Synthesis of oligogalacturonates conjugated to BSA

Article abstract of DOI:10.1016/j.carres.2004.06.012

The synthesis of three oligogalacturonates with an aldehyde spacer attached at the reducing end is described. Trigalacturonates α-D-GalpA-(1→4)- α-D-GalpA-(1→4)-α-D-GalpA-(1→O(CH₂) ₇CHO and α-D-GalpA(Me)-(1→4)-α-D-GalpA(Me)- (1→4)-α-

Full text of DOI:10.1016/j.carres.2004.06.012

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 2159–2169
Synthesis of oligogalacturonates conjugated to BSA
Mads H. Clausen and Robert Madsen*
Department of Chemistry, Building 201, Technical University of Denmark, DK-2800 Lyngby, Denmark
Received 17 December 2003; received in revised form 9 June 2004; accepted 14 June 2004
Available online 23 July 2004
Dedicated to the memory of Professor Christian Pedersen
Abstract—The synthesis of three oligogalacturonates with an aldehyde spacer attached at the reducing end is described. Trigalac-
turonates a-D-GalpA-(1fi4)-a-D-GalpA-(1fi4)-a-D-GalpA-(1fiO(CH2)7CHO and a-D-GalpA(Me)-(1fi4)-a-D-GalpA(Me)-
(1fi4)-a-D-GalpA(Me)-(1fiO(CH2)7CHO as well as hexagalacturonate a-D-GalpA-(1fi4)-[a-D-GalpA-(1fi4)]4-a-D-GalpA-
(1fiO(CH2)7CHO are prepared by stepwise coupling of galactose units followed by oxidation of the 6-positions. The a-linkages
are formed by employing n-pentenyl galactosides as glycosyl donors and N-iodosuccinimide/triethylsilyl triflate as the promoter. De-
protection furnishes the three target oligogalacturonates, which are subsequently linked to bovine serum albumin by reductive ami-
nation. These neoglycoproteins will serve as immunogens for generation of new antibodies that can be used for localization and
characterization of pectin in plants.
Ó 2004 Elsevier Ltd. All rights reserved.
Keywords: Antigen; Galactose; Neoglycoprotein; Pectin; Pentenyl glycoside
1. Introduction
synthetic oligosaccharides conjugated to bovine serum
albumin (BSA) through the reducing end aldehyde. LM5
and LM6 are specific for the galactan and arabinan side-
chains in the hairy regions of pectin. Since homogalac-
turonan is the most abundant of the pectic
polysaccharides, antibodies recognizing parts of this
structure are particularly useful. Unfortunately, it has
so far not been possible to attach small oligogalacturo-
nates directly to BSA through the reducing end alde-
hyde. As a result, we envisioned that well-defined
synthetic oligogalacturonates could be covalently linked
to BSA through a spacer and used for immunization stud-
ies. Three different target structures were chosen for these
studies: a nonesterified trigalacturonate, a nonesterified
hexagalacturonate as well as a fully methyl-esterified tri-
galacturonate. These three structures were selected based on
their difference in size and methyl esterification.
Pectin is an important constituent of the primary cell wall
of higher plants. It is composed of ꢀsmoothꢁ homogalac-
turonan regions and ꢀhairyꢁ rhamnogalacturonan regions.1
The function and chemical composition of pectin in
plant cell walls, however, is still relatively poorly under-
stood. In order to answer these questions, a number of
antibodies binding to various pectic polysaccharides
have been prepared. JIM5,2 JIM7,2 and LM73 recognize
partly methylated homogalacturonan, while PAM14 is
specific for longer stretches of unesterified homogalac-
turonan. The epitopes have not been characterized in
greater detail.5,6 These antibodies have been raised from
complex polysaccharide mixtures, obtained after isola-
tion fromplants. This has given the process a somewhat
randomoutcome, since it has been difficult to control
the characteristics of the antibodies derived fromsuch
immunizations. Recently, however, Knox and co-work-
ers have been able to tailor their immunizations, result-
ing in antibodies against well-defined haptens. Two
antibodies, LM57 and LM6,8 were both raised against
2. Results and discussion
2.1. Synthetic planning
A number of elements required consideration in order to
conjugate oligogalacturonates to BSA. The conjugation
*
Corresponding author. E-mail: rm@kemi.dtu.dk
0008-6215/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.06.012

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M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
acid or methyl ester
O
b
RO
RO
α-configuration resembling
OH
O
76%
homogalacturonan linkages
1: R = H
(α-
D
-GalpA)n
O
3: R = TBDPS
4: R = H
a
c
COOR
O
67%
99%
2: R = TBDPS
acid-labile
aldehyde
protection
HO
O
HO
O
Scheme 1. Reagents and conditions: (a) TBDPSCl, DIPEA, DMF, rt,
3h; (b) (1) Me2SO, (COCl)2, CH2Cl2, ꢀ70°C, 30min, then DIPEA,
O
additional
galacturonates
˚
ꢀ70°C to rt; (2) propane-1,3-diol, TsOH, 4A MS, hexane, rt, 10h;
8-carbon spacer
(c) TBAF, THF, rt, 1h.
Chart 1. Target oligogalacturonate with spacer.
tert-butyldiphenylsilyl chloride in a mixture of DMF
and Hunigꢁs base.15 Swern oxidation16 of the remaining
primary alcohol in 2 gave the aldehyde, which was
trapped by acid-catalyzed acetal formation in the pres-
ence of molecular sieves17 to produce 3. Lastly, the silyl
group was removed quantitatively by treatment with
tetrabutylammonium fluoride.
method should be compatible with the carboxylic acid
and ester groups in the sugar haptens. Furthermore,
conjugation of the methyl-esterified trigalacturonate
should be performed at neutral pH, since basic condi-
tions will degrade this molecule by b-elimination. There-
fore, reductive amination was chosen as the conjugation
method.9 It offers the possibility of conjugating up to 60
sugar moieties to a single protein molecule, because BSA
contains 59 lysine residues as well as the N-terminal ami-
no group.10 The use of a hydrocarbon spacer at the
reducing end of the carbohydrate hapten was pioneered
by Lemieux et al.11 This prevents the alteration of the
reducing end sugar, which occurs when direct conjuga-
tion to the reducing end aldehyde is used. Furthermore,
the method minimizes the risk of introducing a stronger
immunogen close to the sugar, because this type of
spacer contains no functional groups in the vicinity of
the carbohydrate. Hence, it was planned to synthesize
a protected oligogalacturonate with an aglycon spacer
containing a masked aldehyde. This will be prepared
by repeated coupling of galactose glycosyl donors to a
galactose acceptor containing the spacer followed by a
single oxidation of all the 6-positions to carboxylic
acids. The glycosylations will be performed with n-pen-
tenyl galactosides as glycosyl donors in line with our
previously developed strategy for assembling oligogalac-
turonates.12,13 Conjugation to BSA will then be carried
out after removal of the aldehyde protecting group
(Chart 1).
¨
The coupling to galactose was then investigated
(Table 1). With the fully protected pentenyl donor 5,12
a 1:3.5 ratio was obtained under standard conditions
favoring the b-linked product 6b (entry 1). Cooling the
reaction mixture to ꢀ78°C and using stoichiometric
amounts of triflic acid as the promoter provided even
less of the desired 6a (entry 2). This is quite surprising
since donor 5 has previously given good a-selectivities
in couplings to other monosaccharides.12 Adding ether
as co-vent did not significantly alter these ratios. The
anomeric configuration of 6a,b were determined by
1
NMR. The H NMR spectrumof 6a showed H-1 at
1
4.80ppm( JH-1,H-2 3.7Hz) while H in 6b was observed
at 4.37ppm( JH-1,H-2 7.5Hz). The 13C NMR spectrum
of 6a showed C-1 at 97.9ppm( JC-1,H-1 172Hz) while
C-1 in 6b appeared at 102.6ppm( JC-1,H-1 160Hz).18
It seems that 4 is too reactive under these conditions
and therefore it was decided to change the coupling
method. Lemieux et al. showed that glycosyl bromides
could serve as donors when activated with bromide
Table 1. Attaching spacer to galactose
In designing the linker, several elements were taken
into consideration: an a-linkage was desirable because
it would resemble the linkages in native homogalacturo-
nan. The spacer was intended to be eight carbons long,
in order to effectively distance the sugar hapten fromthe
protein. Finally, a 1,3-dioxane was chosen as blocking
group for the aldehyde, because it is quite stable to
acidic conditions14 thus preventing premature unveiling
of the masked aldehyde during the assembly of the
oligogalacturonate.
OR'
O
OR'
O
RO
BnO
RO
BnO
4
O
O
CH2Cl2
O
BnO
BnO
O
5: R = ClAc, R' = Ac
7: R = H, R' = Ac
9: R = H, R' = PMP
6: R = ClAc, R' = Ac
8: R = H, R' = Ac
10: R = H, R' = PMP
Entry Donor Conditions
Yield a:b
(%)
1
2
3
4
5
6
7
5
5
7
7
7
7
9
NIS, TESOTf, ꢀ20°C
68
50
5
1:3.5
NIS, TfOH, ꢀ78°C
1:5.5
95:5
2.2. Attaching the spacer
Br2, then Bu4NI, 20°C
Br2, then Et4NBr, 20°C
Br2, then Et4NBr, 0–20°C
Br2, then Et4NBr, 4A MS, 0–20°C 56
Br2, then Et4NBr, 4A MS, 0–20°C 70
10
25
>95:5
>95:5
>95:5
>95:5
The protected hydroxyaldehyde 4, needed as the agly-
con, was prepared fromoctane-1,8-diol ( 1) (Scheme 1).
Monoprotection was performed by treatment with
˚
˚

M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
2161
ions.19 Under these conditions, an equilibriumis estab-
OAc
O
RO
BnO
lished between the a- and b-glycosyl bromide, which
heavily favors the a-bromide, but the b-bromide is much
more reactive. Consequently, only the equatorial bro-
mide reacts with the acceptor, giving rise to the a-glyco-
side. This method does have limitations, since reaction
times are usually rather long, and sterically hindered
alcohols cannot be glycosylated in this fashion.
However, for the purpose of glycosylating the primary
alcohol 4 it was very appropriate, especially since it
would not be necessary to protect the less reactive
4-hydroxy group of the donor. The glycosyl bromides
were prepared fromthe corresponding pentenyl glyco-
sides by brominolysis20 and then subjected directly to
the halide mediated glycosylation in the same pot. Initial
results with the donor 713 were promising in terms of
a-selectivity, albeit the yields were low (entries 3 and
4). Lowering the temperature gave minor improvements
(entry 5), but a satisfactory yield was reached only when
OAc
O
ClAcO
BnO
BnO
OAc
O
O
O
BnO
BnO
5
a
BnO
OSp
16%
12: R = ClAc
13: R = H
b
78%
OAc
O
HO
OAc
BnO
BnO
BnO
a
BnO
O
OSp
O
15%
8α
BnO
a
15
OAc
BnO
BnO
OAc
BnO
BnO
14%
O
O
BnO
OAc
O
BnO
OAc
O
O
O
BnO
O
BnO
BnO
BnO
OAc
O
11
O
O
O
BnO
14
˚
4A molecular sieves were added (entry 6). The reaction
BnO
Sp =
OSp
was even more efficient in the case of the PMP-protected
donor 913 (PMP=p-methoxyphenyl, entry 7), an obser-
vation that soon proved important (vide infra).
Scheme 2. Reagents and conditions: (a) NIS, TESOTf, CH2Cl2, 0°C,
30min; (b) thiourea, Bu4NI, NaHCO3, THF, 55°C, 24h.
2.3. Synthesis of neoglycoproteins
The product 21, obtained in an excellent 90% yield,
was deprotected to 22 and glycosylated with monosac-
charide donor 23,12 furnishing hexasaccharide 24 in
69% yield. With 20 and 24 in hand, the stage was set
for elaboration to the oligogalacturonates.
Having prepared the required a-linked spacer-monosac-
charide building block 8a, the stage was now set for
targeting the desired tri- and hexasaccharide neoglyco-
proteins. Unfortunately, glycosylation of the acceptor
8a with donors 5 and 1113 proved surprisingly difficult
giving 12 in only 16% yield and 14 in a mere 14%
(Scheme 2). None of the corresponding b-linked saccha-
rides were isolated. An attempt to further elaborate 12
into 14 by selective removal of the chloroacetyl group
and glycosylation of the resulting disaccharide with
1512 resulted in another disappointing result. In all three
cases, the donors and acceptors were protected with an
acetyl group at the 6-position. Unexpectedly, there
seems to be a mismatch between these donors and
acceptors, which required the coupling partners to be
changed.
Fortunately, this could easily be achieved by using the
PMP-protected building blocks (Scheme 3). Initially, di-
saccharide donor 17 was prepared from 9 by Koenigs–
Knorr coupling with bromide 16, which is also obtained
from 9. The PMP-protected monosaccharide 10a was
then glycosylated with 17 affording the desired a-linked
trisaccharide 18 in 71% yield. At this point, bifurcation
of the product gave access to two trisaccharides––one
that could be taken on to conjugation and one that
served as acceptor in the synthesis of the hexasaccha-
ride. Simple deacetylation of 18 yielded alcohol 19,
which upon standard benzylation gave fully protected
trisaccharide 20. Alternatively, alcohol 19 was used as
an acceptor and subjected to another coupling with 17.
CAN-mediated oxidative cleavage of the PMP-groups
of 20 and 24 furnished 25 and 26 and was followed by
oxidations12 and esterifications (Scheme 4). For the tri-
galacturonate both methyl and benzyl esters were de-
sired, giving access to either a fully methylated or a
nonmethylated conjugate. Furthermore, it was antici-
pated that benzyl esterification would facilitate handling
and purification, especially for the hexagalacturonate.
Thus oxidation to carboxylic acid was performed by a
two step procedure using the Dess–Martin periodin-
ane21 followed by sodiumchlorite oxidation. The acids
were not purified, but immediately subjected to esterifi-
cation with cesiumcarbonate and methyl iodide or benz-
yl bromide. Hereby, 27 and 28 were obtained in 37 and
42% yield, respectively. Some degradation was observed
in both cases, and this proved to be an even more severe
problemfor the synthesis of hexabenzyl ester 29, which
could be isolated in only 17% yield under these condi-
tions. For the preparation of 27 and 28, a considerable
improvement could be obtained by performing the este-
rifications under neutral conditions.13 The use of tri-
methylsilyl diazomethane for methyl esterification
afforded 27 in 79% yield while the tribenzyl ester 28
was obtained in 68% yield by applying phenyl diazo-
methane. The neutral esterification conditions only
led to a slight improvement in the yield of 29 and the

2162
M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
OPMP
O
AcO
BnO
a
9
O
Sp =
BnO
16
O
Br
OPMP
O
Table 1
BnO
BnO
71%
b
O
OPMP
O
HO
BnO
23
OPMP
O
AcO
BnO
BnO
OPMP
O
BnO
BnO
BnO
OSp
BnO
OPMP
O
O
OPMP
O
RO
BnO
10α
BnO
OPMP
O
O
BnO
17
O
c
BnO
BnO
OPMP
O
BnO
O
c
71%
OPMP
O
BnO
OPMP
O
RO
BnO
O
BnO
69%
BnO
OPMP
O
BnO
O
BnO
OPMP
O
BnO
OPMP
O
c
O
O
BnO
90%
BnO
BnO
OPMP
BnO
O
BnO
OPMP
O
BnO
OPMP
O
O
O
BnO
O
BnO
BnO
OPMP
O
BnO
O
BnO
BnO
OPMP
O
O
OSp
BnO
18: R = Ac
19: R = H
20: R = Bn
d
82%
BnO
BnO
24
OSp
21: R = Ac
22: R = H
d
89%
e
BnO
OSp
98%
˚
Scheme 3. Reagents and conditions: (a) Ac2O, DMAP, Et3N, CH2Cl2, rt, 16h, then Br2, CH2Cl2, 0°C, 15min; (b) AgOTf, 4A MS, CH2Cl2, ꢀ45°C,
2h; (c) NIS, TESOTf, CH2Cl2, ꢀ20°C, 30min; (d) NaOMe, THF, MeOH; (e) BnBr, NaH, Bu4NI, DMF, rt, 2h.
OH
O
HO
BnO
BnO
OPMP
O
BnO
BnO
BnO
COOR
O
COOR
O
HO
BnO
HO
O
BnO
OH
O
BnO
BnO
OPMP
O
O
a
b or c
d
O
O
COOR
O
COOR
O
HO
BnO
BnO
BnO
n
n
n
n
HO
O
BnO
OH
O
BnO
OPMP
BnO
O
O
O
COOR
O
COOR
O
O
HO
BnO
BnO
BnO
HO
BnO
BnO
BnO
OSp
OSp
OSp
O
OSp
25: n = 1 (75%)
26: n = 4 (54%)
20: n = 1
24: n = 4
27: n = 1, R = Me (79%)
28: n = 1, R = Bn (68%)
29: n = 4, R = Bn (22%)
30: n = 1, R = Me (99%)
31: n = 1, R = H (99%)
32: n = 4, R = H (70%)
Sp =
O
Scheme 4. Reagents and conditions: (a) CAN, MeCN, H2O, 0°C, 20min; (b) Dess–Martin periodinane, CH2Cl2, rt, 45min, then NaClO2, NaH2PO4,
THF, t-BuOH, H2O, rt, 1h, then TMSCHN2, MeOH, rt, 2h; (c) Dess–Martin periodinane, CH2Cl2, rt, 1h, then NaClO2, NaH2PO4, THF, t-BuOH,
H2O, rt, 1h, then PhCHN2, EtOAc, rt, 2h; (d) H2, Pd/C, THF, MeOH, H2O.
multistep sequence was still plagued by considerable
decomposition.
3. Experimental
3.1. General procedures
Hydrogenolysis of the fully protected oligogalacturo-
nates proceeded uneventfully. After treatment with
aqueous acetic acid to unmask the aldehyde the sugars
could be attached to BSA by reductive amination with
sodiumcyanoborohydride ( Scheme 5). According to
MALDI-TOFMS data the resulting glycoconjugates
33, 34, and 35 contained on average 22.6, 11.5, and
3.8molligand/mol protein. The successful synthesis of
the three neoglycoproteins 33–35 will now permit the
first attempts to raise antibodies toward well-defined
homogalacturonan fragments.
CH2Cl2 was distilled fromCaH . Thin-layer chromato-
2
graphy was performed on aluminum plates precoated
with silica gel. Compounds were visualized by heating
after dipping in a soln of Ce(SO4)2 (2.5g) and
(NH4)6Mo7O24 (6.25g) in 10% aq H2SO4 (250mL).
Flash chromatography was performed with Silica Gel
60. NMR spectra were recorded on a Varian Unity Ino-
va 500 or a Varian Mercury 300 spectrometer. Optical
rotations were measured on a Perkin–Elmer 241 pola-

M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
2163
allowed to reach rt. CH2Cl2 (150mL) was added, and
the soln was washed with 1M aq HCl (2·150mL) and
water (150mL). The combined aq layers were extracted
with CH2Cl2 (150mL) and the combined organic phases
were dried and concentrated. The residue was suspended
HO
COOR
O
HO
HO
O
COOR
O
30
31
32
a
HO
n
HO
O
˚
COOR
O
in hexane (150mL) and 4A molecular sieves (5g) was
added, followed by propane-1,3-diol (2.53mL,
35.0mmol) and TsOH (2.58g, 13.6mmol). The mixture
was stirred for 10h, then quenched with id NaHCO3
and filtered through Celite. The pad was rinsed with
hexane (3·75mL). The filtrate was washed with 1M
aq NaOH (200mL), dried, concentrated and purified
by flash chromatography (15:1 hexanes–EtOAc) to give
HO
H
N BSA
HO
O
33: n = 1, R = Me
34: n = 1, R = H
35: n = 4, R = H
Scheme 5. Reagents and conditions: (a) AcOH, H2O, 50°C, 12h, then
BSA, NaCNBH3, H2O, rt, 4d.
1
4.80g (76%) of 3 as a colorless oil. H NMR (300MHz,
CDCl3): d 7.71–7.67 (m, 4H), 7.46–7.35 (m, 6H), 4.52 (t,
1H, J 5.1Hz), 4.15–4.07 (m, 2H), 3.82–3.75 (m, 2H),
3.67 (t, 2H, J 6.6Hz), 2.09 (m, 1H), 1.65–1.52 (m, 4H),
1.45–1.25 (m, 9H), 1.07 (s, 9H). 13C NMR (75MHz,
CDCl3): d 135.66 (4C), 134.30 (2C), 129.55 (2C),
127.66 (4C), 102.56, 67.00 (2C), 64.11, 35.38, 32.69,
29.60, 29.39, 27.04 (3C), 26.03, 25.81, 24.06, 19.37. Anal.
Calcd for C27H40O3Si: C, 73.59; H, 9.15. Found: C,
73.55; H, 9.20.
rimeter. ESI mass spectra were obtained at the Depart-
ment of Biochemistry and Molecular Biology, Univer-
sity of Southern Denmark using an Esquire-LC
instrument. MALDI-TOF mass spectra were obtained
at Danisco Biotechnology in Copenhagen using a Per-
ceptive Biosystems Voyager-De instrument in positive-
ion mode with a-cyano-4-hydroxycinnamic acid as the
matrix. Microanalyses were conducted at the Depart-
ment of Chemistry, University of Copenhagen.
3.4. 7-(1,3-Dioxan-2-yl)-heptan-1-ol (4)
3.2. 8-(tert-Butyldiphenylsilyloxy)-octan-1-ol (2)
A soln of 3 (2.30g, 5.22mmol) in anhyd THF (30mL)
was treated with a 1M soln of TBAF in THF
(7.83mL, 7.83mmol). Concentrated after 1h and puri-
fied by flash chromatography (2:1 hexanes–EtOAc)
yielding 1.05g (99%) of 4 as a colorless oil. Bp 112–
Octane-1,8-diol (1) (5.0g, 34.2mmol) was disved in
DMF (80mL) and DIPEA (60mL, 344mmol).
TBDPSCl (5.84mL, 22.5mmol) was added dropwise
and the reaction mixture was stirred for 3h at rt. Water
(500mL) was added and the mixture was extracted with
Et2O (3·500mL). The combined organic phases were
washed with 2M aq HCl (2 ·300mL) and satd aq NaH-
CO3 (500mL), dried, concentrated and purified by flash
chromatography (3:1 hexanes–EtOAc), yielding 5.81g
(67%) of 2 as a colorless oil. 1H NMR (300MHz,
CDCl3): d 7.73–7.67 (m, 4H), 7.47–7.36 (m, 6H), 3.69
(t, 2H, J 6.4Hz), 3.64 (t, 2H, J 6.6Hz), 1.81 (br s,
1H), 1.65–1.51 (m, 5H), 1.44–1.26 (m, 7H), 1.09 (s,
9H). 13C NMR (75MHz, CDCl3): d 135.69 (4C),
134.33 (2C), 129.58 (2C), 127.67 (4C), 64.14, 63.21,
32.96, 32.72, 29.53, 29.48, 27.06 (3C), 25.87, 25.52,
19.40. Anal. Calcd for C24H36O2Si: C, 74.95; H, 9.43.
Found: C, 74.76; H, 9.45.
1
120°C/0.2mmHg. H NMR (300MHz, CDCl3): d 4.50
(t, 1H, J 5.1Hz), 4.14–4.06 (m, 2H), 3.81–3.70 (m,
2H), 3.63 (t, 2H, J 6.6Hz), 2.07 (m, 1H), 1.63–1.50 (m,
4H), 1.45–1.25 (m, 9H), 1.21 (br s, 1H). 13C NMR
(75MHz, CDCl3): d 102.33, 66.77 (2C), 62.52, 35.09,
32.64, 29.35, 29.23, 25.78, 25.60, 23.78. ESIMS: m/z
203.2 [M+H]+. Anal. Calcd for C11H22O3: C, 65.31; H,
10.96. Found: C, 64.96; H, 11.01.
3.5. General procedure for the pentenyl glycoside
couplings with NIS/TESOTf
A mixture of the donor (6.5mmol) and the acceptor
(5.0mmol) was dried azeotropically with toluene and
subjected to high vacuumfor 2h. The mixture was dis-
ved in anhyd CH2Cl2 (60mL), cooled to ꢀ20°C, fol-
lowed by addition of NIS (1.49g, 6.63mmol) and
TESOTf (0.29mL, 1.3mmol). The reaction mixture
was stirred at ꢀ20°C until TLC revealed full conversion
of the donor (15–45min). The soln was diluted with
CH2Cl2 (60mL) and washed with 10% aq Na2S2O3
(100mL) and satd aq NaHCO3 (100mL). The combined
aq phases were extracted with CH2Cl2 (100mL). The
combined organic phases were dried (MgSO4), concen-
trated, and purified by flash chromatography.
3.3. 2-(7-(tert-Butyldiphenylsilyloxy)-heptyl)-
1,3-dioxane (3)
A soln of anhyd Me2SO (4.11mL, 57.9mmol) in anhyd
CH2Cl2 (50mL) under Ar was cooled to ꢀ70°C and
oxalyl chloride (2.43mL, 27.9mmol) was added drop-
wise. After 15min, a soln of 2 (5.50g, 14.3mmol) in
CH2Cl2 (30mL) was added slowly, keeping the temper-
ature below ꢀ65°C. After 30min, DIPEA (24.9mL,
143mmol) was added, and the reaction mixture was

2164
M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
3.6. 7-(1,3-Dioxan-2-yl)-heptyl 6-O-acetyl-2,3-di-O-benz-
yl-4-O-chloroacetyl-D-galactopyranoside (6)
m/z 609.4 [M+Na]+. Anal. Calcd for C33H46O9: C,
67.56; H, 7.90. Found: C, 67.41; H, 7.94.
Coupling of 5 (2.70g, 4.94mmol) and 4 (1.28g,
6.26mmol) following the general procedure above
afforded 0.50g (15%) of 6a as a colorless oil and 1.73g
3.8. 7-(1,3-Dioxan-2-yl)-heptyl 2,3-di-O-benzyl-6-O-(4-
methoxyphenyl)-a-D-galactopyranoside (10a)
(53%) of 6b as a colorless oil.
Glycoside 9 (10.4g, 21.2mmol) was dried azeotropically
with toluene and subjected to high vacuumfor 2h. It
was then disved in anhyd CH2Cl2 (40mL), cooled to
0°C and titrated with a 1M soln of Br2 in anhyd CH2Cl2
until a faint yellow color persisted. Powdered molecular
20
D
For 6a: ½aꢁ +54.6 (c 1.1, CHCl3). 1H NMR
(300MHz, CDCl3): d 7.37–7.21 (m, 10H), 5.59 (d, 1H,
J 1.7Hz), 4.80 (d, 1H, J 3.7Hz), 4.79 (d, 1H, J
12.1Hz), 4.73 (d, 1H, J 11.0Hz), 4.61 (d, 1H, J
12.1Hz), 4.58 (d, 1H, J 11.0Hz), 4.50 (t, 1H, J 5.1Hz),
4.18–4.04 (m, 5H), 4.10 (br s, 2H), 4.00 (dd, 1H, J
10.1, 3.4Hz), 3.80–3.69 (m, 3H), 3.60 (dt, 1H, J 9.5,
7.0Hz), 3.44 (dt, 1H, J 9.9, 6.6Hz), 2.03 (m, 1H), 2.02
(s, 3H), 1.69–1.53 (m, 4H), 1.42–1.21 (m, 9H). 13C
NMR (75MHz, CDCl3): d 170.58, 167.14, 138.64,
138.11, 128.54 (4C), 128.23 (2C), 128.06 (2C), 127.93,
127.90, 102.58, 97.87 (JCH 172Hz), 76.06, 75.68, 73.63,
72.79, 70.35, 68.89, 67.10 (2C), 66.53, 62.38, 41.01,
35.47, 29.65, 29.58, 29.49, 26.30, 26.11, 24.12, 20.94. ES-
IMS: m/z 663.4 [M+H]+. Anal. Calcd for C35H47ClO10:
C, 63.39; H, 7.14; Cl, 5.35. Found: C, 63.63; H, 6.91; Cl,
˚
sieves 4A (10g) was added, followed by a soln of 4
(2.52g, 12.5mmol) and Et4NBr (5.35g, 25.5mmol) in
anhyd CH2Cl2 (40mL). The mixture was stirred for
40h, quenched with satd aq NaHCO3 (50mL) and then
stirred for an additional 4h. The mixture was filtered
through Celite and the pad was rinsed with CH2Cl2
(50mL). The organic phase was separated and washed
with water. The combined aq phases were extracted with
CH2Cl2. The combined organic phases were dried, con-
centrated and purified by flash chromatography (3:1
hexanes–EtOAc) yielding 10a (5.71g, 70%) as a id. Mp
20
74–76°C (hexanes–EtOAc). ½aꢁ +33.1 (c 1.0, CHCl3).
D
5.10.
1H NMR (300MHz, CDCl3): d 7.39–7.26 (m, 10H),
6.88–6.77 (m, 4H), 4.86–4.62 (m, 5H), 4.49 (t, 1H, J
5.1Hz), 4.19–4.05 (m, 5H), 3.93 (dd, 1H, J 9.9,
3.3Hz), 3.85 (dd, 1H, J 9.9, 3.7Hz), 3.80–3.60 (m,
4H), 3.76 (s, 3H), 3.43 (dt, 1H, J 9.9, 6.6Hz), 2.49 (br
s, 1H), 2.06 (m, 1H), 1.68–1.49 (m, 4H), 1.43–1.24 (m,
9H). 13C NMR (75MHz, CDCl3): d 153.93, 152.72,
138.52, 138.20, 128.40 (2C), 128.29 (2C), 127.84 (3C),
127.77, 127.74, 127.64, 115.54 (2C), 114.58 (2C),
102.33, 97.25, 77.65, 76.01, 73.14, 72.83, 68.27, 67.99,
67.69 (2C), 66.81 (2C), 55.63, 35.22, 29.44, 29.38,
29.33, 26.09, 25.86, 23.94. ESIMS: m/z 673.7
[M+Na]+. Anal. Calcd for C38H50O9: C, 70.13; H,
7.74. Found: C, 70.01; H, 7.77.
20
D
For 6b: ½aꢁ +14.9 (c 1.0, CHCl3). 1H NMR
(300MHz, CDCl3): d 7.37–7.22 (m, 10H), 5.52 (d,
1H, J 1.8Hz), 4.86 (d, 1H, J 11.0Hz), 4.74 (d, 1H,
J 11.3Hz), 4.70 (d, 1H, J 11.0Hz), 4.53 (d, 1H,
J 11.3Hz), 4.48 (t, 1H, J 5.1Hz), 4.37 (br d, 1H, J
7.5Hz), 4.22–4.04 (m, 4H), 4.14 (br s, 2H), 3.92 (dt,
1H, J 9.5, 6.4Hz), 3.82–3.68 (m, 3H), 3.58–3.47 (m,
3H), 2.04 (m, 1H), 2.02 (s, 3H), 1.68–1.52 (m, 4H),
1.44–1.22 (m, 9H). 13C NMR (75MHz, CDCl3): d
170.64, 167.28, 138.64, 137.74, 128.58 (2C), 128.49
(2C), 128.33 (2C), 128.25 (2C), 128.03, 127.87, 103.99,
102.61 (JCH 160Hz), 79.09, 78.87, 75.57, 72.79, 70.73,
70.56, 68.96, 67.12 (2C), 61.88, 41.07, 35.46, 29.91,
29.63, 29.54, 26.23, 26.12, 24.14, 20.96. ESIMS: m/z
663.4 [M+H]+. Anal. Calcd for C35H47ClO10: C, 63.39;
H, 7.14; Cl, 5.35. Found: C, 63.27; H, 6.98; Cl, 5.17.
3.9. Pent-4-enyl 4-O-acetyl-2,3-di-O-benzyl-6-O-(4-
methoxyphenyl)-a-D-galactopyranosyl-(1fi4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-b-D-galactopyranoside
(17)
3.7. 7-(1,3-Dioxan-2-yl)-heptyl 6-O-acetyl-2,3-di-O-benz-
yl-a-D-galactopyranoside (8a)
To a soln of 9 (7.28g, 13.6mmol) in CH2Cl2 (50mL)
were added Ac2O (1.7mL, 18mmol), Et3N (3.5mL,
25mmol), and DMAP (50mg). The mixture was stirred
overnight and then diluted with CH2Cl2 (50mL),
washed with water (50mL), dried, and concentrated.
The syrupy residue was purified by flash chromatogra-
phy (4:1 hexanes–EtOAc) to afford pent-4-enyl 4-O-
acetyl-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-b-D-gal-
Prepared from 7 and 4 as described below for 10a. Col-
20
D
orless oil. ½aꢁ +52.3 (c 1.1, CHCl3). 1H NMR
(300MHz, CDCl3): d 7.41–7.24 (m, 10H), 4.87–4.81
(m, 5H), 4.50 (t, 1H, J 5.1Hz, 1H), 4.38–4.20 (m, 2H),
4.14–4.05 (m, 2H), 4.02–3.69 (m, 6H), 3.60 (m, 1H),
3.42 (dt, 1H, J 9.9, 6.6Hz), 2.50 (br s, 1H), 2.06 (s,
3H), 2.05 (m, 1H), 1.70–1.53 (m, 4H), 1.44–1.25 (m,
9H). 13C NMR (75MHz, CDCl3): d 170.77, 138.55,
138.24, 128.56 (2C), 128.44, 128.34, 128.15, 128.06,
127.97 (3C), 127.84, 102.48, 97.35, 77.55, 75.94, 73.34,
73.04, 68.43, 67.92, 67.57, 66.99 (2C), 63.80, 35.32,
29.56, 29.44 (2C), 26.24, 25.98, 24.02, 20.98. ESIMS:
actopyranoside (6.39g) as a id. Mp 63–65°C (hexanes–
20
D
EtOAc). ½aꢁ
+4.6 (c 1.0, CHCl3). 1H NMR
(300MHz, CDCl3): d 7.38–7.22 (m, 10H), 6.85–6.78
(m, 4H), 5.81 (m, 1H), 5.64 (br s, 1H), 5.07–4.95 (m,
2H), 4.89 (d, 1H, J 11.1Hz), 4.78 (d, 1H, J 10.9Hz),

M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
2165
4.75 (d, 1H, J 10.9Hz), 4.57 (d, 1H, J 11.1Hz), 4.41 (m,
1H), 4.08 (dd, 1H, J 10.5, 7.2Hz), 4.02–3.85 (m, 3H),
3.77 (s, 3H), 3.62–3.54 (m, 3H), 2.18 (m, 2H), 2.09 (s,
3H), 1.77 (m, 2H). 13C NMR (75MHz, CDCl3): d
170.1, 154.4, 152.8, 138.2, 138.0, 137.9, 128.5 (4C),
128.3 (2C), 128.2 (2C), 127.9, 127.8, 116.3 (2C), 114.9
(2C), 114.7, 104.0, 79.5, 78.9, 75.6, 72.3, 71.7, 70.0,
67.8, 67.4, 55.9, 30.3, 29.1, 21.4. Anal. Calcd for
C34H40O8: C, 70.81; H, 6.99. Found: C, 70.86; H, 6.83.
A soln of this compound (6.50g, 11.3mmol) in anhyd
CH2Cl2 (30mL) was titrated at 0°C with a 1M soln of
Br2 in anhyd CH2Cl2 until a faint yellow color persisted.
The soln was then cannulated into a mixture of 9 (4.30g,
8.04mmol), AgOTf (4.40g, 17.1mmol), and flame dried
138.75, 138.64, 138.49, 138.35, 138.30 (2C), 115.60
(2C), 115.28 (2C), 115.25 (2C), 114.81 (2C), 114.57
(2C), 114.41 (2C), 102.42, 100.42, 100.18, 97.60, 78.77,
78.14, 76.82, 76.26, 75.65, 75.18, 74.73, 74.21, 73.56,
73.12, 73.01, 72.89 (2C), 71.77, 69.46, 69.02, 68.43,
67.63, 67.21, 66.92 (2C), 65.90, 65.43, 64.16, 55.80,
55.73 (2C), 35.31, 29.52, 29.49, 29.39, 26.15, 25.96,
24.00, 20.83. ESIMS: m/z 1590.0 [M+H]+.
3.11. 7-(1,3-Dioxan-2-yl)-heptyl 2,3-di-O-benzyl-6-O-(4-
methoxyphenyl)-a-D-galactopyranosyl-(1fi4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-D-galactopyranosyl-
(1fi4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-D-
galactopyranoside (19)
˚
powdered 4A molecular sieves (10g) in anhyd CH2Cl2
(30mL) at ꢀ45°C. The reaction mixture was stirred at
ꢀ45°C for 2h and then quenched by addition of satd
aq NaHCO3 (50mL), allowed to reach room tempera-
ture and filtered through Celite. The pad was rinsed with
CH2Cl2 (50mL). The organic phase was separated and
washed with satd aq NaHCO3 (50mL) and water
(50mL). The combined aq layers were extracted with
CH2Cl2 (25mL). The combined organic phases were
dried, concentrated, and purified by flash chromatogra-
phy (5:1 hexanes–EtOAc) to give 17 (5.83g, 71%) as a
Trisaccharide 18 (3.01g, 1.89mmol) was dissolved in
THF (12mL) and MeOH (24mL) followed by addition
of sodium(100mg). The soln was stirred overnight and
then quenched with Amberlite IRC-50 (H+) ion
exchange resin. The mixture was filtered, concentrated,
and the residue purified by flash chromatography (2:1
hexanes–EtOAc) to afford 19 (2.40g, 82%) as a foam.
20
D
½aꢁ +18.4 (c 1.1, CHCl3). 1H NMR (500MHz, CDCl3):
d 7.41–7.06 (m, 30H), 6.81–6.41 (m, 12H), 5.08–5.01 (m,
2H), 4.91–4.41 (m, 19H), 4.33 (s, 1H), 4.28–4.21 (m,
3H), 4.12–3.86 (m, 8H), 3.80–3.58 (m, 5H), 3.75 (s,
3H), 3.74 (s, 3H), 3.69 (s, 3H), 3.56–3.46 (m, 2H), 2.45
(br s, 1H), 2.06 (m, 1H), 1.66–1.54 (m, 4H), 1.41–1.25
(m, 9H). 13C NMR (75MHz, CDCl3): d 154.02,
153.88, 153.77, 152.71, 152.61, 152.37, 138.82, 138.68,
138.56, 138.51, 138.48, 138.26, 115.42 (2C), 115.34
(4C), 114.84 (2C), 114.61 (2C), 114.51 (2C), 102.49,
100.53, 99.98, 97.64, 78.79, 78.31, 78.20, 76.33, 75.57,
75.46, 75.25, 74.43, 73.60, 73.20, 73.04, 72.94 (2C),
72.72, 72.10, 69.53, 69.09, 68.47, 67.98, 66.97 (2C),
66.71, 66.30, 65.53, 55.84 (2C), 55.81, 35.35, 29.56,
29.53, 29.45, 26.19, 26.00, 24.06. ESIMS: m/z 1569.8
[M+Na]+. Anal. Calcd for C92H106O21: C, 71.39; H,
6.90. Found: C, 71.17; H, 6.80.
20
1
syrup. ½aꢁ +25.6 (c 1.0, CHCl3). H NMR (300MHz,
D
CDCl3): d 7.39–7.12 (m, 20H), 6.77–6.56 (m, 8H), 5.82
(m, 1H), 5.74 (br s, 1H), 5.08–4.91 (m, 3H), 4.83–4.60
(m, 7H), 4.58–4.44 (m, 3H), 4.39 (d, 1H, J 10.3Hz),
4.17 (d, 1H, J 2.1Hz), 4.13 (dd, 1H, J 11.1, 2.0Hz),
4.01–3.90 (m, 2H), 3.79–3.44 (m, 7H), 3.73 (s, 3H),
3.70 (s, 3H), 2.12 (m, 2H), 1.97 (s, 3H), 1.79 (m, 2H).
13C NMR (75MHz, CDCl3): d 169.91, 153.77 (2C),
152.44, 152.22, 138.49, 138.33, 138.17, 138.07, 137.92,
115.49 (2C), 115.22 (2C), 114.84, 114.48 (2C), 114.25
(2C), 104.01, 100.50, 80.59, 78.46, 76.29, 75.06, 74.85,
73.20, 72.90, 72.74 (2C), 71.62, 69.38, 67.69, 67.27,
66.18, 65.29, 55.55 (2C), 30.11, 28.87, 20.73. ESIMS:
m/z 1047.5 [M+Na]+.
3.10. 7-(1,3-Dioxan-2-yl)-heptyl 4-O-acetyl-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-D-galactopyranosyl-
(1fi4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-D-
galactopyranosyl-(1fi4)-2,3-di-O-benzyl-6-O-(4-meth-
oxyphenyl)-a-D-galactopyranoside (18)
3.12. 7-(1,3-Dioxan-2-yl)-heptyl 2,3,4-tri-O-benzyl-6-O-
(4-methoxyphenyl)-a-D-galactopyranosyl-(1fi4)-2,3-di-
O-benzyl-6-O-(4-methoxyphenyl)-a-D-galactopyranosyl-
(1fi4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-D-
galactopyranoside (20)
Coupling of 17 (1.37g, 1.34mmol) and 10a (621mg,
0.954mmol) following the aforementioned general pro-
To a soln of 19 (1.22g, 0.788mmol) in DMF (10mL)
was added NaH (70mg of a ꢂ50% oil dispersion,
1.46mmol) and the mixture was stirred at rt for
15min. BnBr (0.17mL, 1.43mmol) and Bu4NI (15mg,
0.04mmol) were added and the soln was stirred for an
additional 2h. Quenched with MeOH, diluted with
CH2Cl2 (50mL), and washed with water (2·40mL).
The organic phase was dried, concentrated and purified
20
D
cedure gave 1.07g (71%) of 18 as a colorless oil. ½aꢁ
1
+13.1 (c 1.1, CHCl3). H NMR (500MHz, CDCl3): d
7.42–7.00 (m, 30H), 6.82–6.41 (m, 12H), 5.71 (br s,
1H), 5.10–5.01 (m, 2H), 4.93–4.20 (m, 19H), 4.12–3.89
(m, 9H), 3.83–3.40 (m, 9H), 3.73 (s, 3H), 3.72 (s, 3H),
3.71 (s, 3H), 2.09 (m, 1H), 1.91 (s, 3H), 1.70–1.51 (m,
4H), 1.42–1.23 (m, 9H). 13C NMR (75MHz, CDCl3):
d 169.90, 154.03, 153.92, 153.76, 152.53 (2C), 152.33,
by flash chromatography (5:2 hexanes–EtOAc), yielding
20
D
20 (1.27g, 98%) as a foam. ½aꢁ +8.4 (c 1.1, CHCl3). 1H

2166
M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
NMR (300MHz, CDCl3): d 7.42–7.08 (m, 35H), 6.82–
6.42 (m, 12H), 5.09–5.02 (m, 2H), 4.96–4.39 (m, 20H),
4.34–4.22 (m, 3H), 4.16–3.88 (m, 9H), 3.86–3.60 (m,
6H), 3.76 (s, 3H), 3.76 (s, 3H), 3.70 (s, 3H), 3.55–3.42
(m, 2H), 2.08 (m, 1H), 1.70–1.53 (m, 4H), 1.46–1.24
(m, 9H). 13C NMR (75MHz, CDCl3): d 154.17, 153.91
(2C), 152.77 (2C), 152.55, 139.04, 138.99, 138.89,
138.85, 138.76, 138.71, 138.67, 115.49 (4C), 115.39
(2C), 114.98 (2C), 114.75 (2C), 114.66 (2C), 102.63,
100.71, 100.31, 97.79, 79.56, 79.05, 78.32, 76.38, 75.94,
75.46, 75.41, 75.16, 74.61, 74.51, 73.65, 73.40, 73.15,
73.11, 72.83, 72.68, 69.74, 69.26, 69.18, 68.60, 67.11
(2C), 65.67, 65.42, 64.54, 56.00 (2C), 55.96, 35.50,
29.68 (2C), 29.60, 26.34, 26.15, 24.21. ESIMS: m/z
1660.9 [M+Na]+.
6H), 3.98–3.57 (m, 17H), 3.74 (s, 3H), 3.73 (s, 3H),
3.69 (s, 6H), 3.67 (s, 3H), 3.54 (m, 1H), 3.48 (m, 1H),
3.44–3.37 (m, 2H), 2.40 (br s, 1H), 2.06 (m, 1H), 1.66–
1.52 (m, 4H), 1.40–1.23 (m, 9H). 13C NMR (75MHz,
CDCl3): d 153.95, 153.77, 153.70, 153.62, 153.52,
152.60, 152.49, 152.37, 152.34, 152.17, 115.28 (4C),
115.24 (2C), 115.17 (2C), 115.12 (2C), 114.77 (2C),
114.62 (2C), 114.47 (2C), 114.40 (4C), 102.40, 100.38,
100.22 (2C), 99.83, 97.54, 66.89 (2C), 55.75 (5C),
35.27, 29.48 (2C), 29.37, 26.12, 25.92, 23.98. Anal. Calcd
for C146H162O33: C, 71.73; H, 6.68. Found: C, 71.55; H,
6.62.
3.15. 7-(1,3-Dioxan-2-yl)-heptyl 2,3,4-tri-O-benzyl-6-O-
(4-methoxyphenyl)-a-D-galactopyranosyl-(1fi4)-2,3-di-
O-benzyl-6-O-(4-methoxyphenyl)-a-D-galactopyranosyl-
(1fi4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-D-
galactopyranosyl-(1fi4)-2,3-di-O-benzyl-6-O-(4-meth-
oxyphenyl)-a-D-galactopyranosyl-(1fi4)-2,3-di-O-benz-
yl-6-O-(4-methoxyphenyl)-a-D-galactopyranosyl-(1fi4)-
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-D-galactopyr-
anoside (24)
3.13. 7-(1,3-Dioxan-2-yl)-heptyl 4-O-acetyl-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-D-galactopyranosyl-
(1fi4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-D-
galactopyranosyl-(1fi4)-2,3-di-O-benzyl-6-O-(4-meth-
oxyphenyl)-a-D-galactopyranosyl-(1fi4)-2,3-di-O-benz-
yl-6-O-(4-methoxyphenyl)-a-D-galactopyranosyl-(1fi4)-
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-D-galactopyra-
noside (21)
Coupling of 23 (1.55g, 2.48mmol) and 22 (3.03g,
1.24mmol) following the aforementioned general proce-
dure gave 2.54g (69%) of 24 as a foam. 1H NMR
(500MHz, CDCl3): d 7.40–6.88 (m, 65H), 6.77–6.17
(m, 24H), 5.06–4.18 (m, 46H), 4.11–3.82 (m, 12H),
3.80–3.57 (m, 11H), 3.75 (s, 3H), 3.73 (s, 3H), 3.70 (s,
3H), 3.69 (s, 3H), 3.68 (s, 3H), 3.67 (s, 3H), 3.54–3.24
(m, 6H), 2.06 (m, 1H), 1.67–1.51 (m, 4H), 1.41–1.22
(m, 9H). 13C NMR (75MHz, CDCl3): d 153.95,
153.74, 153.69, 153.65 (2C), 153.59, 152.48 (2C),
152.35, 152.30, 152.27, 152.16, 115.23 (2C), 115.14
(2C), 115.10 (6C), 115.06 (2C), 114.76 (2C), 114.58
(6C), 114.44 (2C), 114.39 (2C), 102.38, 100.36, 100.24,
100.20, 100.17, 99.99, 97.54, 66.86 (2C), 55.78, 55.74
(3C), 55.66, 55.59, 35.27, 29.46 (2C), 29.35, 26.11,
25.90, 23.96. Anal. Calcd for C180H196O39: C, 72.46;
H, 6.62. Found: C, 72.33; H, 6.58.
Coupling of 17 (2.11g, 2.06mmol) and 19 (2.40g,
1.55mmol) following the aforementioned general proce-
1
dure gave 3.47g (90%) of 21 as a colorless syrup. H
NMR (300MHz, CDCl3): d 7.41–6.88 (m, 50H), 6.77–
6.27 (m, 20H), 5.62 (m, 1H), 5.08–4.19 (m, 38H), 4.16–
3.30 (m, 23H), 3.73 (s, 3H), 3.72 (s, 3H), 3.68 (s, 9H),
2.05 (m, 1H), 1.85 (s, 3H), 1.65–1.51 (m, 4H), 1.41–
1.22 (m, 9H). 13C NMR (75MHz, CDCl3): d 169.83,
153.98, 153.85, 153.77 (2C), 153.65, 152.49, (2C),
152.38, 152.31, 152.20, 115.53 (2C), 115.24 (2C),
115.20 (2C), 115.14 (4C), 114.79 (2C), 114.63 (4C),
114.50 (2C), 114.36 (2C), 102.42, 100.39, 100.22,
100.17, 100.11, 97.62, 66.91 (2C), 55.81, 55.77 (2C),
55.73 (2C), 35.29, 29.50 (2C), 29.39, 26.14, 25.94,
24.00, 20.79.
3.14. 7-(1,3-Dioxan-2-yl)-heptyl 2,3-di-O-benzyl-6-O-(4-
methoxyphenyl)-a-D-galactopyranosyl-(1fi4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-D-galactopyranosyl-
(1fi4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-D-
galactopyranosyl-(1fi4)-2,3-di-O-benzyl-6-O-(4-meth-
oxyphenyl)-a-D-galactopyranosyl-(1fi4)-2,3-di-O-benz-
yl-6-O-(4-methoxyphenyl)-a-D-galactopyranoside (22)
3.16. 7-(1,3-Dioxan-2-yl)-heptyl 2,3,4-tri-O-benzyl-a-D-
galactopyranosyl-(1fi4)-2,3-di-O-benzyl-a-D-galactopy-
ranosyl-(1fi4)-2,3-di-O-benzyl–D-galactopyranoside (25)
To an ice-cooled soln of trisaccharide 20 (1.64g,
1.00mmol) in MeCN (40mL) was added a soln of
CAN (8.23g, 15.0mmol) in water (10mL). The mixture
was stirred at 0°C for 20min, then diluted with CHCl3
(100mL) and washed twice with water (80mL). The
combined aq layers were extracted with CHCl3
(80mL). The combined organic phases were dried
(MgSO4), filtered, concentrated, and the residue purified
Pentasaccharide 21 (3.45g) was deacetylated as de-
scribed above for 19 to afford 22 (3.03g, 89%) as a foam.
1H NMR (500MHz, CDCl3): d 7.40–6.93 (m, 50H),
6.77–6.26 (m, 20H), 5.04 (d, 1H, J 3.0Hz), 5.02 (d,
1H, J 3.4Hz), 4.99 (d, 1H, J 3.4Hz), 4.97 (d, 1H, J
3.4Hz), 4.94–4.88 (m, 3H), 4.84 (d, 1H, J 12.4Hz),
4.79–4.75 (m, 2H), 4.71–4.21 (m, 25H), 4.18–4.04 (m,
by flash chromatography (1:1 hexanes–EtOAc) to give
20
D
25 (986mg, 75%) as a foam. ½aꢁ +48.2 (c 1.0, CHCl3).
1H NMR (300MHz, CDCl3): d 7.47–7.25 (m, 35H),

M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
2167
5.00–4.89 (m, 4H), 4.86–4.59 (m, 14H), 4.52 (t, 1H, J
5.1Hz), 4.16–3.97 (m, 9H), 3.92 (br s, 1H), 3.90–3.30
(m, 13H), 2.19 (br s, 1H), 2.16 (br s, 1H), 2.09 (m,
1H), 1.81 (br s, 1H), 1.67–1.52 (m, 4H), 1.46–1.26 (m,
9H). 13C NMR (75MHz, CDCl3): d 138.91, 138.75,
138.41, 138.19, 137.91, 137.65, 137.60, 102.47, 100.79,
99.92, 97.54, 79.35, 78.50, 77.77 (2C), 77.49, 76.22,
74.95, 74.76, 74.62, 74.58, 73.22, 73.08, 72.96 (2C),
72.82, 72.20, 71.13, 69.89, 68.35, 66.95 (2C), 62.64,
60.94, 60.66, 60.43, 35.31, 29.49, 29.46, 29.38, 26.14,
25.97, 24.00. ESIMS: m/z 1342.7 [M+Na]+.
centrated to yield 1.08g of the crude triacid. To a soln of
this in MeOH (60mL) was added TMSCHN2 (8.8mL of
a 2.0M soln in hexanes, corresponding to 5.9equiv/
acid). The mixture was stirred at rt for 2h and then
concentrated, and the residue was purified by flash
chromatography (2:1 hexanes–EtOAc) to give 828mg
20
1
(79%) of 27 as a foam. ½aꢁ +41.6 (c 1.1, CHCl3). H
D
NMR (300MHz, CDCl3): d 7.38–7.02 (m, 35H), 5.18
(br s, 1H), 5.06 (d, 1H, J 2.7Hz), 4.93 (d, 1H, J
3.3Hz), 4.89 (d, 1H, J 2.6Hz), 4.81–4.34 (m, 19H),
4.25 (br s, 1H), 4.09 (br s, 1H), 4.05–3.96 (m, 2H),
3.91 (dd, 1H, J 10.3, 3.1Hz), 3.83–3.49 (m, 7H), 3.64
(s, 3H), 3.40 (dt, 1H, J 9.7, 6.8Hz), 3.19 (s, 3H), 3.17
(s, 3H), 1.96 (m, 1H), 1.56–1.43 (m, 4H), 1.34–1.12 (m,
9H). 13C NMR (75MHz, CDCl3): d 169.28, 168.85,
168.72, 138.67, 138.55, 138.46, 138.38 (2C), 138.33,
138.27, 102.40, 99.58, 98.77, 97.34, 78.75, 77.67, 76.67,
76.63, 75.95, 74.59, 74.52, 74.21, 73.29, 73.04 (2C),
72.90, 72.74, 72.49, 72.43, 72.37, 71.75, 71.10, 70.04,
68.89, 66.91 (2C), 53.50, 52.30, 51.74, 35.27, 29.44,
29.35, 29.31, 26.00, 25.92, 23.95. ESIMS: m/z 1424.4
[M+Na]+.
3.17. 7-(1,3-Dioxan-2-yl)-heptyl 2,3,4-tri-O-benzyl-a-D-
galactopyranosyl-(1fi4)-2,3-di-O-benzyl-a-D-galactopy-
ranosyl-(1fi4)-2,3-di-O-benzyl-a-D-galactopyranosyl-
(1fi4)-2,3-di-O-benzyl-a-D-galactopyranosyl-(1fi4)-
2,3-di-O-benzyl-a-D-galactopyranosyl-(1fi4)-2,3-di-O-
benzyl-a-D-galactopyranoside (26)
Hexasaccharide 24 (2.91g) was treated with CAN
(16.1g) as described above for 25 to give 26 (1.24g,
1
54%) as a foam. H NMR (300MHz, CDCl3): d 7.32–
7.20 (m, 65H), 5.04–4.93 (m, 6H), 4.90–4.63 (m, 24H),
4.54 (m, 1H), 4.21–4.00 (m, 13H), 3.98–3.35 (m, 28H),
3.30–3.14 (m, 3H), 2.24 (br s, 2H), 2.21 (br s, 2H),
2.10 (m, 1H), 1.96 (br s, 2H), 1.70–1.57 (m, 4H), 1.50–
1.30 (m, 9H). 13C NMR (75MHz, CDCl3): d 102.40,
100.79, 100.76, 99.94, 99.89, 99.87, 97.47, 66.88 (2C),
35.24, 29.42 (2C), 29.31, 26.07, 25.90, 23.94. Anal. Calcd
for C138H160O33: C, 70.63; H, 6.87. Found: C, 70.17; H,
6.83.
3.19. (Benzyl 2,3,4-tri-O-benzyl-a-D-galactopyranosyl-
uronate)-(1fi4)-(benzyl 2,3-di-O-benzyl-a-D-galactopyr-
anosyluronate)-(1fi4)-(benzyl (7-(1,3-dioxan-2-yl)-hep-
tyl 2,3-di-O-benzyl-a-D-galactopyranosid)uronate) (28)
Prepared from 25 as described above for 27 by using
22
PhCHN2 in Et2O and EtOAc instead of TMSCHN2.
20
D
Colorless oil. ½aꢁ +30.2 (c 0.9, CHCl3). 1H NMR
(300MHz, CDCl3): d 7.50–7.10 (m, 50H), 5.31–5.11
(m, 4H), 5.04 (br s, 1H), 4.97–4.34 (m, 24H), 4.24 (br
s, 1H), 4.18–4.08 (m, 3H), 4.00–3.50 (m, 9H), 2.09 (m,
1H), 1.71–1.57 (m, 4H), 1.49–1.25 (m, 9H). 13C NMR
(75MHz, CDCl3): d 168.33, 167.95, 167.62, 138.46
(2C), 138.29, 138.23, 138.19, 138.15, 138.05, 135.08,
134.99, 134.83, 102.18, 99.52, 98.81, 97.20, 78.61,
77.66, 76.57, 76.49, 76.20, 76.00, 74.28 (2C), 73.89,
73.26, 72.90, 72.56, 72.50, 72.31 (2C), 71.90, 71.51,
70.92, 70.03, 68.76, 66.71, 66.69 (2C), 66.58, 66.47,
35.09, 29.24, 29.16, 29.08, 25.81, 25.74, 23.73. ESIMS:
m/z 1654.8 [M+Na]+.
3.18. (Methyl 2,3,4-tri-O-benzyl-a-D-galactopyranosylur-
onate)-(1fi4)-(methyl 2,3-di-O-benzyl-a-D-galactopyra-
nosyluronate)-(1fi4)-(methyl (7-(1,3-dioxan-2-yl)-heptyl
2,3-di-O-benzyl-a-D-galactopyranosid)uronate) (27)
A soln of trisaccharide 25 (986mg, 0.747mmol) in
anhyd CH2Cl2 (15mL) was added to a suspension of
the Dess–Martin periodinane21 (1.43g, 3.37mmol) in
CH2Cl2 (30mL). The mixture was stirred at rt for
45min and then diluted with Et2O (50mL), quenched
with 10% aq Na2S2O3 (60mL) and stirred for an addi-
tional 30min. The phases were separated and the organ-
ic layer washed with satd aq NaHCO3 (50mL). The
combined aq phases were extracted with Et2O (50mL).
The combined organic phases were dried (MgSO4) and
the solvent removed in vacuo to give 1.01g of the crude
trialdehyde. This was taken up in a mixture of THF
(9mL) and t-BuOH (24mL) followed by addition of 2-
methyl-but-2-ene (12mL) and a soln of NaClO2
(2.03g, 22.4mmol) and NaH2PO4ÆH2O (2.32g,
16.8mmol) in water (11mL). The mixture was stirred
at rt for 1h and then poured into satd aq NaH2PO4
(150mL) and extracted with EtOAc (3·120mL). The
combined organic phases were dried (MgSO4) and con-
3.20. (Benzyl 2,3,4-tri-O-benzyl-a-D-galactopyranosyl-
uronate)-(1fi4)-(benzyl 2,3-di-O-benzyl-a-D-galactopyr-
anosyluronate)-(1fi4)-(benzyl 2,3-di-O-benzyl-a-D-
galactopyranosyluronate)-(1fi4)-(benzyl 2,3-di-O-ben-
zyl-a-D-galactopyranosyluronate)-(1fi4)-(benzyl 2,3-di-
O-benzyl-a-D-galactopyranosyluronate)-(1fi4)-(benzyl
(7-(1,3-dioxan-2-yl)-heptyl 2,3-di-O-benzyl-b-D-galacto-
pyranosid)uronate) (29)
Hexasaccharide 26 (480mg, 0.205mmol) was oxidized
with the Dess–Martin periodinane21 (800mg,
1.89mmol) and then with NaClO2 (1.1g) in the presence

2168
M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
of methyl-but-2-ene (6mL) and NaH2PO4ÆH2O (1.3g) as
described above for 27. The crude hexaacid was dis-
solved in EtOAc (15mL) and titrated over 2h at room
5.02–4.99 (m, 2H), 4.96 (br s, 1H), 4.95 (d, 1H, J 3.8Hz),
4.93 (d, 1H, J 3.4Hz), 4.67 (m, 1H), 4.47 (m, 1H), 4.39–
4.35 (m, 2H), 4.23 (br s, 1H), 4.00–3.89 (m, 3H), 3.82
(dd, 1H, J 10.4, 3.2Hz), 3.72 (m, 1H), 3.67 (dd, 1H, J
10.6, 3.4Hz), 3.64 (dd, 1H, J 10.2, 3.8Hz), 3.61–3.54
(m, 4H), 3.47 (m, 1H), 1.67 (p, 1H, J 6.4Hz), 1.54–
1.32 (m, 4H), 1.26–1.12 (m, 9H). 13C NMR (125MHz,
D2O): d 173.34, 172.64, 172.31, 103.17, 100.75, 100.64,
99.23, 79.10, 78.83, 71.57, 70.81, 70.62, 70.22, 69.71,
69.40, 68.89, 68.72, 68.52 (2C), 68.41, 67.65, 59.34,
34.87, 34.46, 29.22, 28.96, 28.88, 25.84, 23.84. ESIMS:
m/z 753.9 [M+Na]+.
22
temperature with a 1M soln of PhCHN2 in Et2O
until a persisting orange color was obtained. The mix-
ture was concentrated and the residue purified by flash
chromatography (3:2 hexanes–EtOAc) to afford 131mg
1
(22%) of 29 as a colorless syrup. H NMR (500MHz,
CDCl3): d 7.43–6.92 (m, 95H), 5.19 (d, 1H, J 3.4Hz),
5.15 (d, 1H, J 12.4Hz), 5.12 (d, 1H, J 3.4Hz), 5.09 (d,
1H, J 3.4Hz), 5.08 (d, 1H, J 3.4Hz), 5.05–4.92 (m,
4H), 4.84–4.08 (m, 49H), 4.04 (dd, 1H, J 10.2, 3.0Hz),
3.84 (dd, 1H, J 10.7, 3.4Hz), 3.83–3.51 (m, 14H), 3.46
(dt, 1H, J 10.2, 6.8Hz), 2.08 (m, 1H), 1.64–1.50 (m,
4H), 1.42–1.20 (m, 9H). 13C NMR (125MHz, CDCl3):
d 168.52, 168.17, 168.12, 167.93, 167.86, 167.55,
102.47, 99.64, 98.73, 98.70, 98.59, 98.51, 97.37, 66.97
(2C), 35.31, 29.47 (2C), 29.33, 26.01, 25.96, 23.97. ES-
IMS: m/z 2994.4 [M+Na]+. Anal. Calcd for
3.23. 7-(1,3-Dioxan-2-yl)-heptyl (a-D-galactopyranosyl-
uronic acid)-(1fi4)-(a-D-galactopyranosyluronic acid)-
(1fi4)-(a-D-galactopyranosyluronic acid)-(1fi4)-(a-D-
galactopyranosyluronic acid)-(1fi4)-(a-D-galactopyrano-
syluronic acid)-(1fi4)-a-D-galactopyranosiduronic acid
(32)
C180H184O39: C, 72.76; H, 6.24. Found: C, 72.48; H,
6.34.
Prepared from 29 using the same procedure as described
above for 30. White solid. 13C NMR (125MHz, D2O): d
173.67, 173.28, 173.21, 173.15 (2C), 173.11, 103.26,
100.65 (2C), 100.59 (3C), 99.18, 67.67 (2C), 34.47,
29.16, 28.91, 25.82, 25.75, 24.61. MALDI-TOFMS:
m/z 1257.7 [MꢀH]ꢀ.
3.21. (Methyl a-D-galactopyranosyluronate)-(1fi4)-
(methyl a-D-galactopyranosyluronate)-(1fi4)-(methyl (7-
(1,3-dioxan-2-yl)-heptyl a-D-galactopyranosid)uronate)
(30)
Trisaccharide 27 (118mg, 0.084mmol) was dissolved in
THF (2mL) and MeOH (6mL) and Ar was bubbled
through the soln for 5min. Then 10% Pd/C (72mg)
was added and the reaction mixture was stirred at rt
under 1atmof H 2. Water (2mL) was added after
15min and the mixture was stirred under a H2 atmo-
sphere overnight. Filtered through Celite and concen-
3.24. BSA-conjugate (33)
A soln of 30 (65mg, 0.084mmol) in AcOH (4mL) and
water (1mL) was heated at 50°C for 12h. It was then
concentrated and the residue dissolved in water (2mL).
BSA (23mg, 0.35lmol) was added and the mixture
was stirred for 1h. Then NaCNBH3 (13mg, 0.21mmol)
was added, and the stirring was continued at rt for
4days. Purified by filtration through a Centricon YM-
30 filter and lyophilized yielding 18.6mg (65%) of a
white solid. MALDI-TOFMS: m/z 82,541 [M]+ , which
corresponds to 22.6molligand/mol BSA (BSA standard:
66,431).
1
trated to give 30 (65mg, quant) as a white solid. H
NMR (500MHz, CD3OD): d 5.15 (br s, 1H), 5.09 (br
s, 1H), 4.94 (d, 2H, J 3.8Hz), 4.87 (d, 1H, J 3.8Hz),
4.53 (t, 1H, J 5.1Hz), 4.51 (br s, 1H), 4.40–4.36 (m,
2H), 4.35 (t, 1H, J 6.0Hz), 4.20 (dd, 1H, J 3.0,
1.3Hz), 4.04 (br dd, 1H, J 10.7, 5.1Hz), 3.91 (dd, 1H,
J 10.2, 3.0Hz), 3.89 (dd, 1H, J 10.2, 3.0Hz), 3.81 (s,
3H), 3.80–3.63 (m, 7H), 3.78 (s, 3H), 3.75 (s, 3H), 3.52
(m, 1H), 1.99 (m, 1H), 1.75 (p, 1H, J 6.4Hz), 1.69–
1.49 (m, 4H), 1.44–1.26 (m, 8H). 13C NMR (125MHz,
CD3OD): d 171.67, 171.14, 170.76, 106.12, 103.63,
102.01, 100.66, 80.39, 80.13, 72.77, 72.03, 71.85, 71.30,
70.75, 70.14, 69.91 (2C), 69.74, 69.72, 67.92 (2C),
60.09, 53.04, 52.86, 52.54, 36.27, 33.71, 30.46, 30.38,
27.08, 27.01, 25.54. ESIMS: m/z 795.5 [M+Na]+.
3.25. BSA-conjugate (34)
A soln of 31 (43mg, 0.059mmol) in AcOH (2.8mL)
and water (0.7mL) was heated at 50°C for 12h. It
was then concentrated and the residue taken up in
water (2mL) and the pH was adjusted to 9 with
NaHCO3. BSA (13mg, 0.20lmol) was added and
the mixture was stirred for 1h. Then NaCNBH3
(7.5mg, 0.12mmol) was added, and the stirring was
continued at rt for 2days. Purified by filtration through
a Centricon YM-30 filter and lyophilized yielding
11.7mg (81%) of a white solid. MALDI-TOFMS: m/z
73,980 [M]+, which corresponds to 11.5molligand/mol
BSA.
3.22. 7-(1,3-Dioxan-2-yl)-heptyl (a-D-galactopyranosyl-
uronic acid)-(1fi4)-(a-D-galactopyranosyluronic acid)-
(1fi4)-a-D-galactopyranosiduronic acid (31)
Prepared from 28 using the same procedure as described
above for 30. White solid. 1H NMR (500MHz, D2O): d

M. H. Clausen, R. Madsen / Carbohydrate Research 339 (2004) 2159–2169
2169
4. Willats, W. G. T.; Gilmartin, P. M.; Mikkelsen, J. D.;
Knox, J. P. Plant J. 1999, 18, 57–66.
3.26. BSA-conjugate (35)
5. Willats, W. G. T.; Limberg, G.; Buchholt, H. C.; van
Alebeek, G.-J.; Benen, J.; Christensen, T. M. I. E.; Visser,
J.; Voragen, A.; Mikkelsen, J. D.; Knox, J. P. Carbohydr.
Res. 2000, 327, 309–320.
6. Clausen, M. H.; Willats, W. G. T.; Knox, J. P. Carbohydr.
Res. 2003, 328, 1797–1800.
7. Jones, L.; Seymour, G. B.; Knox, J. P. Plant Physiol. 1997,
113, 1405–1412.
8. Willats, W. G. T.; Marcus, S. E.; Knox, J. P. Carbohydr.
Res. 1998, 308, 149–152.
9. Wong, S. S. Chemistry of Protein Conjugation and Cross-
Linking; CRC: Boca Raton, 1991.
10. Peters, T., Jr. All About Albumin––Biochemistry, Genetics
and Medicinal Applications; Academic: San Diego, 1996.
11. Lemieux, R. U.; Bundle, D. R.; Baker, D. A. J. Am. Chem.
Soc. 1975, 97, 4076–4083.
A soln of 32 (15mg, 0.012mmol) in AcOH (1.25mL)
and water (0.25mL) was heated at 50°C for 12h. The
solvent was removed under diminished pressure and
the residue taken up in water (1mL). pH was adjusted
to 9 with NaHCO3 and BSA (5mg, 0.08lmol) was
added. The soln was stirred for 1h followed by addition
of NaCNBH3 (30mg, 0.48mmol). The mixture was
stirred at rt for 4days. Purified by filtration through
a Centricon YM-30 filter and lyophilized yielding
5.0mg (94%) of a white solid. MALDI-TOFMS: m/z
70,996 [M]+, which corresponds to 3.8molligand/mol
BSA.
12. Clausen, M. H.; Jørgensen, M. R.; Thorsen, J.; Madsen,
R. J. Chem. Soc., Perkin. Trans. 1, 2001, 543–551.
13. Clausen, M. H.; Madsen, R. Chem. Eur. J. 2003, 9,
3821–3832.
14. Greene, T. W.; Wuts, P. G. M.. In Protective Groups in
Organic Chemistry, 3rd ed.; John Wiley and Sons: New
York, 1997, pp 724–727.
15. Yu, C.; Liu, B.; Hu, L. Tetrahedron Lett. 2000, 41,
4281–4285.
16. Mancuso, A. J.; Huang, S.-L.; Swern, D. J. Org. Chem.
1978, 43, 2480–2482.
Acknowledgements
We thank Dr. Kudzai E. Mutenda, Department of Bio-
chemistry and Molecular Biology, University of South-
ern Denmark for conducting analyses by mass
spectrometry. Financial support from the Danish Re-
search Agency Center Contract Programand the Lund-
beck Foundation are gratefully acknowledged.
17. Roelofsen, D. P.; Wils, E. R. J.; van Bekkum, H. Rec.
Trav. Chim. Pays-Bas. 1971, 90, 1141–1152.
18. For assignment of anomeric configuration based on
C-1, H-1 coupling constants, see: Bock, K.; Pedersen, C.
J. Chem. Soc., Perkin. Trans. 2, 1974, 293–297.
19. Lemieux, R. U.; Hendriks, K. B.; Stick, R. V.; James, K.
J. Am. Chem. Soc. 1975, 97, 4056–4062.
20. Konradsson, P.; Fraser-Reid, B. J. Chem. Soc., Chem.
Commun., 1989, 1124–1125.
21. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277–7287.
22. Creary, X. Org. Synth. 1986, 64, 207–216.
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