 Hz, 1H), 4.04 (dd, J ˆ 12.4, 5.8 Hz, 1H),
3.98 (q, J ˆ 10.2 Hz, 1H), 3.81 (s, 3H), 2.70 (dd, J ˆ 13.0, 4.6 Hz, 1H), 2.17
(s, 3H), 2.13 (s, 3H), 2.03 (s, 3H), 2.01 (dd, J ˆ 13.0, 11.6 Hz, 1H) ppm;
13C NMR (100 MHz, CDCl3): d ˆ 171.48 (C), 171.01 (C), 170.81 (C), 170.36
(C), 170.30 (C), 169.67 (C), 167.81 (C), 167.61 (C), 98.33 (C), 72.46 (CH),
68.37 (CH), 67.98 (CH), 67.21 (CH), 62.75 (CH2), 62.33 (CH2), 61.74 (CH2),
53.21 (CH3), 49.53 (CH), 37.44 (CH2), 21.11 (CH3), 20.80 (CH3), 20.75
(CH3), 20.71 (CH3), 20.66 (CH3) ppm; LRMS (FAB): m/z (%): 630 (100)
Compound 9: 1H NMR (400 MHz, CDCl3): d ˆ 7.33 7.25 (m, 6H), 5.50
(dd, J ˆ 3.5, 2.0 Hz, 1H), 5.46 (dd, J ˆ 10.0, 2.0 Hz, 1H), 4.80 4.77 (m, 1H),
4.79 (d, J ˆ 8.0 Hz, 1H), 4.44 (d, J ˆ 8.8 Hz, 1H), 4.34 (dd, J ˆ 12.6, 2.0 Hz,
1H), 4.29 (dd, J ˆ 12.6, 3.5 Hz, 1H), 3.97 (d, J ˆ 10.0 Hz, 1H), 3.70 (s, 3H),
2.77 (dd, J ˆ 12.5, 4.6 Hz, 1H), 2.64 (t, J ˆ 10.0 Hz, 1H), 2.19 (s, 3H), 2.18 (s,
3H), 2.11 (s, 3H), 2.07 (s, 3H), 1.78 (t, J ˆ 12.5 Hz, 1H) ppm; 13C NMR
(100 MHz, CDCl3): d ˆ 171.09 (C), 170.68 (C), 170.42 (C), 170.08 (C),
168.05 (C), 137.34 (C), 128.17 (CH), 127.75 (CH), 127.59 (CH), 98.35 (C),
74.12 (CH), 71.21(CH), 67.99 (CH), 66.83 (CH), 66.60 (CH2), 61.98 (CH2),
52.54 (CH3), 51.17 (CH), 37.74 (CH2), 21.11 (CH3), 20.97 (CH3), 20.93
(CH3), 20.77 (CH3), 20.71 (CH3) ppm; LRMS (FAB): m/z (%): 580 (33)
‡
‡
[M‡Na] , 608 (7) [M‡H] , 548 (22), 472 (73), 430 (11), 310 (6), 254 (16),
‡
193 (12), 136 (46), 101 (31); HRMS (FAB, MH ): found 608.1818;
C24H34O17N calcd 608.1827.
‡
‡
[M‡41] , 540 (35) [M‡H] , 480 (8), 432 (5), 372 (5), 192 (8), 126 (8), 91
1
Compound 12: H NMR (400 MHz, CDCl3): d ˆ 5.32 5.40 (m, 2H), 5.08
‡
(100); HRMS (FAB, MH ): found 540.2090; C25H34O12N calcd 540.2081.
(ddd, J ˆ 12.4, 11.6, 4.6 Hz, 1H), 4.97 (d, J ˆ 9.6 Hz, 1H), 4.88 (d, J ˆ
12.1 Hz, 1H), 4.46 (d, J ˆ 12.1 Hz, 1H), 4.35 (d, J ˆ 16.7 Hz, 1H), 4.25
(dd, J ˆ 12.1, 1.8 Hz, 1H), 4.23 (d, J ˆ 16.7 Hz, 1H), 4.12 (d, J ˆ 11.6, Hz,
1H), 4.09 (dd, J ˆ 12.1, 4.5 Hz, 1H), 3.80 (s, 3H), 3.60 (q, J ˆ 11.6 Hz, 1H),
2.74 (dd, J ˆ 13.0, 4.6 Hz, 1H), 2.14 (s, 3H), 2.12(s, 3H), 2.03 (s, 3H), 2.01 (s,
3H), 1.95 (dd, J ˆ 13.0, 12.4 Hz, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d ˆ 171.58 (C), 170.81 (C), 170.36 (C), 170.19 (C), 167.46(C), 154.08 (C),
98.18 (C), 95.34 (C), 74.52 (CH2), 72.06 (CH), 68.24 (CH), 68.06 (CH), 67.20
(C), 62.20 (CH2), 61.79 (CH2), 61.34 (CH2), 53.20 (CH3), 51.57 (CH), 37.46
(CH2), 21.04 (CH3), 20.81 (CH3), 20.72 (CH3) ppm; MS (MALDI-TOF):
Compound 10: 1H NMR (400 MHz, CDCl3): d ˆ 7.34 7.25 (m, 6H), 5.24
(d, J ˆ 10.0 Hz, 1H), 5.12 (dd, J ˆ 8.5, 1.9 Hz, 1H), 4.86 (ddd, J ˆ 11.8, 10.5,
4.8 Hz, 1H), 4.80 (d, J ˆ 11.8 Hz, 1H), 4.48 (d, J ˆ 11.8 Hz, 1H), 4.20 4.03
(m, 4H), 3.92 (dd, J ˆ 10.5, 1.9 Hz, 1H), 3.76 (s, 3H), 2.75 (dd, J ˆ 13.1,
4.8 Hz, 1H), 2.13 2.06 (m, 7H), 2.02 (s, 3H), 1.86 (s, 3H) ppm; 13C NMR
(100 MHz, CDCl3): d ˆ 170.94 (C), 170.36 (C), 170.29 (C), 169.61 (C),
136.64 (C), 128.37 (CH), 127.94 (CH), 127.62 (CH), 98.73 (C), 72.85 (CH),
69.42 (CH), 68.93 (CH), 68.21 (CH), 66.58 (CH2), 64.69 (CH2), 53.52 (CH3),
49.12 (CH), 37.41 (CH2), 23.12 (CH3), 20.82 (CH3) ppm; MS (MALDI-
‡
‡
TOF): m/z: found 562.1883; [C25H33O12NNa] calcd 562.1901.
m/z: found 704.0513; [C23H30Cl3NO16Na] calcd 704.0527.
Methyl [2-(2'-benzyloxy-2'-oxoethyl)-5-acetoxyacetamido-4,7,8,9-tetra-O-
Elongation of the a-(2 ! 5)-linked oligosaccharides from reducing end
(4)–preparation of 13, 15, and 17: Freshly activated zinc dust (2.04 g) was
added to a solution of compound 4 (0.60 g, 0.84 mmol) in glacial acetic acid
(7.5 mL). The mixture was stirred at room temperature for 1.5 h, diluted
with ethyl acetate (200 mL), and filtered. The filtrate was washed with
saturated aqueous NaHCO3 solution, dried over Na2SO4, filtered, and
concentrated. Without purification, compound 12 (0.482 g, 0.71 mmol) in
acetonitrile (8.0 mL), NaHCO3 (140 mg, 1.67 mmol), HOBt (20.2 mg,
0.15 mmol), and EDC (241 mg, 1.26 mmol) were added sequentially to the
residue 9. The resulting mixture was stirred at room temperature for
acetyl-3,5-dideoxy-a-d-glycero-d-galacto-2-nonulopyranosid]onate
(2):
Freshly activated zinc dust (2.16 g) was added to a solution of compound
3 (0.585 g, 0.76 mmol) in glacial acetic acid (5.0 mL). The mixture was
stirred at room temperature for 1.5 h, diluted with ethyl acetate (100 mL),
and filtered. The filtrate was washed with saturated aqueous NaHCO3
solution, dried over Na2SO4, filtered, and concentrated. The residue was
dissolved in CH2Cl2 (4.0 mL) and cooled to 08C, and acetoxyacetyl chloride
(0.10 mL, 0.93 mmol) was added, followed by EtNiPr2 (0.25 mL,
1.44 mmol). After the mixture had been stirred for 2 h it was quenched
with saturated aqueous NaHCO3 (2.0 mL), diluted with CH2Cl2 (50 mL),
washed with saturated aqueous NaHCO3 (8 mL), dried over Na2SO4,
filtered, and concentrated. The residue was purified by flash silica gel
4
18 h. The solvent was removed under reduced pressure, and the
residue was purified by flash silica gel chromatography (20 100% gradient
EtOAc in hexane) to give compound 13 (0.548 g, 54%) as a colorless
powder.
chromatography (50 100% gradient EtOAc in hexane) to afford
2
(422 mg, 80%). Alternatively, 7 (151 mg, 0.25 mmol) was acetoxyacety-
lated under the same reaction conditions to yield 2 (168 mg, 96%).
By the same procedure, 13 (400 mg, 0.33 mmol) afforded 15 (327 mg,
58%), and 15 (262 mg, 0.16 mmol) afforded 17 (188 mg, 56%) as colorless
powders.
Compound 2: 1H NMR (400 MHz, CDCl3): d ˆ 7.35 7.29 (m, 5H), 5.85 (d,
J ˆ 9.3 Hz, 1H), 5.35 (ddd, J ˆ 8.2, 5.4, 2.9 Hz, 2H), 5.22 (dd, J ˆ 8.2, 1.3 Hz,
1H), 5.16 (dd, J ˆ 12.3, 23.9 Hz, 2H), 5.00 (m, 1H), 4.56 (d, J ˆ 15.3 Hz,
1H), 4.34 (d, J ˆ 16.4 Hz, 1H), 4.27 (d, J ˆ 15.3 Hz, 1H), 4.25 (d, J ˆ
16.4 Hz, 1H), 4.24 (dd, J ˆ 12.4, 2.9 Hz, 1H), 4.05 (dd, J ˆ 12.4, 5.4 Hz,
1H), 4.03 3.99 (m, 2H), 3.73 (s, 3H), 2.71 (dd, J ˆ 12.9, 4.7 Hz, 1H), 2.16
(s, 3H), 2.10(s, 3H), 2.09 (s, 3H), 2.02 1.96 (m, 7H) ppm; 13C NMR
(100 MHz, CDCl3): d ˆ 171.02 (C), 170.60 (C), 170.26 (C), 170.00 (C),
169.62 (C), 169.09 (C), 167.63 (C), 167.57 (C), 135.41 (C), 128.53 (CH),
128.35 (CH), 98.23 (C), 72.45 (CH), 68.39 (CH), 68.05 (CH), 67.23 (CH),
66.65 (CH2), 62.76 (CH2), 62.22 (CH2), 61.90 (CH2), 53.02 (CH3), 49.43
(CH), 37.69 (CH2), 21.01 (CH3), 20.83 (CH3), 20.74 (CH3), 20.68
Compound 13: 1H NMR (400 MHz, [D6]benzene): d ˆ 7.42 (d, J ˆ 7.4 Hz,
2H), 7.15 7.12 (m, 2H), 7.04 (t, J ˆ 7.4 Hz, 1H), 6.42 (d, J ˆ 10.0 Hz, 1H),
5.93 (dt, J ˆ 7.2, 2.7 Hz, 1H), 5.70 (dd, J ˆ 7.2, 2.2 Hz, 1H), 5.64 (ddd, J ˆ
7.8, 5.4, 2.5 Hz, 1H), 5.51 (dd, J ˆ 7.8, 1.9 Hz, 1H), 5.28 (ddd, J ˆ 12.0, 10.3,
4.7 Hz, 1H), 5.12 (d, J ˆ 12.1 Hz, 1H), 4.78 4.69 (m, 4H), 4.65 (d, J ˆ
10.2 Hz, 1H), 4.63 (d, J ˆ 10.6, Hz, 1H), 4.49 (dd, J ˆ 10.6, 2.2 Hz, 1H),
4.37 4.26 (m, 4H), 4.16 (d, J ˆ 14.5 Hz, 1H), 4.00 3.96 (m, 2H), 3.88 (q,
J ˆ 10.2 Hz, 1H), 3.48 (s, 3H), 3.18 (s, 3H), 2.93 (dd, J ˆ 12.4, 4.6 Hz, 1H),
2.58 (dd, J ˆ 12.4, 4.6 Hz, 1H), 2.19 (t, J ˆ 12.4 Hz, 1H), 2.07 (s, 3H), 2.05 (s,
3H), 1.98 (s, 3H), 1.96 (s, 3H), 1.83 (s, 3H), 1.76 (t, J ˆ 12.4 Hz, 1H), 1.74 (s,
3H), 1.72 (s, 3H), 1.61 (s, 3H) ppm; 13C NMR (100 MHz, [D6]benzene):
d ˆ 170.49 (C), 170.36 (C), 170.13 (C), 170.02 (C), 169.95 (C), 169.86 (C),
169.56 (C), 168.79 (C), 168.68 (C), 168.02 (C), 154.48 (C), 137.93 (C),
128.43(CH), 128.17 (C), 99.28 (C), 98.79 (C), 96.08 (C), 74.77 (CH2), 73.51
(CH), 73.04 (CH), 69.81 (CH), 69.45 (CH), 69.01 (CH), 68.56 (CH), 68.26
(CH), 67.48 (CH), 67.23 (CH2), 64.48 (CH2), 63.17 (CH2), 62.66 (CH2), 52.84
(CH3), 52.09 (CH3), 51.22 (CH), 49.62 (CH), 38.88 (CH2), 37.33 (CH2), 21.12
(CH3), 20.97 (CH3), 20.74 (CH3), 20.55 (CH3), 20.45 (CH3), 20.38 (CH3),
‡
‡
(CH3) ppm; LRMS (FAB): m/z (%): 720 (2) [M‡Na] , 699 (1) [M‡H‡1] ,
‡
698 (2) [M‡H] , 638 (18), 532 (7), 472 (56), 430 (4), 351 (3), 254 (9), 193 (5),
‡
137 (6), 91 (100); HRMS (FAB): found 698.2295 [M‡H] ; C31H40O17N
calcd 698.2296.
General procedure for the catalytic hydrogenation of benzyl esters–
preparation of the carboxyl compounds 11 and 12: Pd on carbon (10%,
18 mg) was added to a solution of compound 2 (0.456 g, 0.65 mmol) in
EtOAc (20 mL). The mixture was stirred under hydrogen at one
‡
20.27 (CH3) ppm; LRMS (FAB): m/z (%): 1227 (2) [M‡Na‡2] , 1225 (2)
Chem. Eur. J. 2003, 9, No. 5
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/03/0905-1091 $ 20.00+.50/0
1091

S.-H. Wu et al.
FULL PAPER
‡
‡
‡
[M‡Na] , 1207 (8) [M‡H‡2] , 1205 (7) [M‡H] , 1145 (8), 1143 (7), 1037
(CH2), 37.49 (CH2), 21.13 (CH3), 20.97 (CH3), 20.73 (CH3), 20.47 (CH3),
‡
(10), 1035 (9), 598 (6), 430 (28), 368 (6), 366 (6), 154 (55), 136 (43), 91 (100);
HRMS (FAB): found 1203.2588 [M‡H] ; C48H62Cl3O27N2 calcd 1203.2606.
20.40 (CH3), 20.14 (CH3) ppm; LRMS (FAB): m/z (%): 1151 (6) [M‡Na] ,
‡
‡
1129 (12) [M‡H] , 1069 (14), 961 (16), 598 (4), 532 (9), 472 (71), 430 (31),
307 (10), 254 (15), 154 (60), 91 (100); HRMS (FAB): found 1129.3732
Compound 15: 1H NMR (400 MHz, CDCl3): d ˆ 7.40 7.24 (m, 5H), 6.11
(d, J ˆ 9.5 Hz, 3H), 5.46 (ddd, J ˆ 8.7, 6.4, 2.8 Hz, 1H), 5.38 5.29 (m, 3H),
5.26 5.21 (m, 2H), 5.02 (ddd, J ˆ 10.3, 7.2, 3.1 Hz, 1H), 4.97 4.86 (m, 4H),
4.79 (d, J ˆ 12.0 Hz, 1H), 4.48 (d, J ˆ 12.1 Hz, 1H), 4.43 (d, J ˆ 12.0 Hz,
1H), 4.32 (dd, J ˆ 12.3, 2.9 Hz, 1H), 4.29 4.20 (m, 3H), 4.20 4.03 (m,
9H), 3.87 (s, 3H), 3.85 (s, 3H), 3.81 (d, J ˆ 14.8 Hz, 2H), 3.68 (s, 3H), 3.64
(q, J ˆ 10.3 Hz, 1H), 2.75 2.66 (m, 3H), 2.14 2.10 (m, 18H), 2.05 1.93
(m, 21H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 170.63 (C), 170.53 (C),
170.44 (C), 170.29 (C), 170.20 (C), 170.03 (C), 169.96 (C), 169.78 (C), 168.60
(C), 168.56 (C), 167.78 (C), 167.53 (C), 154.08 (C), 137.23 (C), 128.24 (CH),
127.82 (CH), 127.68 (CH), 98.63 (C), 98.55 (C), 98.39 (C), 95.36 (C), 74.58
(CH2), 73.16 (CH), 72.70 (CH), 72.42 (CH), 68.73 (CH), 68.62 (CH), 68.42
(CH), 68.23 (CH), 67.57 (CH), 67.44 (CH), 67.33 (CH), 66.87 (CH2), 63.92
(CH2), 62.66 (CH2), 62.50 (CH2), 62.08 (CH2), 53.27 (CH3), 52.62 (CH3),
51.51 (CH), 48.92 (CH), 48.66 (CH), 38.26 (CH2), 37.65 (CH2), 21.14 (CH3),
21.00 (CH3), 20.96 (CH3), 20.87 (CH3), 20.75 (CH3) ppm; MS (MALDI-
‡
[M‡H] ; C49H65O28N2 calcd 1129.3724.
Compound 20: 1H NMR (400 MHz, CDCl3): d ˆ 7.36 7.24 (m, 5H), 6.13 (t,
J ˆ 8.3 Hz, 2H), 5.85 (d, J ˆ 10.0 Hz, 1H), 5.46 (ddd, J ˆ 8.5, 6.1, 2.8 Hz,
1H), 5.36 5.29 (m, 2H), 5.26 5.20 (m, 3H), 5.02 4.86 (m, 3H), 4.81(d,
J ˆ 12.0 Hz, 1H), 4.58 (d, J ˆ 15.3 Hz, 1H), 4.42 (d, J ˆ 12.0 Hz, 1H), 4.34
4.01 (m, 15H), 3.87 (s, 3H), 3.86 (s, 3H), 3.81 (d, J ˆ 14.9 Hz, 1H), 3.80(d,
J ˆ 14.9 Hz, 1H), 3.68 (s, 3H), 2.73 2.64 (m, 3H), 2.17 (s, 3H), 2.15(s, 3H),
2.12 (s, 6H), 2.11 (s, 3H), 2.10 (s, 6H), 2.04 1.97 (m, 21H) ppm; 13C NMR
(100 MHz, CDCl3): d ˆ 170.95 (C), 170.55 (C), 170.22 (C), 170.10 (C),
169.97 (C), 169.83 (C), 169.62 (C), 168.61 (C), 167.71 (C), 137.22 (C), 128.24
(CH), 127.82 (CH), 127.69 (C), 98.56 (C), 98.56 (C),73.14 (CH), 72.92 (CH),
72.68 (CH), 68.62 (CH), 68.38 (CH), 67.94 (CH), 67.57 (CH), 67.45 (CH),
67.32 (CH), 66.87 (CH2), 63.91 (CH2), 62.80 (CH2), 62.66 (CH2), 62.52
(CH2), 62.19 (CH2), 53.34 (CH3), 53.29 (CH3), 52.63 (CH3), 49.37 (CH),
48.91 (CH), 48.68 (CH), 38.25 (CH2), 37.60 (CH2), 21.14 (CH3), 21.03 (CH3),
20.72 (CH3) ppm; MS (MALDI-TOF): m/z: found 1640.5023;
‡
TOF): m/z: found 1714.3930; [C68H88Cl3N3O40Na] calcd 1714.3905.
‡
[C69H91O41N3Na] calcd 1640.5026.
Compound 17: 1H NMR (400 MHz, CDCl3): d ˆ 7.40 7.24 (m, 5H), 6.12 (t,
J ˆ 7.9 Hz, 3H), 5.46 (ddd, J ˆ 8.5, 6.3, 2.8 Hz, 1H), 5.38 5.29 (m, 4H),
5.26 5.21 (m, 3H), 5.02 (ddd, J ˆ 10.3, 7.2, 3.1 Hz, 1H), 4.97 4.86 (m, 6H),
4.81 (d, J ˆ 12.0 Hz, 1H), 4.47 (d, J ˆ 12.1 Hz, 1H), 4.42 (d, J ˆ 12.0 Hz,
1H), 4.34 4.02 (m, 21H), 3.87 (s, 3H), 3.86 (s, 3H), 3.85 (s, 3H), 3.81(d, J ˆ
14.8 Hz, 3H), 3.68 (s, 3H), 3.64 (q, J ˆ 10.3 Hz, 1H), 2.75 2.66 (m, 4H),
2.15 2.10 (m, 25H), 2.05 1.93 (m, 27H) ppm; 13C NMR (100 MHz,
CDCl3): d ˆ 170.62 (C), 170.52 (C), 170.42 (C), 170.27 (C), 170.16 (C),
170.00 (C), 169.94 (C), 169.73 (C), 168.59 (C), 168.48 (C), 167.73 (C), 167.49
(C), 154.05 (C), 137.20 (C), 128.21 (CH), 127.80 (CH), 127.66 (CH), 98.60
(C), 98.53 (C), 98.36 (C), 95.33 (C),74.55 (CH2), 73.14 (CH), 72.67 (CH),
72.39 (CH), 68.66 (CH), 68.59 (CH), 68.35 (CH), 68.20 (CH), 67.53 (CH),
67.40 (CH), 67.30 (CH), 66.85 (CH2), 63.96 (CH2), 62.63 (CH2), 62.48 (CH2),
62.05 (CH2), 53.26 (CH3), 52.60 (CH3), 51.48 (CH), 48.89 (CH), 48.66 (CH),
38.24 (CH2), 37.57 (CH2), 21.12 (CH3), 21.00 (CH3), 20.95 (CH3), 20.85
(CH3), 20.72 (CH3) ppm; MS (MALDI-TOF): m/z: found 2203.5376;
Compound 21: 1H NMR (400 MHz, CDCl3): d ˆ 7.40 7.24 (m, 5H), 6.14 (d,
J ˆ 8.4 Hz, 2H), 6.12 (d, J ˆ 9.0 Hz, 1H), 5.85 (d, J ˆ 9.8 Hz, 1H), 5.46 (ddd,
J ˆ 8.2, 5.8, 3.4 Hz, 1H), 5.35 5.29 (m, 3H), 5.27 5.21 (m, 4H), 5.03 4.86
(m, 4H), 4.81 (d, J ˆ 12.0 Hz, 1H), 4.58 (d, J ˆ 15.3 Hz, 1H), 4.42 (d, J ˆ
12.0 Hz, 1H), 4.34 4.24 (m, 4H), 4.23 4.02 (m, 16H), 3.87 (s, 3H), 3.86 (s,
6H), 3.82 (d, J ˆ 14.8 Hz, 2H), 3.81 (d, J ˆ 14.8 Hz, 1H), 3.68 (s, 3H), 2.73
2.64 (m, 4H), 2.17 (s, 3H), 2.14 (s, 3H), 2.12 2.09 (m, 21H), 2.05 1.97 (m,
28H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 170.93 (C), 170.54 (C), 170.44
(C), 170.18 (C), 170.06 (C), 169.96 (C), 169.83 (C), 169.78 (C), 169.60 (C),
168.60 (C), 168.49 (C), 167.69 (C), 137.21 (C), 128.22 (CH), 127.81 (CH),
127.67 (CH), 98.62 (C), 98.55 (C),73.14 (CH), 72.91 (CH), 72.67 (CH), 68.62
(CH), 68.35 (CH), 67.93 (CH), 67.55 (CH), 67.42 (CH), 67.30 (CH), 66.86
(CH2), 63.88 (CH2), 62.79 (CH2), 62.64 (CH2), 62.50 (CH2), 62.17 (CH2),
53.33 (CH3), 53.28 (CH3), 52.61 (CH3), 49.36 (CH), 48.90 (CH), 48.67 (CH),
38.24 (CH2), 37.58 (CH2), 21.13 (CH3), 21.02 (CH3), 20.85 (CH3), 20.79
(CH3), 20.71 (CH3) ppm; MS (MALDI-TOF): m/z: found 2129.6506;
‡
[C88H115Cl3N4O53Na] calcd 2203.5388.
‡
[C89H118O54N4Na] calcd 2129.6508.
General procedure for the conversion of N-Troc-containing oligomers 13,
15, and 17 into fully protected a-(2 ! 5)-linked Neu5Gc oligomers–
preparation of 19 21: Freshly activated zinc dust (1.03 g) was added to a
solution of compound 13 (0.201 g, 0.17 mmol) in glacial acetic acid
(3.0 mL). The mixture was stirred at room temperature for 1.5 h, diluted
with ethyl acetate (100 mL), and filtered. The filtrate was washed with
saturated aqueous NaHCO3 solution, dried over Na2SO4, filtered, and
concentrated. The residue was dissolved in CH2Cl2 (4.0 mL) and cooled to
08C, and acetoxyacetyl chloride (0.05 mL, 0.46 mmol) was added, followed
by EtNiPr2 (0.15 mL, 0.86 mmol). After the mixture had been stirred for 2 h
it was quenched with saturated aqueous NaHCO3 (2.0 mL), diluted with
CH2Cl2 (50 mL), washed with saturated aqueous NaHCO3 (8 mL), dried
over Na2SO4, filtered, and concentrated. The residue was purified by flash
silica gel chromatography (50 100% gradient EtOAc in hexane) to afford
19 (0.136 g, 72%).
Elongation of the a-(2 ! 5)-linked oligosaccharides from non-reducing end
(11)–preparation of 22, 24, and 26: NaHCO3 (84 mg, 1.00 mmol), HOBt
(12 mg, 0.09 mmol), and EDC (142 mg, 0.74 mmol) were added sequen-
tially to a solution of compound 11 (0.30 g, 0.49 mmol) and compound 7
(0.31 g, 0.51 mmol) in acetonitrile (8.0 mL). The resulting mixture was
stirred at room temperature for 4 18 h. The solvent was removed under
reduced pressure, and the residue was purified by flash silica gel
chromatography (20 100% gradient EtOAc in hexane) to give compound
22 (0.465 g, 79%) as a colorless powder.
By the same procedure, 23 (318 mg, 0.290 mmol) afforded 24 (357 mg,
73.5%), and 25 (360 mg, 0.227 mmol) afforded 26 (366 mg, 74.5%) as
colorless powders.
Compound 22: 1H NMR (400 MHz, CDCl3): d ˆ 7.38 7.26 (m, 5H), 6.12
(d, J ˆ 10.0 Hz, 1H), 5.86 (d, J ˆ 9.8 Hz, 1H), 5.37 (ddd, J ˆ 8.2, 6.2, 2.8 Hz,
1H), 5.31 (ddd, J ˆ 8.0, 5.4, 2.4 Hz, 1H), 5.24 (dd, J ˆ 8.0, 2.0 Hz, 1H), 5.20
(dd, J ˆ 8.2, 2.1 Hz, 1H), 5.16 (dd, J ˆ 23.9, 12.3 Hz, 2H), 5.01 4.92 (m,
2H), 4.57 (d, J ˆ 15.3 Hz, 1H), 4.37 4.18 (m, 6H), 4.14 3.97 (m, 6H), 3.86
(s, 3H), 3.80 (d, J ˆ 14.9 Hz, 1H), 3.74 (s, 3H), 2.74 (dd, J ˆ 12.8, 4.6 Hz,
1H), 2.66 (dd, J ˆ 12.8, 4.6 Hz, 1H), 2.17 (s, 3H), 2.11 (s, 6H), 2.10 (s, 3H),
2.09 (s, 3H), 2.04 1.97 (m, 14H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ
170.96 (C), 170.52 (C), 170.19 (C), 169.94 (C), 169.14 (C), 168.57 (C),
167.70(C), 135.40(C), 128.53 (CH), 128.35 (CH), 98.51 (C), 98.26 (C), 72.87
(CH), 72.68 (CH), 68.63 (CH), 68.42 (CH), 67.94 (CH), 67.38 (CH), 67.29
(CH), 66.64 (CH), 63.85 (CH2), 62.78 (CH2), 62.57 (CH2), 62.17 (CH2),
61.92 (CH2), 53.34 (CH3), 52.97 (CH3), 49.34 (CH), 48.80 (CH), 37.70 (CH2),
37.56 (CH2), 21.09 (CH3), 21.04 (CH3), 20.72(CH3) ppm; LRMS (FAB): m/z
By the same procedure, 15 (200 mg, 0.12 mmol) afforded 20 (139 mg,
73%), and 17 (98 mg, 0.045 mmol) afforded 21 (70 mg, 74%) as colorless
powders.
Compound 19: 1H NMR (400 MHz, [D6]benzene): d ˆ 7.42 (d, J ˆ 7.4 Hz,
2H), 7.15 7.12 (m, 2H), 7.04 (t, J ˆ 7.4 Hz, 1H), 6.53 (d, J ˆ 10.0 Hz, 1H),
5.92 (dt, J ˆ 7.2, 2.7 Hz, 1H), 5.70 (dd, J ˆ 7.2, 2.1 Hz, 1H), 5.67 (m, 1H),
5.57 (dd, J ˆ 6.8, 2.2 Hz, 1H), 5.56 (d, J ˆ 9.1 Hz, 1H), 5.29 (ddd, J ˆ 12.1,
4.6 Hz, 1H), 5.12 5.03 (m, 2H), 4.77 4.62 (m, 4H), 4.51 4.47 (m, 2H),
4.42 4.32 (m, 4H), 4.28 (dd, J ˆ 10.6, 2.0 Hz, 1H), 4.22 4.16 (m, 2H), 3.46
(s, 3H), 3.19 (s, 3H), 2.92 (dd, J ˆ 12.4, 4.6 Hz, 1H), 2.58 (dd, J ˆ 12.4,
4.6 Hz, 1H), 2.18 (t, J ˆ 12.4 Hz, 1H), 2.08 (s, 3H), 2.03 (s, 3H), 2.00 (s,
3H), 1.96 (s, 3H), 1.90 (t, J ˆ 12.4 Hz, 1H), 1.83 (s, 3H), 1.82 (s, 3H), 1.74 (s,
6H), 1.73 (s, 3H) ppm; 13C NMR (100 MHz, [D6]benzene): d ˆ 170.94 (C),
170.48 (C), 170.35 (C), 170.16 (C), 169.64 (C), 168.82 (C), 168.70 (C), 168.16
(C), 167.62(C), 137.92 (C), 128.45(CH), 128.17 (C), 99.31 (C), 99.00 (C),
73.59 (CH), 73.54 (CH), 70.98 (CH2), 70.05 (CH), 69.87 (CH), 69.01 (CH),
68.37 (CH), 67.74 (CH), 67.20 (CH2), 64.46 (CH2), 63.23 (CH2), 62.95 (CH2),
62.74 (CH2), 52.91 (CH3), 52.13 (CH3), 49.63 (CH), 49.41 (CH), 38.86
‡
‡
(%): 1209 (8) [M‡Na] , 1187 (15) [M‡H] , 1127 (22), 961 (50), 663 (5),
532 (15), 472 (93), 430 (58), 254 (26), 154 (75), 136 (61), 91 (100); HRMS
(FAB): found 1187.3789 [M‡H] ; C51H67O30N2 calcd 1187.3779.
‡
1
Compound 23: H NMR (400 MHz, CDCl3): d ˆ 6.20 (d, J ˆ 9.4 Hz, 1H),
5.86 (d, J ˆ 9.8 Hz, 1H), 5.37 (ddd, J ˆ 8.2, 6.1, 2.2 Hz, 1H), 5.31 (ddd, J ˆ
8.1, 5.4, 2.2 Hz, 1H), 5.25 (dd, J ˆ 8.2, 1.3 Hz, 1H), 5.20 (d, J ˆ 8.1 Hz, 1H),
1092
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/03/0905-1092 $ 20.00+.50/0
Chem. Eur. J. 2003, 9, No. 5

a-(2 ! 5)Neu5Gc Oligomers
1085 1095
5.05 4.95 (m, 2H), 4.58 (d, J ˆ 15.3 Hz, 1H), 4.38 4.16 (m, 7H), 4.14
4.00 (m, 6H), 3.86 (s, 3H), 3.84 3.77 (m, 5H), 2.73 (dd, J ˆ 13.0, 4.6 Hz,
1H), 2.66 (dd, J ˆ 13.0, 4.6 Hz, 1H), 2.17 (s, 3H), 2.12 (s, 12H), 2.05 1.99
(m, 14H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 170.98 (C), 170.71 (C),
170.44 (C), 170.20 (C), 170.00 (C), 169.65 (C), 168.75 (C), 167.81 (C),
167.69(C), (C), 98.55 (C), 98.41 (C), 72.88 (CH), 72.75 (CH), 68.69 (CH),
68.53 (CH), 68.41 (CH), 67.99 (CH), 67.41 (CH), 67.32 (CH), 63.82 (CH2),
62.79 (CH2), 62.69 (CH2), 62.19 (CH2), 61.84 (CH2), 53.33 (CH3), 53.15
(CH3), 49.35 (CH), 48.94 (CH), 37.50 (CH2), 37.44 (CH2), 21.07 (CH3), 21.02
(CH3), 20.77 (CH3), 20.72(CH3) ppm; LRMS (FAB): m/z (%): 1119 (32)
(C), 170.53 (C), 170.39 (C), 170.31 (C), 170.16 (C), 170.04 (C), 169.93 (C),
169.83 (C), 169.71 (C), 169.61 (C), 168.58 (C), 167.80 (C), 167.74 (C), 167.68
(C), 167.58 (C), 98.91 (C), 98.66 (C), 98.05 (C), 97.93 (C), 73.09 (CH), 72.91
(CH), 72.62 (CH), 69.01 (CH), 68.89 (CH), 68.75 (CH), 68.68 (CH), 68.48
(CH), 68.24 (CH), 67.87 (CH), 67.36 (CH), 67.21 (CH), 64.18 (CH2), 63.99
(CH2), 63.31 (CH2), 62.78 (CH2), 62.62 (CH2), 62.47 (CH2), 62.17 (CH2),
61.03 (CH2), 53.37 (CH3), 53.25 (CH3), 53.19 (CH3), 53.10 (CH3), 49.30
(CH), 48.95 (CH), 48.32 (CH), 47.76 (CH), 37.79 (CH2), 37.67 (CH2), 37.47
(CH2), 37.07 (CH2), 21.00 (CH3), 20.69 (CH3), 20.62 (CH3) ppm; MS
‡
(MALDI-TOF): m/z: found 2097.6091; [C84H114O56N4Na] calcd 2097.6094.
‡
‡
[M‡Na] , 1097 (8) [M‡H] , 1037 (7), 961 (7), 532 (7), 472 (48), 430 (17),
Synthesis of the fully protected octamer 28: Freshly activated zinc dust
(1.0 g) was added to a solution of compound 17 (0.251 g, 0.12 mmol) in
glacial acetic acid (3.0 mL). The mixture was stirred at room temperature
for 1.5 h, diluted with ethyl acetate (100 mL), and filtered. The filtrate was
washed with saturated aqueous NaHCO3 solution, dried over Na2SO4,
filtered, and concentrated. The residue and compound 27 (0.204 g,
0.10 mmol) were dissolved in acetonitrile (4.0 mL), and NaHCO3 (19 mg,
0.23 mmol), HOBt (4 mg, 0.03 mmol), and EDC (52 mg, 0.27 mmol) were
added sequentially. The resulting mixture was stirred at room temperature
for 18 h. The solvent was removed under reduced pressure, and the residue
was purified by flash silica gel chromatography (0 10% gradient MeOH in
EtOAc) to give relatively pure compound 28 (0.274 g). This material was
recrystallized from 20% EtOAc in hexane to afford 28 (0.231 g, 49%) as a
colorless powder.
307 (15), 154 (100), 136 (70), 69 (50); HRMS (FAB): found 1097.3315
‡
[M‡H] ; C44H61O30N2 calcd 1097.3309.
Compound 24: 1H NMR (400 MHz, CDCl3): d ˆ 7.36 7.31 (m, 5H), 6.10
(d, J ˆ 8.6 Hz, 1H), 6.08 (d, J ˆ 9.6 Hz, 1H), 5.82 (d, J ˆ 9.8 Hz, 1H), 5.37
(ddd, J ˆ 8.2, 5.4, 2.9 Hz, 2H), 5.34 5.29 (m, 2H), 5.26 5.18 (m, 3H), 516
(dd, J ˆ 23.7, 12.3 Hz, 2H), 5.01 4.90 (m, 3H), 4.58(d, J ˆ 15.3 Hz, 1H),
4.35 (d, J ˆ 16.4 Hz, 1H), 4.30 4.00 (m, 18H), 3.98 (dd, J ˆ 10.8, 2.2 Hz,
1H), 3.86 (s, 6H), 3.82 (d, J ˆ 14.8 Hz, 1H), 3.81 (d, J ˆ 14.8 Hz, 1H), 3.74
(s, 3H), 2.74 (dd, J ˆ 12.8, 4.6 Hz, 1H), 2.70 (dd, J ˆ 12.8, 4.6 Hz, 1H), 2.66
(dd, J ˆ 12.8, 4.6 Hz, 1H), 2.17 (s, 3H), 2.11(s, 6H), 2.10 (s, 9H), 2.09 (s,
3H), 2.05 1.98 (m, 20H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 170.93
(C), 170.61 (C), 170.45 (C), 170.15 (C), 170.06 (C), 169.89 (C), 169.79 (C),
169.59 (C), 169.12 (C), 168.58 (C), 167.68 (C), 167.61 (C), 135.49 (C), 128.52
(CH), 128.33 (CH), 98.54 (C), 98.27 (C),73.12 (CH), 72.92 (CH), 72.71
(CH), 68.69 (CH), 68.63 (CH), 68.40 (CH), 67.92 (CH), 67.39 (CH), 67.30
(CH), 66.61 (CH2), 63.87 (CH2), 62.79 (CH2), 62.56 (CH2), 62.49 (CH2),
62.17 (CH2), 61.92 (CH2), 53.33 (CH3), 53.26 (CH3), 52.95 (CH3), 49.36
(CH), 48.82 (CH), 48.67 (CH), 37.72 (CH2), 37.58 (CH2), 21.07 (CH3), 21.01
(CH3), 20.79 (CH3), 20.70 (CH3) ppm; MS (MALDI-TOF): m/z: found
Compound 28: 1H NMR (400 MHz, CDCl3): d ˆ 7.40 7.24 (m, 5H), 6.13
(d, J ˆ 9.3 Hz, 6H), 6.11 (d, J ˆ 9.0 Hz, 1H), 5.83 (d, J ˆ 9.8 Hz, 1H), 5.46
(ddd, J ˆ 8.2, 5.8, 3.4 Hz, 1H), 5.36 5.28 (m, 7H), 5.28 5.19 (m, 8H),
5.03 4.86 (m, 8H), 4.81 (d, J ˆ 12.0 Hz, 1H), 4.58 (d, J ˆ 15.3 Hz, 1H), 4.43
(d, J ˆ 12.0 Hz, 1H), 4.34 4.24 (m, 8H), 4.23 4.02 (m, 28H), 3.87 (s, 21H),
3.82(d, J ˆ 10.9 Hz, 7H), 3.68 (s, 3H), 2.73 2.64 (m, 8H), 2.17 (s, 3H), 2.15
(s, 3H), 2.14 2.06 (m, 42H), 2.06 1.94 (m, 59H) ppm; 13C NMR
(100 MHz, CDCl3): d ˆ 170.90 (C), 170.52 (C), 170.40 (C), 170.13 (C),
170.01 (C), 169.82 (C), 169.58 (C), 168.67 (C), 168.47 (C), 167.69 (C), 137.21
(C), 128.20 (CH), 127.78 (CH), 127.64 (CH), 98.53 (C), 73.11 (CH), 72.89
(CH), 72.65 (CH), 68.61 (CH), 68.33 (CH), 67.91 (CH), 67.54 (CH), 67.39
(CH), 66.83 (CH2), 63.85 (CH2), 62.77 (CH2), 62.62 (CH2), 62.47 (CH2),
62.16 (CH2), 53.25 (CH3), 52.58 (CH3), 49.34 (CH), 48.88 (CH), 48.65 (CH),
38.22 (CH2), 37.56 (CH2), 21.10 (CH3), 21.00 (CH3), 20.77 (CH3), 20.69
(CH3) ppm; MS (MALDI-TOF): m/z: found 4085.2420; [C169H225O106N8-
‡
1698.5078; [C71H93O43N3Na] calcd 1698.5081.
Compound 25: 1H NMR (400 MHz, CDCl3): d ˆ 6.29 (br, 2H), 5.86 (d, J ˆ
9.6 Hz, 1H), 5.41 (m, 1H), 5.37 5.21 (m, 5H), 5.16 (d, J ˆ 7.5 Hz, 1H), 5.06
(m, 1H), 4.97 4.85 (m, 2H), 4.57(d, J ˆ 15.3 Hz, 1H), 4.35 4.02 (m, 21H),
3.87 (s, 3H), 3.84 (s, 3H), 3.82 (s, 3H), 3.82 3.77 (m, 2H), 2.76 (dd, J ˆ 13.0,
4.6 Hz, 1H), 2.67 (dd, J ˆ 12.8, 4.6 Hz, 1H), 2.62 (dd, J ˆ 12.8, 3.9 Hz, 1H),
2.17 (s, 3H), 2.11 2.12 (m, 12H), 2.10 (s, 9H), 2.09 (s, 3H), 2.07 1.94 (m,
21H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 170.91 (C), 170.82 (C), 170.66
(C), 170.57 (C), 170.42 (C), 170.32 (C), 170.22 (C), 170.10 (C), 170.02 (C),
169.87 (C), 169.62 (C), 168.68 (C), 168.14 (C), 167.82 (C), 167.73 (C), 167.68
(C), 98.71 (C), 98.27 (C),72.97 (CH), 72.89 (CH), 72.69 (CH), 68.68 (CH),
68.34 (CH), 68.24 (CH), 67.38 (CH), 67.33 (CH), 67.19 (CH), 63.98 (CH2),
63.61 (CH2), 62.80 (CH2), 62.55 (CH2), 62.18 (CH2), 61.50 (CH2), 53.34
(CH3), 53.21 (CH3), 53.17 (CH3), 49.14 (CH), 48.90 (CH), 48.29 (CH), 37.60
(CH2), 37.44 (CH2), 37.36 (CH2), 21.01 (CH3), 20.75 (CH3), 20.66
‡
Na] calcd 4085.2356.
Deprotection of 29 with MeONa in methanol–preparation of 31 and 32: A
methanolic sodium methoxide solution (0.05 mL, 1n) was added under N2
at room temperature to a solution of 29 (79 mg, 0.07 mmol) in dry MeOH
‡
(3.0 mL). After 2 h, Amberlite IR-120 (H ) ion exchange resin was added.
The mixture was filtered, and the resin was washed with methanol (2 Â
5 mL). The combined filtrate was concentrated, and the residue was
purified by flash silica gel chromatography (0 20% MeOH in EtOAc) to
give 31 (10mg, 17%) and 32 (36 mg, 67%) as colorless powders.
‡
(CH3) ppm; MS (MALDI-TOF) m/z: found 1608.4609; [C64H87O43N3Na]
calcd 1608.4611.
Compound 26: 1H NMR (400 MHz, CDCl3): d ˆ 7.36 7.31 (m, 5H), 6.10
(d, J ˆ 9.5 Hz, 2H), 6.08 (d, J ˆ 10.2 Hz, 1H), 5.82 (d, J ˆ 9.8 Hz, 1H), 5.37
(ddd, J ˆ 8.0, 4.8, 2.4 Hz, 1H), 5.34 5.29 (m, 3H), 5.26 5.18 (m, 4H), 516
(dd, J ˆ 23.9, 12.3 Hz, 2H), 4.98 4.90 (m, 4H), 4.58 (d, J ˆ 15.3 Hz, 1H),
4.35 (d, J ˆ 16.4 Hz, 1H), 4.30 4.21 (m, 6H), 4.19 3.96 (m, 14H), 3.86 (s,
9H), 3.81 (d, J ˆ 14.8 Hz, 2H), 3.80 (d, J ˆ 14.8 Hz, 1H), 3.73 (s, 3H), 2.74
(dd, J ˆ 12.8, 4.6 Hz, 1H), 2.72 2.64 (m, 3H), 2.17 (s, 3H), 2.11 (s, 6H),
2.10 (s, 15H), 2.09 (s, 3H), 2.05 1.98 (m, 28H) ppm; 13C NMR (100 MHz,
CDCl3): d ˆ 170.92 (C), 170.61 (C), 170.53 (C), 170.43 (C), 170.13 (C),
170.04 (C), 169.88 (C), 169.80 (C), 169.59 (C), 169.12 (C), 168.58 (C), 167.68
(C), 167.61 (C), 135.49 (C), 128.52 (CH), 128.33 (CH), 98.54 (C), 98.26
(C),73.14 (CH), 72.92 (CH), 72.71 (CH), 68.64 (CH), 68.44 (CH), 68.34
(CH), 67.92 (CH), 67.39 (CH), 67.29 (CH), 66.61 (CH2), 63.86 (CH2), 62.79
(CH2), 62.56 (CH2), 62.48 (CH2), 62.16 (CH2), 61.92 (CH2), 53.33 (CH3),
53.27 (CH3), 52.95 (CH3), 49.36 (CH), 48.82 (CH), 48.66 (CH), 37.72 (CH2),
37.58 (CH2), 21.08 (CH3), 21.02 (CH3), 20.80 (CH3), 20.70 (CH3) ppm; MS
Compound
[C31H46N2O18Na] calcd 757.2643.
31:
MS (MALDI-TOF):
m/z:
found
757.2665;
‡
Compound 32: MS
[C31H46N2O18Na] calcd 757.2643.
(MALDI-TOF): m/z: found 757.2635;
‡
For NMR data for compounds 31 and 32 see Table 1.
General procedure for deprotection of methyl esters with LiCl–prepara-
tion of the lithium salts 33 35: LiCl (21 mg, 0.50 mmol) was added to a
solution of 19 (174 mg, 0.15 mmol) in pyridine (4.0 mL). The solution was
heated at 1208C for 16 h, and the solvent was removed under reduced
pressure. The darkbrown residue was purified by chromatography over
Sephadex LH-20 with MeOH to afford the dilithium salt 33 as a light brown
powder (147 mg, 85%). By the same procedure, 21 (150 mg, 0.07 mmol)
afforded 34 (131 mg, 89%), and 28 (99 mg, 0.02 mmol) afforded 35 (87 mg,
89%) as light brown powders.
Compound 33: 1H NMR (400 MHz, D2O): d ˆ 7.56 7.44 (m, 5H), 5.57
(ddd, J ˆ 8.0, 5.3, 2.6 Hz, 1H), 5.50 (dd, J ˆ 8.1, 1.8 Hz, 1H), 5.49 5.45 (m,
1H), 5.43 (dd, J ˆ 8.4, 1.8 Hz, 1H), 5.10 (dt, J ˆ 11.4, 4.7 Hz, 2H), 4.88 (d,
J ˆ 10.9 Hz, 1H), 4.77 (d, J ˆ 10.9 Hz, 1H), 4.66 4.59 (m, 4H), 4.53 (dd,
J ˆ 10.2, 2.1 Hz, 1H), 4.44 (dd, J ˆ 12.6, 2.4 Hz, 1H), 4.38 4.32 (m, 3H),
4.12 (t, J ˆ 10.5 Hz, 1H), 4.07 (t, J ˆ 10.5 Hz, 1H), 3.98 (d, J ˆ 15.0 Hz, 1H),
2.87 (dd, J ˆ 12.4, 4.7 Hz, 1H), 2.79 (dd, J ˆ 12.4, 4.7 Hz, 1H), 2.30 (s, 3H),
2.29 (s, 6H), 2.28 (s, 3H), 2.27 (s, 3H), 2.20 (s, 3H), 2.19 (s, 3H), 2.16 (s,
‡
(MALDI-TOF): m/z: found 2187.6560; [C91H120O56N4Na] calcd 2187.6563.
Compound 27: 1H NMR (400 MHz, CDCl3): d ˆ 6.56 (br, 1H), 6.42 (br,
1H), 6.21 (d, J ˆ 10.0 Hz, 1H), 5.84 (d, J ˆ 9.8 Hz, 1H), 5.46 (m, 1H), 5.35
5.23 (m, 6H), 5.14 5.05 (m, 2H), 4.98 (dt, J ˆ 11.6, 4.6 Hz, 1H), 4.89 4.72
(m, 2H), 4.58 (d, J ˆ 15.3 Hz, 1H), 4.30 4.00 (m, 21H), 3.88 (s, 3H), 3.86 (s,
3H), 3.83 3.80 (m, 7H), 3.72 (d, J ˆ 15.3 Hz, 2H), 2.80 (dd, J ˆ 13.0,
4.6 Hz, 1H), 2.70 2.57 (m, 3H), 2.17 (s, 3H), 2.12 2.07 (m, 27H), 2.04
1.94 (m, 25H) ppm; 13C NMR (100 MHz, CDCl3): d ˆ 170.92 (C), 170.63
Chem. Eur. J. 2003, 9, No. 5
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1093

S.-H. Wu et al.
FULL PAPER
Table 1. 500 MHz 1H NMR data (300 K) and 100 MHz 13C NMR data (300 K) for the disaccharides 31 and 32. Two-dimensional COSY, NOESY, HMQC,
and HMBC experiments were used for the assignments.
1H NMR chem. shift [ppm]
13C NMR chem. shift [ppm]
31
32
31
32
unit
A
B
A
B
unit
A
B
A
B
H3eq
H3ax
H4
H5
H6
H7
H8
H9
NH
2.56
1.71
3.60
3.61
3.44
3.59
3.27
3.39
7.68
2.54
1.63
3.71
3.68
3.67
3.67
3.30
3.63
7.97
1.87
4.19, 3.98
3.73
2.55
1.65
3.71
3.73
3.76
3.28
3.68
3.65
8.01
2.71
1.47
3.50
3.50
3.50
3.34
3.55
3.41
8.05
1.89
4.24, 4.10
3.74
C1
C2
C3
C4
C5
C6
C7
C8
169.15
98.17
40.47
66.13
51.78
73.42
70.27
68.73
63.43
65.04
168.55
98.28
40.15
65.65
51.91
72.93
71.01
68.73
63.32
62.67
169.88
171.38
22.61
52.63
169.08
98.33
40.70
65.29
52.68
72.69
69.13
71.10
63.13
65.70
168.46
98.93
39.63
65.75
52.30
73.99
67.85
71.00
63.57
60.45
169.45
172.27
22.53
51.54
C9
Ac-CH3
CH2
CO2Me
C21
C22
C51
C52
CH3
4.75, 4.45
3.75
4.75, 4.45
3.64
52.63
52.67
6H), 1.99 (t, J ˆ 12.4 Hz, 1H), 1.89 (dt, J ˆ 12.4, 1.8 Hz, 1H) ppm; 13C NMR
(100 MHz, D2O): d ˆ 174.49 (C), 174.40 (C), 173.74 (C), 173.66 (C), 173.46
(C), 173.17 (C), 173.00 (C), 172.74 (C), 171.15 (C), 138.04 (C), 129.30 (CH),
129.22 (CH), 128.84 (CH), 101.60 (C), 101.01 (C), 71.81 (CH), 71.73 (CH),
71.06 (CH), 70.67 (CH), 69.75 (CH), 69.00 (CH), 68.89 (CH), 68.43 (CH),
67.75 (CH2), 63.95 (CH2), 63.35 (CH2), 63.01 (CH2), 49.58 (CH), 49.49
(CH), 38.75 (CH2), 37.86 (CH2), 21.35 (CH3), 21.08 (CH3), 21.06 (CH3),
20.94 (CH3), 20.86 (CH3), 20.63 (CH3) ppm; MS (MALDI-TOF): m/z:
found 1105.50; [C47H58O28N2Li]À calcd 1105.33.
Compound 36: 1H NMR (400 MHz, D2O): d ˆ 7.56 7.42 (m, 5H), 4.88 (d,
J ˆ 10.9 Hz, 1H), 4.65 (d, J ˆ 10.9 Hz, 1H), 4.43 (d, J ˆ 15.3 Hz, 1H), 4.24
(d, J ˆ 15.3 Hz, 1H), 4.24 (s, 2H), 4.08 3.90 (m, 12H), 3.79 3.67 (m, 4H),
2.90 (ddd, J ˆ 12.1, 7.7, 4.6 Hz, 2H), 1.93 (t, J ˆ 12.1 Hz, 1H), 1.81 (t, J ˆ
12.0 Hz, 1H) ppm; 13C NMR (100 MHz, D2O): d ˆ 176.40 (C), 174.17 (C),
173.75 (C), 173.54 (C), 137.78 (C), 129.33 (CH), 129.30 (CH), 128.88 (CH),
101.62 (C), 101.05 (C), 73.11 (CH), 72.98 (CH), 72.52 (CH), 72.20 (CH),
68.80 (CH), 68.73 (CH), 68.61 (CH), 68.56 (CH), 67.66 (CH2), 63.73 (CH2),
63.25 (CH2), 61.64 (CH2), 52.46 (CH), 52.09 (CH), 41.13 (CH2), 40.17
(CH2) ppm; MS (MALDI-TOF): m/z: found 743.42; [C29H40O19N2Na]À
calcd 743.21.
Compound 34: 1H NMR (400 MHz, D2O): d ˆ 7.56 7.44 (m, 5H), 5.54
(ddd, J ˆ 8.0, 5.3, 2.6 Hz, 1H), 5.49 5.38 (m, 7H), 5.11 5.01 (m, 4H), 4.84
(d, J ˆ 10.5 Hz, 1H), 4.74 (dd, J ˆ 10.5, 2.1 Hz, 1H), 4.70 4.53 (m, 8H),
4.49 (dd, J ˆ 12.5, 2.6 Hz, 1H), 4.42 4.26 (m, 10H), 4.11 3.87 (m, 8H),
2.86 2.71 (m, 4H), 2.26 (s, 12H), 2.25 (s, 3H), 2.24 (s, 9H), 2.23 (s, 3H),
2.16 (s, 9H), 2.15 (s, 3H), 2.14 (s, 3H), 2.13 (s, 3H), 2.12 (s, 3H), 2.11 (s,
3H), 2.10 (s, 3H), 1.95 (dt, J ˆ 12.1, 3.2 Hz, 1H), 1.83 (t, J ˆ 12.4 Hz,
1H) ppm; 13C NMR (100 MHz, D2O): d ˆ 174.40 (C), 173.73 (C), 173.40
(C), 173.01 (C), 172.68 (C), 171.13 (C), 137.96 (C), 129.25 (CH), 128.81
(CH), 101.59(C), 100.97 (C), 71.68 (CH), 70.93 (CH), 70.60 (CH), 69.63
(CH), 68.82 (CH), 68.47 (CH), 68.37 (CH), 67.76 (CH2), 63.91 (CH2), 63.30
(CH2), 63.13 (CH2), 62.97 (CH2), 49.55 (CH), 49.47 (CH), 49.27 (CH), 38.76
(CH2), 37.83 (CH2), 21.33 (CH3), 21.04 (CH3), 20.91 (CH3), 20.81 (CH3),
Compound 37: 1H NMR (400 MHz, D2O): d ˆ 7.54 7.42 (m, 5H), 4.87 (d,
J ˆ 10.9 Hz, 1H), 4.64 (d, J ˆ 10.9 Hz, 1H), 4.41 (d, J ˆ 15.2 Hz, 3H), 4.22
(d, J ˆ 15.2 Hz, 3H), 4.22 (s, 2H), 4.06 3.88 (m, 24H), 3.77 3.65 (m, 8H),
2.92 2.84 (m, 4H), 1.90 (dt, J ˆ 12.2, 4.9 Hz, 3H), 1.79 (t, J ˆ 12.0 Hz,
1H) ppm; 13C NMR (100 MHz, D2O): d ˆ 176.37 (C), 174.15 (C), 173.75
(C), 173.48 (C), 137.73 (C), 129.32 (CH), 129.29 (CH), 128.88 (CH), 101.58
(C), 101.01 (C), 73.07 (CH), 72.97 (CH), 72.48 (CH), 72.32 (CH), 72.17
(CH), 68.67 (CH), 68.57 (CH), 68.51 (CH), 68.44 (CH), 67.63 (CH2), 63.68
(CH2), 63.28 (CH2), 63.21 (CH2), 61.61 (CH2), 52.45 (CH), 52.26 (CH),
52.06 (CH), 41.09 (CH2), 40.09 (CH2) ppm; MS (MALDI-TOF): m/z:
found 1401.15; [C51H72O37N4Na3]À calcd 1401.36.
‡
‡
20.56 (CH3) ppm; MS (ESI): m/z: found 2051.93 [M‡H] ; [C85H111N4O54]
Compound 38: 1H NMR (400 MHz, D2O): d ˆ 7.52 7.42 (m, 5H), 4.84 (d,
J ˆ 10.9 Hz, 1H), 4.61 (d, J ˆ 10.9 Hz, 1H), 4.39 (d, J ˆ 15.2 Hz, 7H), 4.18
(d, J ˆ 15.2 Hz, 9H), 4.03 3.88 (m, 48H), 3.72 3.63 (m, 16H), 2.89 2.80
(m, 8H), 1.89 (t, J ˆ 12.2 Hz, 1H), 1.87 (t, J ˆ 12.2 Hz, 6H), 1.76 (t, J ˆ
12.0 Hz, 1H) ppm; 13C NMR (100 MHz, D2O): d ˆ 176.37 (C), 174.15 (C),
173.74 (C), 173.44 (C), 137.72 (C), 129.32 (CH), 129.28 (CH), 128.87 (CH),
101.58(C), 101.01 (C), 73.06 (CH), 72.96 (CH), 72.50 (CH), 72.33 (CH),
72.17 (CH), 68.66 (CH), 68.57 (CH), 68.42 (CH), 67.64 (CH2), 63.68 (CH2),
63.30 (CH2), 61.60 (CH2), 52.44 (CH), 52.25 (CH), 52.04 (CH), 40.10 (CH2),
calcd 2051.61; MS (MALDI-TOF): m/z: found 2031.75; [C83H103N4O53Li4]À
calcd 2031.61.
Compound 35: 1H NMR (400 MHz, D2O): d ˆ 7.58 7.46 (m, 5H), 5.58
5.40 (m, 16H), 5.16 5.02 (m, 8H), 4.86 (d, J ˆ 11.5 Hz, 1H), 4.76 4.53 (m,
13H), 4.52 4.27 (m, 29H), 4.15 3.89 (m, 16H), 2.88 2.73 (m, 8H), 2.30
2.22 (m, 51H), 2.20 2.13 (m, 48H), 2.01 1.92 (m, 7H), 1.86 (t, J ˆ 12.0 Hz,
1H) ppm; 13C NMR (100 MHz, D2O): d ˆ 174.45 (C), 173.76 (C), 173.53
(C), 173.46 (C), 173.19 (C), 173.06 (C), 172.71 (C), 171.16 (C), 138.06 (C),
129.33 (CH), 129.24 (CH), 129.07 (CH), 128.86 (CH), 101.61 (C), 100.97
(C), 71.75 (CH), 71.01 (CH), 70.68 (CH), 69.77 (CH), 68.91 (CH), 68.56
(CH), 67.75 (CH2), 63.96 (CH2), 63.37 (CH2), 61.59 (CH2), 49.60 (CH), 49.50
(CH), 49.30 (CH), 38.46 (CH2), 37.92 (CH2), 21.39 (CH3), 21.11 (CH3), 20.98
‡
38.52 (CH2) ppm; MS (ESI, MH ): m/z: found 2566.43; [C95H145N8O73]À
calcd 2565.79.
Neuraminidase hydrolysis: a-(2 ! 5)Neu5Gc octamer 38 (20 mg) dissolved
in 30 mL of 100 mm ammonium acetate buffer (pH 5) was digested with
neuraminidase (0.1 mU) from Anthrobacter ureafaciens at 378C for
different time intervals. The progress of hydrolysis was monitored by
HPCE at each time interval.
(CH3), 20.89 (CH3), 20.67 (CH3) ppm; MS (ESI): m/z: found 3954.25
‡
[M‡H] ; [C161H211N8O106
]
À calcd 3954.39.
General procedure for the alkaline hydrolysis of lithium salts 33 35–
preparation of the oligomer sodium salts 36 28: Compound 33 (147 mg,
0.13 mmol) was treated with aqueous NaOH (0.1m, 15 mL) at room
temperature for 3 h. The mixture was lyophilized to give the crude product
as a light brown solid. The crude product was purified by chromatography
over Sephadex LH-20 with water, and the fractions containing the product
were lyophilized to afford the disodium salt 36 as a light brown powder
(88 mg, 87%). By the same procedure, 34 (130 mg, 0.06 mmol) afforded 37
(79 mg, 88%), and 35 (87 mg, 0.02 mmol) afforded 38 (52 mg, 86%) as light
brown powders.
High-performance capillary electrophoresis chromatographic conditions
(HPCE): Capillary electrophoresis (CE) was performed on a Beckman
capillary electrophoresis system (P/ACE 2100) on a fused silica capillary
(107 cm  75 mm (inner diameter)) and carried out by applying 20 kV at
258C. Phosphate buffer (50 mm, pH 7.0) was used as the running buffer.
The spectra were monitored by their UV absorption at 200 nm. Samples
were injected into the capillary under high nitrogen pressure (1.3 bar) for
3 s. The capillary was regenerated by washing with 0.1n NaOH for 7 min
and then with double distilled water for 5 min.
1094
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Chem. Eur. J. 2003, 9, No. 5

a-(2 ! 5)Neu5Gc Oligomers
1085 1095
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Received: August 9, 2002 [F4334]
Chem. Eur. J. 2003, 9, No. 5
¹ 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/03/0905-1095 $ 20.00+.50/0
1095