Synthesis of hexasaccharide fragments of pectin

Article abstract of DOI:10.1002/chem.200204636

Abstract: Short syntheses of partially methyl-esterified hexagalacturonates 1-5 are described as part of the development of strategies for the preparation of larger pectic oligosaccharides. The methodology is based on the repeated coupling of galactose mo

Full text of DOI:10.1002/chem.200204636

FULL PAPER
Synthesis of Hexasaccharide Fragments of Pectin
Mads H. Clausen and Robert Madsen*[a]
Abstract: Short syntheses of partially
methyl-esterified hexagalacturonates
5 are described as part of the devel-
yields of the desired a anomers. Penten-
yl disaccharide donors are prepared by
the coupling of two pentenyl galacto-
sides controlled by either the armed
disarmed effect or by converting one
pentenyl galactoside into the corre-
sponding galactosyl bromide or fluoride.
Two orthogonal protecting groups are
employed at C6,which makes it possible
to oxidize these positions to either the
carboxylic acid or to the methyl ester.
Each hexagalactan is therefore able to
bifurcate into two different hexagalac-
turonates with a reverse methyl-esterifi-
cation pattern. The methyl ester distri-
bution in the hexagalacturonates is con-
firmed by tandem mass spectrometry.
1
opment of strategies for the preparation
of larger pectic oligosaccharides. The
methodology is based on the repeated
coupling of galactose mono- and disac-
charide donors onto a galactose accept-
or until a hexagalactan is obtained. All
glycosylations are carried out with n-
pentenyl glycosides to provide good
Keywords: carbohydrates
¥ glyco-
sylation ¥ oligosaccharides ¥ pectin
¥ synthetic methods
methyl esters.[1,4] Chromatographic and mass spectrometric
analyses of the degradation products have shown that
polygalacturonases cleave preferentially between two uronic
acids.[5] The structural requirements for lyases and methyl
esterases are less certain,but it appears that they often prefer
small sequences of acid or ester units.[5] A major problem in
this regard is the lack of pectin fragments with a well-defined
methyl ester pattern which could serve to elucidate the
substrate specificity of these enzymes. With a better know-
ledge of their specificity,the utility of these enzymes for the
analysis of pectin would be greatly enhanced.[6]
Monoclonal antibodies are also important for the molecular
analysis of pectin. The binding of several antibodies is
dependent on methyl esterification state although for most
the detailed structure of the epitopes is not fully defined.[7]
The availability of well-defined oligogalacturonates would be
valuable for the elucidation of epitope structures.
We envisioned to make these pectin fragments available by
chemical synthesis,which required synthetic methods tailored
for this class of compounds. Although the common problems
in complex oligosaccharide synthesis have been recognized,[8]
a general solution to these challenges has not yet emerged and
the synthesis of larger saccharides still requires strategies
suited for the specifics of the target. We have recently
developed a procedure for synthesis of smaller pectin frag-
ments with a defined esterification pattern,which led to the
preparation of three different monomethylated trigalacturo-
nates.[9] While useful as reference compounds these trisac-
charides are in general too short to give reasonable rates when
employed as substrates for pectic enzymes.[10] In fact,recent
studies of several polygalacturonases and pectate lyases have
Introduction
Pectin is a complex polysaccharide,which is a major
component of the primary cell walls in higher plants.[1] In
the food industry pectin is used as a gelling and stabilizing
agent.[2] The main structural feature in pectin is a linear a1 !
4-linked oligomer of d-galacturonic acid which is partially
methyl-esterified. The functionality of pectin,both in planta
and in commercial samples,is largely determined by the
extent and pattern of methyl esterification. However,methyl
esterification states are typically highly heterogeneous and
present a considerable challenge for detailed analysis.[3] The
development of molecular tools is important for the better
understanding of the biological roles of pectin and to enhance
its industrial applications.
Several pectin-degrading enzymes are essential for the
elucidation of pectin structure because their activity is
influenced by the extent and pattern of methyl esterification.
These enzymes include pectin and pectate lyase as well as
[1,4]
polygalacturonase,which all cleave the oligomer chain.
Pectin and pectate lyase operate by a b-elimination mecha-
nism,whilst polygalacturonase is a hydrolase. Another
important enzyme is pectin methyl esterase,which hydrolyzes
[a] Prof. R. Madsen,Dr. M. H. Clausen
Department of Chemistry,Building 201
Technical University of Denmark
2800 Lyngby (Denmark)
Fax : (‡45)45933968
E-mail: rm@kemi.dtu.dk
Supporting information for this article is available on the WWW under
http://www.wiley-vch.de/home/chemeurj.org/ or from the author.
Chem. Eur. J. 2003, 9,3821 3832
DOI:10.1002/chem.200204636
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
3821

R. Madsen,M. H. Clausen
FULL PAPER
revealed that in most cases four to six galacturonic acids are
accommodated in the active site.[11] As a result,substrates should
be at least hexameric to provide meaningful information about
the cleavage pattern of these enzymes. However,selectively
methyl-esterified oligogalacturonates larger than trisacchar-
ides have never previously been obtained pure on a prepa-
rative scale by synthesis or isolation from natural sources.[12,13]
Accordingly,hexagalacturonates 1 5 will be prepared from
hexagalactans 6 8 in which an ester group and a p-methoxy-
phenyl (PMP) ether group serve as orthogonal C6 protecting
groups (Figure 1). Hereby,C6 can be oxidized to either the
carboxylic acid or to the corresponding methyl ester. By
switching the order of these two oxidations hexagalacturonate
1 and 2 can arise from the same galactan 6,while 3 and 4 can
both originate from 7. Hexagalactans 6 and 7 will be prepared
from the same tetrasaccharide 11 by coupling with disacchar-
ides 9 and 10,respectively. This ability of the key intermedi-
ates to diverge into two products is an important feature,
which reduces the number of steps for preparation of 1 4.
Hexagalacturonate 5 will arise from hexagalactan 8 con-
taining an alternating ether ester protecting group sequence
(Figure 1). The preparation of 8 will involve two consecutive
couplings with pentenyl disaccharide 13. An allyl group is
used as a temporary protecting group at C4' because this
group is stable to pentenyl glycosylations and can be removed
in the presence of ester and ether groups. We plan to assemble
this disaccharide by controlled coupling of two pentenyl
monosaccharides 15 and 16. The donor pentenyl glycoside 15
is fully protected with ether protecting groups,while the
acceptor pentenyl glycoside 16 has an ester group at C6 and
ether groups at C2 and C3. Electron-withdrawing ester groups
Herein,we report the synthesis of hexagalacturonates 1
5
(Figure 1). Compounds 1 4 all contain a block of three to
four acid or ester residues. As many pectic enzymes appear to
prefer small blocks of acids or esters these four molecules
should provide valuable information about their substrate
specificity. Compound 5 with an alternating acid ester
sequence serves as a reference,since it should not be cleaved
readily by any of the pectic enzymes.
Results and Discussion
Retrosynthesis: In our earlier work,we assembled small
oligogalacturonates by the coupling of galactose glycosyl
donors and acceptors followed by oxidation at C6.[9] The
glycosylations were carried out with n-pentenyl glycosyl
donors[14] possessing two different protecting groups at C6.[9]
OAc
OAc
BnO
HO
BnO
BnO
COOR1
O
O
O
HO
BnO
HO
O
BnO
OR
O
BnO
OR
O
2
O
O
COOR
O
HO
BnO
BnO
O
+
HO
BnO
OPMP
O
BnO
3
O
O
COOR
O
9: R = Ac
HO
BnO
OPMP
O
HO
10: R = PMP
HO
BnO
OPMP
O
3
O
O
COOR
BnO
O
HO
BnO
BnO
OPMP
O
O
HO
BnO
OPMP
O
3
O
O
COOR
BnO
O
HO
BnO
BnO
OPMP
O
O
HO
BnO
OAc
O
1
O
O
1: R1 = R2 = Me, R3 = H
2: R1 = R2 = H, R3 = Me
3: R1 = Me, R2 = R3 = H
4: R1 = H, R2 = R3 = Me
COOR
BnO
O
6: R = Ac
HO
BnO
OBn
BnO
OAc
O
O
OH
7: R = PMP
HO
BnO
BnO
OBn
11
BnO
OAc
OAc
O
BnO
BnO
12
HO
BnO
COOMe
O
O
O
HO
BnO
BnO
HO
BnO
OPMP
O
O
O
COOH
+
OPMP
O
O
O
HO
BnO
OPMP
O
O
HO
BnO
Oester
O
BnO
O
O
O
COOMe
BnO
O
BnO
HO
BnO
15
BnO
Oester
O
O
HO
O
BnO
OPMP
O
O
+
COOH
O
BnO
13
O
HO
BnO
Oester
O
HO
BnO
HO
BnO
Oester
O
2
O
O
COOMe
+
BnO
16
O
O
HO
BnO
BnO
OPMP
O
HO
HO
O
BnO
OPMP
O
O
COOH
O
BnO
14
OBn
HO
BnO
OBn
5
8
BnO
OH
HO
BnO
Figure 1. Retrosynthesis for partially methyl-esterified hexagalacturonates 1 5.
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
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Chem. Eur. J. 2003, 9,3821 3832

Hexasaccharide Fragments of Pectin
3821 3832
are known to decrease the reactivity of glycosyl donors. It is
also known that two pentenyl glycosides can be selectively
coupled if the donor glycoside is fully protected with ether
groups while the acceptor is protected with ester groups at all
positions but one. This phenomenon has been termed the
™armed disarmed effect∫ where the donor is armed and the
acceptor disarmed toward the coupling reaction.[15] However,
this effect has never been extended to an acceptor which
contains only one ester group at C6 and two ether groups at
C2 and C3. The question is whether one ester group alone is
sufficiently electron-withdrawing to allow this selective cou-
pling to occur.
Pentenyl disaccharide 13: To probe this modification of the
armed disarmed coupling protocol we carried out a number
of glycosylations between donor 15 and ester-protected
acceptors 16. All the glycosylations were performed with N-
iodosuccinimide (NIS) and a catalytic amount of triethylsilyl
trifluoromethanesulfonate (TESOTf) in CH2Cl2. The weaker
promoter iodonium dicollidine perchlorate (IDCP)[14] was not
reactive enough and led to incomplete conversion of the
coupling partners. In the first reaction,acetate protected
acceptor 16a was employed giving rise to a 27% yield (based
on 15) of the desired disaccharide 13a (Table 1,entry 1). Small
esters. Three acceptors containing benzoate groups at C6 were
prepared and glycosylated with 15: 4-nitrobenzoate 16d,35, -
dinitrobenzoate 16e,and pentafluorobenzoate (PFBz) 16 f
(Table 1,entries 4 6). Interestingly,these all gave rise to
higher yields of disaccharide 13 and the best result was
obtained with the pentafluorobenzoate at C6. The 52% yield
of 13 f is an acceptable result for further synthetic applica-
tions. No other coupling products were observed by TLC,but
some unreacted acceptor 16 f was recovered. The controlled
coupling of 15 and 16 f represents a new application of the
armed disarmed effect in glycoside synthesis. The electronic
properties of the different ester groups are evident from the
chemical shift of the carbonyl group in the 13C NMR spectra
of 16a 16 f. The disaccharides 13a 13 f were in all cases
obtained as a 6:1 mixture of a and b anomers,regardless of the
ester protecting group. Unfortunately,the anomeric mixture
could not be separated by silica gel chromatography in any of
the six cases.
Pentenyl disaccharides 9 and 10: We would also like to
prepare disaccharides 9 and 10 from the corresponding
pentenyl monosaccharides 12, 16a,and 17 (Scheme 1). The
OAc
BnO
Table 1. Coupling of pentenyl galactosides 15 and 16 controlled by the
armed disarmed effect.
O
BnO
OPMP
O
F
BnO
O
18
a
BnO
b
Oester
O
Oester
O
OAc
O
BnO
BnO
13
O
OAc
HO
BnO
74%
BnO
BnO
HO
BnO
NIS
55%
O
15
+
O
O
O
O
TESOTf
CH2Cl2
9
BnO
BnO
16
BnO
12
BnO
16a
OAc
d
α:β = 6:1
c
40%
Entry
Acceptor
ester (dC O[ppm])
Product
Yield [%]
ˆ
BnO
BnO
O
1
2
3
4
5
6
16a
16b
16c
16d
16e
16 f
Ac (170.9)
ClAc (167.1)
TCA (161.8)
4-NO2Bz (164.6)
3,5-di-NO2Bz (162.5)
PFBz (158.7)
13a
13b
13c
13d
13e
13 f
27
33
35
42
46
52
BnO
Br
19
d
10
61%
OPMP
O
HO
BnO
O
BnO
17
amounts of oligomers originating from the initial homocou-
pling of 16a were also obtained,but were not further
characterized. Disaccharide 13a was obtained as a 6:1 a:b
mixture,which could not be separated. After removing the
acetate,the anomers were separated and fully characterized.
Although the yield of disaccharide 13a was not syntheti-
cally useful it did provide some hope that the armed dis-
armed coupling would indeed be feasible. Several more
electron-withdrawing ester groups were therefore investigat-
ed. In fact,glycosylation with chloroacetate (ClAc) and
trichloroacetate (TCA) protected acceptors 16b and 16c
afforded a slightly higher yield of the corresponding disac-
charides 13b and 13c (Table 1,entries 2 and 3). It was also
attempted to prepare 16 with a trifluoroacetate at C6.
Unfortunately,this group was very sensitive to hydrolysis
upon purification by chromatography giving rise to low yields
in the preparation of 16 and the coupling to 15. Consequently,
attention then shifted toward electron-withdrawing aromatic
Scheme 1. a) NBS,DAST,CH 2Cl2, 08C; b) SnCl2,AgClO 4,4 ä MS,
CH2Cl2, À308C; c) Br2,CH 2Cl2, 08C; d) AgOTf,4 ä MS,CH 2Cl2, À508C.
armed disarmed protocol cannot be employed in this case
because donor 12 has an ester group at C6. Instead, 12 should
be converted into another glycosyl donor that can be coupled
under conditions not affecting a pentenyl moiety. Therefore,
the transformation of 12 into the corresponding glycosyl
fluoride and bromide was investigated. The conversion of
pentenyl glycosides into glycosyl bromides is a well-known
reaction,[16] while the conversion into glycosyl fluorides has
not been achieved before. On the other hand,thioglycosides
are known to undergo reaction into glycosyl fluorides in the
presence of N-bromosuccinimide (NBS) and (diethylamino)-
sulfur trifluoride (DAST).[17] In fact,subjecting pentenyl
glycoside 12 to these conditions gave glycosyl fluoride 18 as
a 3:1 a:b mixture which was purified by flash chromatography.
Chem. Eur. J. 2003, 9,3821 3832
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R. Madsen,M. H. Clausen
FULL PAPER
The anomeric configurations were assigned on the basis of
1H NMR spectra. For the a anomer H1 appeared as a double
doublet at d ˆ 5.60 ppm with JH1,F ˆ 54 Hz and JH1,H2 ˆ 2.8 Hz,
while in the b anomer H1 was observed at d ˆ 5.19 ppm with
JH1,F ˆ 53 Hz and JH1,H2 ˆ 6.6 Hz. Subsequent coupling of this
anomeric mixture to acceptor 16a was performed under
Mukaiyama conditions[18] to afford a-linked disaccharide 9 in
55% yield and the corresponding b anomer in 18% yield. In
this case,the anomeric configuration was determined from
13C NMR spectra. For the a anomer,the C1 ' signal appeared
at d ˆ 100.5 ppm with JC1,H1 ˆ 167.8 Hz,while C1 ' for the b
Hexagalacturonates 1 4: Having finished the preparation of
the necessary building blocks,the stage was now set for
synthesis of hexamers 1 4. Compounds 1 and 2 were first
prepared and the synthesis commenced with the coupling of 9
and 11 (Scheme 3). In the workup the three primary acetyl
¬
groups were removed by Zemplen deacetylation and hexa-
saccharide 23 was obtained in a satisfying 71% overall yield as
the pure a anomer. Oxidation to uronic acid and esters was
achieved by a one-pot procedure. The primary alcohols were
oxidized by the Dess Martin periodinane[20] and the crude
trialdehyde further oxidized with sodium chlorite. The
[21]
anomer was observed at d ˆ 103.3 ppm with JC1,H1
ˆ
obtained triacid was then esterified with either TMSCHN2
159.6 Hz.[19]
to give 24 (44% yield from 23) or with PhCHN2[22] to afford 25
(59% yield from 23). The latter benzyl protection reaction
should be noticed. It is a very mild and efficient method for
masking the carboxylic acids until they will be liberated again
after the final hydrogenolysis. Furthermore,the benzyl
protection of the acids facilitates handling and purification
of the intermediates.
To explore the alternative method,pentenyl galactoside 12
was titrated with bromine,which formed glycosyl bromide 19
very cleanly as judged by 1H NMR spectroscopy (Scheme 1).
Only the a anomer of 19 was observed with H1 as a doublet at
d ˆ 6.41 ppm (JH1,H2 ˆ 2.0 Hz). Bromide 19 was too unstable to
be isolated and the crude reaction mixture was cannulated
into a solution of AgOTf,4ä molecular sieves,and acceptor
16a. Hereby,the a anomer of disaccharide 9 was obtained in
40% yield based on acceptor 16a and only traces of the
corresponding b anomer were observed. All in all,this latter
synthesis of 9 is more convenient than the glycosyl fluoride
route. It involves only one chromatographic purification and
the overall yield from 12 is approximately the same in both
cases. Thus,bromide 19 was also adopted for coupling to
acceptor 17 to furnish a-linked disaccharide 10 in 61% yield.
To complete the syntheses the PMP ethers in 24 and 25
were removed and the obtained triols oxidized and esterified
to the corresponding benzyl and methyl esters. In this way, 26
was obtained from 24 in 48% overall yield,while 27 was
prepared from 25 in 30% yield. Deprotection of the benzyl
ethers and esters in 26 and 27 then provided selectively
trimethyl-esterified hexagalacturonates 1 and 2. The struc-
tures of 1 and 2 were confirmed by tandem mass spectrometry
using the electrospray ionization technique with an ion-trap
mass spectrometer.[5a] Fragmentation of the molecular ion of 1
and 2 occurred in both cases from the reducing end liberating
one galacturonic acid unit at the time,as shown with 2 in
Figure 2. Hereby,the ions of the corresponding penta-,tetra-,
tri-,and digalacturonates were observed which indicated the
Tetragalactan 11: The tetrasaccharide 11 was prepared by two
consecutive couplings of monosaccharide donor 21 to di-
saccharide acceptor 20 (Scheme 2). Both 20 and 21 were
1
positions of the methyl ester groups in 1 and 2. The H and
13C NMR spectra of 1 and 2 were more complex due to the
similarity of the six galacturonic acid units and the anomeric
mixture at the reducing end.
OPMP
O
OPMP
O
HO
BnO
HO
BnO
BnO
OAc
O
BnO
OPMP
O
O
O
A similar series of reactions were employed for preparation
of 3 and 4 (Scheme 3). Coupling of 10 and 11 followed by
deacetylation gave hexasaccharide 28 in a very good 93%
yield. Oxidation and esterification of the two primary alcohols
was then carried out using the same one-pot procedure as
described above. Hereby,dimethyl ester 29 was obtained in
44% yield from 28 while dibenzyl ester 30 was prepared in a
slightly higher 52% yield. The four PMP groups between
these two ester moieties were then removed followed by
another round of oxidations and esterifications. This gave rise
to tetrabenzyl ester 31 in 42% yield from 29,while tetra-
methyl ester 32 was prepared in 31% yield from 30. Finally,
hydrogenolysis of 31 and 32 furnished the target hexagalac-
turonates 3 and 4,respectively. Again,the analysis by tandem
mass spectrometry verified the structures of these two
hexagalacturonates.
a
BnO
20
OBn
BnO
88%
BnO
BnO
OAc
O
O
BnO
OBn
BnO
OPMP
O
ClAcO
BnO
22
O
a
BnO
21
90%
11
Scheme 2. a) NIS,TESOTf,CH 2Cl2, À208C,then thiourea,NaHCO
Bu4NI,THF,55 8C.
,
3
prepared in our earlier study.[9] In the donor 21 a chloroace-
tate at C4 serves as a temporary protecting group during the
glycosylations. It is removed after the coupling with thiourea
leaving the primary acetate unaffected. The glycosylation
between 20 and 21 was performed with NIS and TESOTf to
furnish the desired trisaccharide,which was subsequently
treated with thiourea to afford alcohol 22 in 88% overall yield
as a pure a anomer. Coupling again to donor 21 followed by
chloroacetate removal gave tetrasaccharide 11 in 90% yield.
Hexagalacturonate 5: The alternating acid ester sequence in
hexamer 5 required a different synthetic strategy than
employed for hexamers 1 4. Two consecutive couplings
should now be performed with disaccharide 13 f to mono-
saccharide acceptor 14. Unfortunately,it was impossible to
3824
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Chem. Eur. J. 2003, 9,3821 3832

Hexasaccharide Fragments of Pectin
3821 3832
OH
1
1
BnO
BnO
BnO
COOR
O
COOR
O
O
BnO
BnO
BnO
BnO
OR
O
BnO
BnO
O
2
2
O
O
COOR
O
R
O
BnO
BnO
BnO
BnO
OPMP
O
BnO
OPMP
O
BnO
O
3
O
O
COOR
O
BnO
a
BnO
BnO
BnO
OPMP
O
BnO
OPMP
O
BnO
O
3
O
O
COOR
O
9
d, then
b or c
b or c
or
BnO
BnO
BnO
+
11
BnO
OPMP
O
BnO
OPMP
BnO
O
3
10
O
O
COOR
O
O
BnO
BnO
BnO
BnO
OH
BnO
BnO
O
1
1
O
O
COOR
COOR
O
O
O
BnO
OBn
BnO
OBn
BnO
OBn
e
BnO
BnO
BnO
24: R1 = Me, R2 = COOMe (44%)
25: R1 = Bn, R2 = COOBn (59%)
29: R1 = Me, R2 = CH2OPMP (44%)
30: R1 = Bn, R2 = CH2OPMP (52%)
26: R1 = R2 = Me, R3 = Bn (48%)
27: R1 = R2 = Bn, R3 = Me (30%)
31: R1 = Me, R2 = R3 = Bn (42%)
32: R1 = Bn, R2 = R3 = Me (31%)
1
23: R = H (71%)
28: R = PMP (93%)
e
e
e
2
3
4
Scheme 3. a) NIS,TESOTf,CH 2Cl2, À208C,then NaOMe,MeOH,THF; b) Dess Martin periodinane,CH 2Cl2,then NaClO 2,NaH PO4,THF, tBuOH,
2
H2O,then TMSCHN 2,THF,MeOH; c) Dess Martin periodinane,CH 2Cl2,then NaClO 2,NaH 2PO4,THF, tBuOH,H 2O,then PhCHN 2,EtOAc; d) CAN,
MeCN,H O; e) H2,Pd/C,THF,MeOH,H 2O.
2
Figure 2. ESI MS/MS spectrum of hexagalacturonate 2.
obtain 13 f anomerically pure. Coupling with an anomeric
mixture of 13 f did cause some concern with regard to
purification of the coupling products. However,all the
previous pentenyl couplings in Scheme 2 and Scheme 3
proceeded with very high a-selectivity and separating the
coupling products was not a problem. Consequently,acceptor
14 was glycosylated with the anomeric mixture of 13 f
followed by removal of the allyl protecting group at C4''
(Scheme 4). Gratifyingly,it was possible to isolate a good
yield of anomerically pure trisaccharide 33. Initially,removal
of the allyl group did create some problems. Excess Pd(OAc)2
in aqueous acidic acid is often employed as a protocol for allyl
group removal in carbohydrate chemistry.[23] Unfortunately,
when these conditions were applied in our case a substantial
amount of the propan-2-on-1-yl substituted trisaccharide was
obtained as a by-product most likely arising from a competing
Wacker oxidation. This prompted us to look for other
methods. In this regard,several transition metal complexes
are known to catalyze isomerization of allyl ethers to prop-2-
en-1-yl ethers.[23] Wilkinson×s catalyst [ClRh(PPh3)3] is often
used for this purpose,but in our case it reacted too slowly.
Instead,we treated Wilkinson×s catalyst with butyl lithium
Chem. Eur. J. 2003, 9,3821 3832
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
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R. Madsen,M. H. Clausen
FULL PAPER
1
BnO
R
O
BnO
OPMP
O
HO
BnO
BnO
2
O
R
O
BnO
BnO
OPFBz
O
O
OPMP
O
BnO
HO
BnO
1
O
R
BnO
O
BnO
BnO
OPMP
O
O
BnO
BnO
OPFBz
O
2
O
O
R
b
a
e
a
O
BnO
13f
+ 14
5
BnO
BnO
BnO
OPFBz
62%
51%
O
41%
BnO
BnO
OPMP
1
O
O
R
O
O
O
BnO
BnO
BnO
OBn
BnO
OPMP
O
BnO
BnO
2
O
R
O
O
BnO
OBn
33
BnO
OBn
BnO
34
BnO
35: R1 = CH2OH, R2 = CH2OPMP
36: R1 = COOMe, R2 = CH2OPMP
37: R1 = COOMe, R2 = COOBn
c
54%
d
31%
‡
Scheme 4. a) NIS,TESOTf,CH 2Cl2, À208C,then [ClRh(PPh 3)3], nBuLi,THF,67 8C,then Amberlite IR-120 (H ),MeOH,50 8C; b) NIS,TESOTf,CH 2Cl2,
À208C,then NaOMe,MeOH,THF; c) Dess Martin periodinane,CH 2Cl2,then NaClO 2,NaH PO4,THF, tBuOH,H O,then TMSCHN 2,THF,MeOH;
2
2
d) CAN,MeCN,H 2O,then Dess Martin periodinane,CH 2Cl2,then NaClO 2,NaH 2PO4,THF, tBuOH,H 2O,then PhCHN 2,EtOAc; e) H 2,Pd/C,THF,
MeOH,H O.
2
prior to the isomerization which is known to generate a more
Preliminary experiments have revealed that hexagalactur-
onates 1 and 3 are cleaved by endo-polygalacturonase I and II
from Aspergillus niger while hexamers 2, 4,and 5 are not
substrates for these two enzymes.[25] These observations
confirm that endo-polygalacturonase preferentially cleaves
the oligomer chain between two galacturonic acid residues.
The results are promising and show that the synthetic
hexagalacturonates can be used as substrates for studying
the cleavage pattern of pectic enzymes. Details of the
enzymatic studies will be published elsewhere.
reactive catalyst in situ,presumably [HRh(PPh )3].[24] With
3
this method the allyl group was isomerized completely in 1 h
to provide a 2:1 mixture of (Z) and (E)-prop-2-enyl ethers as
determined by 1H NMR spectroscopy. These were then
cleaved by addition of methanol and acidic ion-exchange
resin followed by stirring for 24 h. Despite the long reaction
time this procedure gave very clean removal of the allyl group
without affecting the pentafluorobenzoyl group.
Trisaccharide 33 was then ready for another glycosylation
with the anomeric mixture of disaccharide 13 f. Again,the
coupling followed by allyl group removal generated a pure
coupling product 34. In this case,cleavage of the allyl ether by
isomerization and methanolysis was slower than with the
trisaccharide above,but the reaction still proceeded very
cleanly. Pentasaccharide 34 was then coupled with mono-
saccharide donor 12. The crude product was subjected to
alkaline methanol solution to remove the acetyl group and the
two pentafluorobenzoyl groups. Hereby,hexasaccharide 35
was isolated in 41% overall yield. The three primary hydroxy
groups were then oxidized to methyl esters to give triester 36.
Oxidative cleavage of the PMP ethers followed by another
round of alcohol oxidations then afforded protected hexaga-
lacturonate 37. Ultimately,hydrogenolysis gave the desired
trimethyl esterified hexamer 5. The alternating acid ester
sequence was confirmed by tandem mass spectrometry where
fragmentation again occurred from the reducing end with one
monosaccharide at the time. Finally,a general trend should be
noticed about the 13C NMR data for all the galactose and
galacturonic acid containing compounds in this study. The
anomeric carbon atom in an a-glycosidic linkage is always
found in the d ˆ 98 101 ppm range,while C1 for a b-linkage
appears between d ˆ 102 and 105 ppm. These values are in
accordance with earlier observations.[9]
Conclusion
In conclusion,we have developed a general and divergent
protocol for synthesis of selectively methyl-esterified oligo-
mers of galacturonic acid. The syntheses feature several
different aspects of pentenyl glycoside chemistry particularly
coupling of two pentenyl glycosides controlled by either the
armed disarmed effect or by prior conversion of the donor
glycoside into a glycosyl halide. This has resulted in the
assembly of hexagalacturonates 1 5,where about 100 mg has
been obtained of each oligomer. These constitute the most
complex oligogalacturonates with a well-defined esterifica-
tion pattern prepared to date. Currently,hexamers 1 5 are
being investigated as substrates for several pectic enzymes
and as tools for the elucidation of the precise epitope
structures of a number of monoclonal antibodies.
Experimental Section
General: Thin-layer chromatography was performed on aluminum plates
precoated with silica gel. Compounds were visualized by heating after
3826
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9,3821 3832

Hexasaccharide Fragments of Pectin
3821 3832
dipping in a solution of Ce(SO4)2 (2.5 g) and (NH4)6Mo7O24 (6.25 g) in 10%
aqueous H2SO4 (250 mL). Flash chromatography was performed using
silica gel 60. Optical rotations were measured with a Perkin-Elmer 241
polarimeter. NMR spectra were recorded on a Varian Unity Inova 500 or a
Varian Mercury 300 spectrometer. Unless otherwise stated 1H and
13C NMR spectra were recorded in CDCl3 solution at 500 and 75 MHz,
respectively. Mass spectrometry was carried out at the Department of
Biochemistry and Molecular Biology,University of Southern Denmark.
Tandem mass spectra were acquired on an Esquire-LC quadrupole ion-trap
mass spectrometer (Bruker Daltonik) using electrospray ionization in the
negative-ion mode. Microanalyses were conducted by the Department of
Chemistry at the University of Copenhagen.
(50 mL). Sodium (12 mg,0.5 mmol) was added followed by stirring at 20 8C
until full conversion was observed by TLC (1 24 h). The reaction was
‡
quenched with Amberlite IR-120 (H ) (5 mL) and stirred for an additional
30 min. The resin was filtered off,washed with CH 2Cl2,and the filtrate was
concentrated and purified by flash chromatography.
General procedure for removal of the 4-methoxyphenyl group (Scheme 3
and 4): The protected saccharide (0.5 mmol) was dissolved in MeCN
(20 mL) and cooled to 08C. A solution of cerium ammonium nitrate (CAN;
5 equiv/PMP group) in water (5 mL) was added. The reaction mixture was
stirred for 15 min,then diluted with CHCl 3 and washed twice with H2O. The
combined aqueous phases were extracted with CHCl3. The combined
organic phases were dried,concentrated,and purified by flash chromatog-
raphy.
General procedure for acylations to prepare 16a 16 f: Pent-4-enyl 2,3-di-
O-benzyl-b-d-galactopyranoside[9] (4.28 g,10.0 mmol) was dissolved in
anhydrous CH2Cl2 (40 mL). The solution was cooled to 08C followed by
addition of Et3N (2.23 mL,16.0 mmol) and the acyl anhydride or chloride
(11 mmol). The mixture was stirred at 08C until TLC revealed full
conversion (0.5 2 h). Silica gel (10 g) was added and the mixture was
concentrated and purified by flash chromatography.
General procedure for oxidation to uronic acid (Scheme 3 and 4): To a
suspension of the Dess Martin periodinane (1.5 equiv/alcohol) in anhy-
drous CH2Cl2 (36 mL) was added
a solution of the hexasaccharide
(0.5 mmol) in CH2Cl2 (24 mL). The reaction was stirred for 45 60 min,
then diluted with Et2O (120 mL),quenched with 10% aqueous Na 2S2O3
(120 mL),and stirred for an additional 30 min. The organic phase was
separated and washed with saturated aqueous NaHCO3. The combined
aqueous phases were extracted with Et2O (100 mL). The combined organic
phases were dried and concentrated. The crude aldehyde was taken up in
THF (11 mL) followed by addition of tBuOH (26 mL),2-methylbut-2-ene
General procedure for armed disarmed glycosylation reactions (Table 1):
A mixture of 15 (1.0 mmol) and the acceptor (1.3 mmol) was dried
azeotropically with toluene and subjected to high vacuum for 2 h. The
mixture was dissolved in anhydrous CH2Cl2 (13 mL),cooled to À208C,
followed by addition of NIS (293 mg,1.3 mmol) and TESOTf (0.06 mL,
0.26 mmol). The reaction mixture was stirred at À208C for 30 min. The
solution was diluted with CH2Cl2 and washed with 10% aqueous Na2S2O3
and saturated aqueous NaHCO3. The combined aqueous phases were
extracted with CH2Cl2. The combined organic phases were dried,
concentrated,and purified by flash chromatography.
(50 equiv/aldehyde),and a solution of NaClO (10 equiv/aldehyde) and
2
NaH2PO4 ¥ H2O (7.5 equiv/aldehyde) in H2O (11 mL). The reaction was
stirred at 208C until full conversion was observed by TLC (1 2 h). The
mixture was concentrated to half the volume and acidified with 1m aqueous
HCl. The aqueous phase was extracted three times with EtOAc. The
combined organic phases were dried and concentrated to afford the crude
acid.
General procedure for glycosylations via glycosyl bromides (Scheme 1):
Pentenyl glycoside 12[9] (9.0 mmol) was dissolved in anhydrous CH2Cl2
(30 mL),cooled to 0 8C,and titrated with a 1 m solution of Br2 in CH2Cl2
until a faint yellow color persisted. The solution was cannulated into a
mixture of the acceptor (5.0 mmol),4ä MS (10 g),and AgOTf (3.47 g,
13.5 mmol) in anhydrous CH2Cl2 (15 mL) at À508C. The reaction was
stirred at À508C and monitored by TLC (2 4 h). It was then quenched by
addition of saturated aqueous NaHCO3 (50 mL),allowed to reach room
temperature and filtered through Celite. The pad was rinsed with CH2Cl2.
The phases were separated and the aqueous phase extracted with CH2Cl2.
The combined organic phases were dried,concentrated,and purified by
flash chromatography.
General procedure for methyl esterification (Scheme 3 and 4): The crude
product from the oxidation was taken up in THF (2.8 mL) and MeOH
(25 mL) followed by addition of TMSCHN2 (2m solution in hexanes,
6 equiv/acid). The reaction was stirred for 12 24 h and monitored by TLC
(more TMSCHN2 added if necessary). When full conversion was observed,
the reaction mixture was concentrated and purified by flash chromatog-
raphy.
General procedure for benzyl esterification (Scheme 3 and 4): The crude
product from the oxidations was taken up in EtOAc (30 mL) and titrated
[22]
with PhCHN2
(0.5m solution in Et2O,1.5 2 equiv/acid) until full
conversion was observed by TLC (1 2 h). The reaction mixture was
concentrated and purified by flash chromatography. Note: PhCHN2 is
potentially explosive and may burn violently when exposed to air. Can be
redistilled under high vacuum at room temperature and should not be
subjected to temperatures above 308C. Obtained as a red liquid,which
solidifies at temperatures below À308C.
General procedure for hydrogenolysis (Scheme 3 and 4): The protected
hexagalacturonate (0.1 mmol) was dissolved in THF (5 mL) and MeOH
(15 mL). Then 10% Pd/C (125 mg) was added and an atmosphere of H2
(1 atm) was installed. The reaction mixture was stirred for 2 3 h followed
by addition of H2O (5 mL). The stirring under the H2 atmosphere was then
continued until TLC revealed only one spot (12 30 h). The mixture was
filtered through Celite,and lyophilized yielding a white solid.
General procedure for glycosylations via pentenyl glycosides (Schemes 2
4): A mixture of the donor (6.5 mmol) and the acceptor (5.0 mmol) was
dried azeotropically with toluene and subjected to high vacuum for 2 h. The
mixture was dissolved in anhydrous CH2Cl2 (60 mL),cooled to À208C,
followed by addition of NIS (1.49 g,6.63 mmol) and TESOTf (0.29 mL,
1.3 mmol). The reaction mixture was stirred at À208C until TLC revealed
full conversion of the donor (15 45 min). Workup as described above.
General procedure for removal of chloroacetyl group (Scheme 2): The
protected saccharide (5 mmol) was dissolved in THF (50 mL). Thiourea
(1.14 g,15 mmol),NaHCO
(1.39 g,16.5 mmol),and Bu 4NI (369 mg,
3
1 mmol) were added and the suspension heated to 558C. The reaction
was monitored by TLC until full conversion was observed (12 24 h). The
mixture was cooled,filtered,concentrated,and purified by flash chroma-
tography.
Pent-4-enyl 6-O-acetyl-2,3,4-tri-O-benzyl-a-d-galactopyranosyl-(1 ! 4)-6-
O-acetyl-2,3-di-O-benzyl-b-d-galactopyranoside (9): [a]2D0 ˆ ‡38.3 (c ˆ
0.5,CHCl 3); 1H NMR: d ˆ 7.46 7.18 (m,25H),5.82 (m,1H),5.03 (dq,
General procedure for removal of allyl group (Scheme 4): Wilkinson×s
catalyst [ClRh(PPh3)3] (463 mg,1.0 mmol,prepared from RhCl 3 ¥ 3H2O[26]
)
J ˆ 16.8,1.5 Hz,1H),4.99 4.66 (m,11H),4.59 (d,
J ˆ 11.2 Hz,1H),4.44
J ˆ 9.7,
was dissolved in anhydrous THF (27 mL) and the solution was degassed.
nBuLi (0.75 mL,1.6 m in hexanes,1.2 mmol) was added,and the mixture
was stirred for 10 min. A degassed solution of the protected saccharide
(5 mmol) in anhydrous THF (40 mL) was heated to reflux,and the solution
of the catalyst was added. The reaction mixture was refluxed until 1H NMR
spectroscopy revealed full conversion into the vinyl ether (1 6 h). It was
then cooled to 508C followed by addition of MeOH (140 mL) and
4.29 (m,4H),4.13 4.02 (m,3H),3.98 3.86 (m,4H),3.64 (dd,
7.6 Hz,1H),3.55 (dt, J ˆ 9.7,7.1 Hz,1H),3.49 (t, J ˆ 6.6 Hz,1H),3.38 (dd,
J ˆ 10.2,3.1 Hz,1H),2.17 (m,2H),2.01 (s,3H),1.81 (s,3H),1.77 ppm (m,
2H); 13C NMR: d ˆ 170.59,170.22,138.83,138.79,138.60,138.54,138.43,
138.17,115.03,104.07,100.46,80.23,79.14,79.05,76.54,75.57,75.15,74.71,
74.60,73.99,73.10,72.78,72.41,69.69,69.01,62.70,62.65,30.37,29.14,21.02,
20.89 ppm; ESI MS (944.4): m/z 967.5 [M ‡ Na].
‡
Amberlite IR-120 (H ) (15 mL). The resulting mixture was stirred at 508C
Pent-4-enyl 6-O-acetyl-2,3,4-tri-O-benzyl-a-d-galactopyranosyl-(1 ! 4)-
until TLC revealed full conversion (24 36 h). The resin was filtered off,
washed with CH2Cl2,and the filtrate was concentrated and purified by flash
chromatography.
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-b-d-galactopyranoside
(10):
[a]2D0 ˆ ‡33.0 (c ˆ 1.2,CHCl 3); 1H NMR: d ˆ 7.40 7.21 (m,25H),6.73
6.64 (m,4H),5.84 (m,1H),5.04 (dq,
3.4 Hz,1H),4.98 (bd, J ˆ 10.2 Hz,1H),4.94 (d, J ˆ 11.1 Hz,1H),4.93 (d,
J ˆ 17.1,1.7 Hz,1H),5.03 (d,
J ˆ
General procedure for removal of acyl groups (Scheme 3 and 4): The
protected saccharide (1 mmol) was dissolved in THF (25 mL) and MeOH
J ˆ 11.1 Hz,1H),4.82 (d, J ˆ 11.9 Hz,1H),4.81 (d, J ˆ 12.4 Hz,1H),4.80
Chem. Eur. J. 2003, 9,3821 3832
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
3827

R. Madsen,M. H. Clausen
FULL PAPER
(d, J ˆ 11.1 Hz,1H),4.79 (d, J ˆ 11.9 Hz,1H),4.73 (d, J ˆ 12.6 Hz,1H),
4.71 (d, J ˆ 12.6 Hz,1H),4.58 (d, J ˆ 12.4 Hz,1H),4.57 (d, J ˆ 11.1 Hz,
1H),4.43 4.35 (m,3H),4.13 (d, J ˆ 3.0 Hz,1H),4.09 (dd, J ˆ 10.2,7.7 Hz,
10.2 Hz,1H),4.83 4.68 (m,7H),4.44 (d,
J ˆ 11.1 Hz,1H),4.43 (ddt,
J ˆ 12.8,5.1,1.3 Hz,1H),4.35 (d, J ˆ 7.3 Hz,1H),4.22 (d, J ˆ 2.6 Hz,1H),
4.13 (dd, J ˆ 9.4,7.7 Hz,1H),4.07 (bdd, J ˆ 12.8,6.4 Hz,1H),4.01 (dd, J ˆ
1H),4.08 3.89 (m,6H),3.74 (s,3H),3.69 (dd,
J ˆ 9.8,7.7 Hz,1H),3.63
9.8,5.1 Hz,1H),3.97 (m,1H),3.94 (dt,
9.8,7.7 Hz,1H),3.77 (s,3H),3.74 (d,
J ˆ 9.4,6.6 Hz,1H),3.92 (dd, J ˆ
J ˆ 3.0 Hz,1H),3.73 3.67 (m,2H),
(dd, J ˆ 7.7,6.0 Hz,1H),3.58 (dt,
J ˆ 9.4,6.8 Hz,1H),3.46 (dd,
J ˆ 9.8,
3.0 Hz,1H),2.19 (m,2H),1.80 (s,3H),1.79 ppm (m,2H);
13C NMR: d ˆ
3.62 (m,1H),3.56 (dd, J ˆ 9.8,3.0 Hz,1H),3.52 (dt, J ˆ 9.4,6.8 Hz,1H),
3.50 3.44 (m,2H),3.41 (bs,1H),2.15 (m,2H),1.76 ppm (m,2H);
13C NMR: d ˆ 154.53,152.30,139.29,138.86,138.73,138.63,138.23,135.21,
170.19,153.87,152.29,138.71,138.52,138.43,138.36,138.32,137.94,115.36
(2C),114.87,114.54 (2C),103.89,99.92,80.24,78.89,78.74,76.29,74.84,
74.47,74.38,74.33,73.30,72.87,72.73,72.39,69.21,68.61,65.87,62.47,55.52,
30.18,28.99,20.63 ppm; ESI MS (1008.5): m/z 1031.6 [M ‡ Na].
117.17,115.97 (2C),114.94,114.91 (2C),104.90 (
JC,H ˆ 161.9 Hz),104.05
(JC,H ˆ 161.3 Hz),81.66,81.29,80.34,79.53,75.42,75.12,73.99,73.85,73.81,
73.59,73.31,73.14,72.83,69.55,67.96,59.79,55.88,30.36,29.11 ppm;
elemental analysis calcd (%) for C55H64O12: C 72.03,H 7.03; found: C 71.81,
H 7.20.
Benzyl
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-
(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-
(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-
1
(1 ! 4)-6-O-acetyl-2,3-di-O-benzyl-b-d-galactopyranoside (11): H NMR:
Pent-4-enyl 4-O-allyl-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-b-d-galac-
topyranoside (15): Alcohol 17 (5.35 g,10 mmol) was dissolved in anhydrous
THF (30 mL). NaH (720 mg,30 mmol) was added and the resulting
suspension stirred for 2 h before addition of allyl bromide (1.3 mL,
15 mmol). The mixture was stirred 24 h,then diluted with Et 2O and
washed twice with H2O. The combined aqueous phases were extracted with
Et2O,and the combined organic phases were dried,concentrated,and
purified by flash chromatography to afford a white solid (6.32 g,91%).
[a]2D0 ˆ À36.3 (c ˆ 0.9,CHCl 3); m.p. 52.5 538C; 1H NMR: d ˆ 7.40 7.26
d ˆ 7.76 7.08 (m,45H),6.75 6.42 (m,12H),5.12 (d, J ˆ 3.0 Hz,1H),5.09
(d, J ˆ 3.4 Hz,1H),5.04 (d, J ˆ 3.8 Hz,1H),5.00 (d, J ˆ 11.5 Hz,1H),4.97
(d, J ˆ 11.5 Hz,1H),4.93 (d, J ˆ 11.9 Hz,1H),4.90 (d, J ˆ 11.9 Hz,1H),
4.88 4.23 (m,25H),4.10 (dd, J ˆ 10.7,2.1 Hz,1H),4.01 (dd,
J ˆ 10.7,
3.4 Hz,1H),3.98 (d, J ˆ 2.6 Hz,1H),3.95 3.87 (m,3H),3.84 3.68 (m,
4H),3.76 (s,3H),3.71 (s,3H),3.69 (s,3H),3.65 (dd,
J ˆ 8.1,5.1 Hz,1H),
3.58 (t, J ˆ 6.8 Hz,1H),3.50 (dd, J ˆ 8.5,4.3 Hz,1H),3.44 (dd,
J ˆ 9.8,
2.6 Hz,1H),2.49 (s,1H),2.12 ppm (s,3H);
13C NMR: d ˆ 170.55,153.81,
153.76 (2C),152.67,152.53,152.33,115.34 (4C),115.29 (2C),114.68 (2C),
114.58 (2C),114.46 (2C),102.81,100.91,100.28,100.00,80.75,79.12,78.70,
78.35,78.28,75.79 (2C),75.42,75.38,75.07,74.66,73.97,73.58,73.30,73.05,
72.90,72.76,72.67,72.38,72.08,71.09,69.67,69.44,67.94,66.62,66.12,64.43,
(m,10H),6.91 6.81 (m,4H),5.90 5.78 (m,2H),5.17 (bd,
J ˆ 17.5 Hz,
1H),5.07 (bd, J ˆ 10.2 Hz,1H),5.02 (dq, J ˆ 17.1,2.1 Hz,1H),4.99 (bd, J ˆ
10.2 Hz,1H),4.94 (d, J ˆ 11.1 Hz,1H),4.79 (d, J ˆ 12.2 Hz,1H),4.78 (d,
J ˆ 11.1 Hz,1H),4.77 (d, J ˆ 12.2 Hz,1H),4.39 (d, J ˆ 7.7 Hz,1H),4.37
(dd, J ˆ 12.4,5.6 Hz,1H),4.16 (t, J ˆ 8.3 Hz,1H),4.14 (dd, J ˆ 12.8,6.8 Hz,
64.25,62.44,55.80,55.75 (2C),21.05 ppm; ESI MS (1837.8):
[M ‡ Na]; elemental analysis calcd (%) for C110H116O25: C 71.88,H 6.36;
found: C 71.64,H 6.41.
m/z 1860.8
1H),4.07 (dd, J ˆ 9.0,5.1 Hz,1H),3.99 3.92 (m,2H),3.80 (dd,
J ˆ 9.8,
7.7 Hz,1H),3.78 (s,3H),3.70 (dd, J ˆ 7.3,5.6 Hz,1H),3.58 3.51 (m,2H),
Pent-4-enyl 4-O-allyl-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galac-
topyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-pentafluorobenzoyl-b-d-galacto-
2.17 (m,2H),1.76 ppm (m,2H);
13C NMR: d ˆ 154.14,152.63,138.86,
138.52,138.18,135.43,128.39,128.29 (3C),128.10 (3C),127.60 (2C),127.55,
117.22,115.61 (2C),114.85,114.74 (2C),104.05,81.96,79.68,75.29,73.97,
73.08,73.00,72.76,69.37,66.67,55.74,30.32,29.06 ppm; elemental analysis
calcd (%) for C35H42O7: C 73.15,H 7.37; found: C 73.12,H 7.37.
pyranoside (13 f): [a]2D0 ˆ ‡32.5 (c ˆ 1.7,CHCl 3); IR(KBr): nÄ ˆ 1742 cmÀ1
1H NMR: d ˆ 7.44 7.17 (m,20H),6.76 6.63 (m,4H),5.87 5.77 (m,2H),
;
5.13 (dq, J ˆ 17.1,1.7 Hz,1H),5.06 4.99 (m,3H),4.96 (bd,
J ˆ 10.2 Hz,
1H),4.88 (d, J ˆ 11.5 Hz,1H),4.86 (d,
J ˆ 11.5 Hz,1H),4.82 (d,
J ˆ
J ˆ
Pent-4-enyl 6-O-acetyl-2,3-di-O-benzyl-b-d-galactopyranoside (16a):
11.9 Hz,1H),4.82 (d, J ˆ 11.9 Hz,1H),4.78 4.62 (m,6H),4.43 (dd,
[a]D20 ˆ ‡2.0 (c ˆ 2.8,CHCl 3); IR(KBr): nÄ ˆ 1723 cmÀ1
;
1H NMR: d ˆ
9.0,4.7 Hz,1H),4.36 4.31 (m,2H),4.12 4.02 (m,4H),3.97 3.89 (m,
7.40 7.26 (m,10H),6.83 (m,1H),5.03 (dq,
J ˆ 17.1,1.7 Hz,1H),4.98
3H),3.72 (s,3H),3.69 (dd, J ˆ 8.1,4.7 Hz,1H),3.66 (dd, J ˆ 10.8,8.3 Hz,
1H),3.60 (t, J ˆ 6.4 Hz,1H),3.54 (dt, J ˆ 9.4,6.8 Hz,1H),3.41 (dd, J ˆ 9.8,
2.6 Hz,1H),2.17 (m,2H),1.77 ppm (m,2H); 13C NMR: d ˆ 158.36,153.86,
152.66,145.40 (bd, JC,F ˆ 263 Hz,2C),143.20 (bd, JC,F ˆ 253 Hz,1C),138.70,
138.57,138.50,138.26,137.98,137.59 (bd, JC,F ˆ 242 Hz,2C),135.43,116.97,
115.31 (2C),114.87,114.53 (2C),107.91 (m,1C),103.99,101.17,80.67,79.07,
78.76,76.98,76.51,75.18,74.39,74.21,73.99,73.16,72.58,72.31,69.67,69.46,
(bd, J ˆ 10.2 Hz,1H),4.93 (d, J ˆ 11.1 Hz,1H),4.76 (d, J ˆ 11.7 Hz,1H),
4.75 (d, J ˆ 11.1 Hz,1H),4.72 (d, J ˆ 11.7 Hz,1H),4.39 4.31 (m,3H),3.96
(dt, J ˆ 9.4,6.4 Hz,1H),3.93 (bs,1H),3.65 (dd,
J ˆ 9.4,7.7 Hz,1H),3.61
3.54 (m,2H),3.51 (dd, J ˆ 9.4,3.4 Hz,1H),2.49 (bs,1H),2.18 (m,2H),2.08
(s,3H),1.77 ppm (m,2H);
13C NMR: d ˆ 170.88,138.66,138.18,137.92,
128.62 (2C),128.44 (2C),128.22,128.08 (2C),127.96 (2C),127.77,115.01,
103.80,80.56,78.96,75.33,72.80,71.88,69.46,66.85,63.17,30.36,29.13,
21.02 ppm; elemental analysis calcd (%) for C27H34O7: C 68.92,H 7.27;
found: C 68.84,H 7.30.
65.79,64.79,55.57,30.23,28.95 ppm; ESI MS (1110.4):
Na]; elemental analysis calcd (%) for C62H63F5O13: C 67.02,H 5.71; found:
C 66.68,H 5.58.
m/z 1133.2 [M ‡
Pent-4-enyl
2,3-di-O-benzyl-6-O-chloroacetyl-b-d-galactopyranoside
Pent-4-enyl 4-O-allyl-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galac-
topyranosyl-(1 ! 4)-2,3-di-O-benzyl-b-d-galactopyranoside: [a]2D0 ˆ ‡32.7
(c ˆ 0.7,CHCl 3); 1H NMR: d ˆ 7.44 7.13 (m,20H),6.78 6.69 (m,4H),
5.89 5.76 (m,2H),5.16 (dq, J ˆ 17.1,1.7 Hz,1H),5.09 5.04 (m,2H),5.01
(dq, J ˆ 17.1,1.7 Hz,1H),4.96 (bd, J ˆ 10.2 Hz,1H),4.91 (d, J ˆ 11.5 Hz,
(16b): [a]2D0 ˆ ‡0.7 (c ˆ 0.9,CHCl 3); m.p. 62 63.58C; IR (KBr): nÄ ˆ
1
1745 cmÀ1; H NMR: d ˆ 7.39 7.26 (m,10H),5.83 (m,1H),5.03 (dq,
J ˆ
17.1,1.7 Hz,1H),4.98 (bd, J ˆ 10.2 Hz,1H),4.92 (d, J ˆ 11.1 Hz,1H),4.76
(d, J ˆ 11.7 Hz,1H),4.76 (d, J ˆ 11.1 Hz,1H),4.71 (d, J ˆ 11.7 Hz,1H),
4.49 (dd, J ˆ 11.1,6.8 Hz,1H),4.42 (dd, J ˆ 11.5,5.1 Hz,1H),4.34 (d, J ˆ
8.1 Hz,1H),4.08 (s,2H),3.94 (dt, J ˆ 9.8,6.6 Hz,1H),3.92 (m,1H),3.66
3.61 (m,2H),3.56 (dt, J ˆ 9.4,6.8 Hz,1H),3.51 (dd, J ˆ 9.4,3.4 Hz,1H),
1H),4.82 (d, J ˆ 11.1 Hz,1H),4.81 (d,
J ˆ 11.5 Hz,1H),4.76 (d,
J ˆ
11.5 Hz,1H),4.69 (d, J ˆ 11.9 Hz,1H),4.68 (d, J ˆ 11.5 Hz,1H),4.67 (d,
J ˆ 11.1 Hz,1H),4.64 (d, J ˆ 11.9 Hz,1H),4.38 (dd, J ˆ 8.5,5.1 Hz,1H),
4.34 (ddq, J ˆ 12.8,6.8,1.3 Hz,1H),4.31 (d, J ˆ 7.3 Hz,1H),4.10 (bdd, J ˆ
2.46 (bs,1H),2.17 (m,2H),1.76 ppm (m,2H);
13C NMR: d ˆ 167.12,
138.56,138.08,137.78,128.56,128.37 (2C),128.12 (3C),128.06,127.90 (2C),
127.71,114.98,103.72,80.34,78.80,75.24,72.82,71.61,69.44,66.81,64.82,
40.81,30.29,29.05 ppm; elemental analysis calcd (%) for C 27H33ClO7: C
64.22,H 6.59; found: C 64.14,H 6.53.
12.4,6.0 Hz,1H),4.08 4.02 (m,4H),3.96 (t,
J ˆ 8.7 Hz,1H),3.91 (dt, J ˆ
9.8,6.4 Hz,1H),3.79 3.72 (m,2H),3.72 (s,3H),3.64 (m,1H),3.62 (dd,
J ˆ 9.8,7.3 Hz,1H),3.51 (dt, J ˆ 9.8,6.8 Hz,1H),3.47 (bt, J ˆ 7.0 Hz,1H),
3.42 (dd, J ˆ 9.8,2.6 Hz,1H),3.27 (bs,1H),2.15 (m,2H),1.74 ppm (m,
2H); 13C NMR: d ˆ 153.88,152.72,138.60,138.46,138.41,138.02,137.58,
Pent-4-enyl
2,3-di-O-benzyl-6-O-trichloroacetyl-b-d-galactopyranoside
(16c): [a]2D0 ˆ ‡0.2 (c ˆ 1.0,CHCl 3); m.p. 52.5 54.58C; IR (KBr): nÄ ˆ
135.30,117.14,115.45 (2C),114.90,114.58 (2C),104.05 (
JC,H ˆ 156.7 Hz),
100.39 (JC,H ˆ 168.1 Hz),81.23,79.08,78.69,77.54,75.83,75.02,74.84,74.13,
74.07,73.97,72.96,72.31,69.59,69.57,66.18,60.18,55.66,30.21,28.98 ppm;
elemental analysis calcd (%) for C55H64O12: C 72.03,H 7.03; found: C 71.69,
H 7.10.
1
1757 cmÀ1; H NMR: d ˆ 7.39 7.27 (m,10H),5.81 (m,1H),5.01 (dd,
J ˆ
17.1,1.7 Hz,1H),4.97 (bd, J ˆ 10.2 Hz,1H),4.93 (d, J ˆ 11.1 Hz,1H),4.78
(d, J ˆ 11.5 Hz,1H),4.74 (d, J ˆ 11.1 Hz,1H),4.70 (d, J ˆ 11.5 Hz,1H),
4.68 (dd, J ˆ 11.5,7.7 Hz,1H),4.54 (dd, J ˆ 11.5,5.1 Hz,1H),4.36 (d, J ˆ
Pent-4-enyl 4-O-allyl-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-b-d-galac-
topyranosyl-(1 ! 4)-2,3-di-O-benzyl-b-d-galactopyranoside: [a]2D0 ˆ ‡10.1
(c ˆ 1.1,CHCl 3); 1H NMR: d ˆ 7.46 7.13 (m,20H),6.81 6.67 (m,4H),
5.91 5.76 (m,2H),5.23 (d, J ˆ 11.1 Hz,1H),5.22 (dq, J ˆ 17.1,1.7 Hz,1H),
8.1 Hz,1H),3.93 (dt, J ˆ 9.8,6.4 Hz,1H),3.91 (m,1H),3.71 (dd,
J ˆ 7.7,
5.1 Hz,1H),3.64 (dd, J ˆ 9.4,8.1 Hz,1H),3.54 3.50 (m,2H),2.48 (bs,
1H),2.15 (m,2H),1.74 ppm (m,2H); 13C NMR: d ˆ 161.77,138.57,138.05,
137.76,128.65,128.43 (3C),128.17 (2C),127.97 (3C),127.78,115.07,103.73,
80.24,78.77,75.27,73.09,71.40,69.44,67.63,66.91,53.55,30.33,29.01 ppm;
5.14 (bd, J ˆ 10.2 Hz,1H),5.00 (dq, J ˆ 17.1,1.7 Hz,1H),4.95 (bd,
J ˆ
3828
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9,3821 3832

Hexasaccharide Fragments of Pectin
3821 3832
elemental analysis calcd (%) for C27H31Cl3O7: C 56.51,H 5.44,Cl 18.53;
found: C 56.65,H 5.54,Cl 18.48.
21.00 ppm; elemental analysis calcd (%) for C83H88O19: C 71.74,H 6.38;
found: C 71.59,H 6.36.
Pent-4-enyl 2,3-di-O-benzyl-6-O-(4-nitrobenzoyl)-b-d-galactopyranoside
Benzyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-
a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-
d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-
galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-
(16d): [a]2D0 ˆ À5.4 (c ˆ 0.9,CHCl 3); m.p. 97 98.58C; IR (KBr): nÄ ˆ
À1
1718,1527,1296 cm
;
1H NMR: d ˆ 8.30 (m,2H),8.21 (m,2H),7.39
7.26 (m,10H),5.80 (m,1H),5.01 (dq,
J ˆ 17.1,1.7 Hz,1H),4.95 (bd, J ˆ
10.2 Hz,1H),4.94 (d, J ˆ 11.1 Hz,1H),4.78 (d, J ˆ 11.7 Hz,1H),4.75 (d,
J ˆ 11.1 Hz,1H),4.72 (d, J ˆ 11.7 Hz,1H),4.66 4.63 (m,2H),4.38 (d, J ˆ
8.1 Hz,1H),3.97 (m,1H),3.93 (dt, J ˆ 9.4,6.4 Hz,1H),3.75 (t, J ˆ 6.2 Hz,
1H),3.67 (dd, J ˆ 9.4,8.1 Hz,1H),3.58 3.52 (m,2H),2.52 (bs,1H),2.16
(m,2H),1.76 ppm (m,2H); 13C NMR: d ˆ 164.55,150.79,138.57,138.05,
137.85,135.43,130.92 (2C),128.65 (2C),128.47,128.21 (3C),128.15,127.97
(2C),127.82,123.68 (2C),115.06,103.88,80.48,78.88,75.35,73.04,71.87,
69.51,67.10,64.62,30.34,29.11 ppm; elemental analysis calcd (%) for
C32H35NO9: C 66.54,H 6.11,N 2.42; found: C 66.31,H 6.05,N 2.45.
galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-b-d-galactopyranoside
1H NMR: d ˆ 7.40 7.05 (m,70H),6.70 6.32 (m,12H),5.11 (d,
(23):
J ˆ
3.4 Hz,1H),5.07 (d, J ˆ 3.0 Hz,1H),5.06 (d, J ˆ 3.0 Hz,1H),5.01 (d,
J ˆ 3.0 Hz,1H),4.95 4.28 (m,38H),4.21 (m,1H),4.14 (dd, J ˆ 9.8,8.5 Hz,
1H),4.09 4.00 (m,4H),3.97 3.88 (m,3H),3.85 3.61 (m,13H),3.68 (s,
3H),3.67 (s,3H),3.66 (s,3H),3.50 3.39 (m,5H),3.29 3.11 ppm (m,4H);
13C NMR: d ˆ 153.85,153.81,153.63,152.59,152.39,152.19,115.29 (2C),
115.25 (2C),115.23 (2C),114.69 (2C),114.62 (2C),114.47 (2C),103.02,
100.80,100.31 (2C),100.02,99.37,55.74 ppm (3C); ESI MS (2571.1):
m/z
2593.9 [M ‡ Na]; elemental analysis calcd (%) for C155H164O34: C 72.41,H
Pent-4-enyl 2,3-di-O-benzyl-6-O-(3,5-dinitrobenzoyl)-b-d-galactopyrano-
6.43; found: C 72.06,H 6.44.
side (16e): [a]2D0 ˆ ‡2.5 (c ˆ 1.0,CHCl ); m.p. 119 1218C; IR (KBr): nÄ ˆ
3
À1
1736,1545,1345 cm
;
1H NMR: d ˆ 9.24 (t, J ˆ 2.1 Hz,1H),9.16 (d, J ˆ
(Methyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(methyl
2,3-di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-2,3-di-O-benzyl-6-
O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-
(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-(4-
methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-(methyl (benzyl 2,3-di-O-
benzyl-b-d-galactopyranosid)uronate) (24): 1H NMR: d ˆ 7.41 6.88 (m,
2.1 Hz,2H),7.39 7.28 (m,10H),5.80 (m,1H),5.00 (dq,
J ˆ 17.1,1.7 Hz,
1H),4.94 (d, J ˆ 11.1 Hz,1H),4.93 (bd, J ˆ 10.2 Hz,1H),4.81 4.68 (m,
5H),4.41 (d, J ˆ 7.7 Hz,1H),4.00 3.94 (m,2H),3.79 (t, J ˆ 6.2 Hz,1H),
3.68 (dd, J ˆ 9.4,7.7 Hz,1H),3.61 3.55 (m,2H),2.56 (bs,1H),2.17 (m,
2H),1.77 ppm (m,2H);
13C NMR: d ˆ 162.48,148.92,138.62,138.14,
137.83,133.89,129.67 (2C),128.76 (2C),128.55 (2C),128.29 (2C),128.22,
128.15 (2C),128.06,127.92,122.69,115.10,103.95,80.38,78.91,75.45,73.16,
71.87,69.69,67.25,65.81,30.39,29.19 ppm; elemental analysis calcd (%) for
C32H34N2O11: C 61.73,H 5.50,N 4.50; found: C 61.58,H 5.48,N 4.50.
70H),6.79 6.41 (m,12H),5.16 (d,
1H),5.07 5.01 (m,2H),5.01 (d,
4.10 4.05 (m,2H),4.01 (dd, J ˆ 10.2,2.6 Hz,1H),3.99 (bs,1H),3.91 (dd,
J ˆ 7.3,3.4 Hz,1H),3.89 (dd, J ˆ 7.3,3.4 Hz,1H),3.79 3.62 (m,6H),3.72
(s,3H),3.70 (s,3H),3.66 (s,3H),3.63 (s,3H),3.61 3.49 (m,4H),3.47 (dd,
J ˆ 3.5 Hz,1H),5.09 (d, J ˆ 3.4 Hz,
J ˆ 3.4 Hz,1H),4.94 4.15 (m,42H),
Pent-4-enyl 2,3-di-O-benzyl-6-O-pentafluorobenzoyl-b-d-galactopyrano-
J ˆ 9.8,3.0 Hz,1H),3.17 (s,3H),3.04 ppm (s,3H);
13C NMR: d ˆ 169.07,
side (16 f): [a]2D0 ˆ À2.3 (c ˆ 0.9,CHCl 3); m.p. 94 958C; IR (KBr): nÄ ˆ
168.54,168.39,153.69 (3C),152.45,152.24,151.97,115.14 (4C),115.02 (2C),
114.57 (4C),114.50 (2C),102.71,99.92,99.72,99.58,99.47,98.93,55.69 (2C),
1
1729 cmÀ1; H NMR: d ˆ 7.39 7.27 (m,10H),5.81 (m,1H),5.01 (dq,
J ˆ
17.1,1.7 Hz,1H),4.96 (m,1H),4.94 (d,
J ˆ 11.1 Hz,1H),4.77 (d, J ˆ
55.62,52.26,51.57,51.49 ppm; ESI MS (2655.1):
m/z: 2678.0 [M ‡ Na].
11.7 Hz,1H),4.74 (d, J ˆ 11.1 Hz,1H),4.72 (d, J ˆ 11.7 Hz,1H),4.69
(dd, J ˆ 11.5,7.3 Hz,1H),4.59 (dd, J ˆ 11.5,5.1 Hz,1H),4.36 (d, J ˆ 7.7 Hz,
1H),3.98 3.92 (m,2H),3.71 (t, J ˆ 6.2 Hz,1H),3.65 (dd, J ˆ 9.2,7.9 Hz,
1H),3.55 (dt, J ˆ 9.4,6.4 Hz,1H),3.53 (dd, J ˆ 9.4,3.4 Hz,1H),2.48 (bs,
(Benzyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl
2,3-di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-2,3-di-O-benzyl-6-
O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-
(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-(4-
methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-(benzyl (benzyl 2,3-di-O-
benzyl-b-d-galactopyranosid)uronate) (25): 1H NMR: d ˆ 7.41 6.81 (m,
1H),2.17 (m,2H),1.76 ppm (m,2H);
13C NMR: d ˆ 158.73,145.68 (bd,
JC,F ˆ 258 Hz,2C),143.27 (bd, JC,F ˆ 259 Hz,1C),138.62,138.07,138.02 (bd,
JC,F ˆ 251 Hz,2C),137.81,128.58 (2C),128.39 (2C),128.14 (2C),128.07,
127.91 (2C),127.73,114.91,108.06 (dt,
JC,F ˆ 11,4 Hz,1C),103.76,80.34,
85H),6.70 6.43 (m,12H),5.18 (d,
J ˆ 3.4 Hz,1H),5.10 4.98 (m,4H),
78.84,75.26,72.89,71.72,69.34,66.91,65.40,30.30,29.02 ppm; elemental
analysis calcd (%) for C32H31F5O7: C 61.73,H 5.02; found: C 61.71,H 4.91.
5.09 (d, J ˆ 3.4 Hz,1H),4.94 4.84 (m,4H),4.81 4.13 (m,42H),4.07
3.99 (m,4H),3.97 (dd, J ˆ 10.2,3.4 Hz,1H),3.89 (dd, J ˆ 10.2,3.4 Hz,1H),
3.82 3.62 (m,7H),3.72 (s,3H),3.69 (s,3H),3.68 (s,3H),3.57 3.46 ppm
(m,5H); 13C NMR: d ˆ 168.43,167.77,167.62,153.82 (2C),153.77,152.56,
152.34,152.09,115.25 (4C),115.10 (2C),114.66 (2C),114.59 (2C),114.58
(2C),102.73,99.94,99.78 (2C),99.11,98.93,55.81 (2C),55.75 ppm; ESI MS
(2883.2): m/z: 2905.9 [M ‡ Na].
Pent-4-enyl 2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-b-d-galactopyrano-
side (17): Prepared from pent-4-enyl 2,3-di-O-benzyl-b-D-galactopyrano-
side according to our previous procedure.[9] [a]D20 ˆ ‡9.7 (c ˆ 2.7,CHCl 3);
1H NMR: d ˆ 7.42 7.26 (m,10H),6.92 6.83 (m,4H),5.85 (m,1H),5.05
(dq, J ˆ 17.1,1.7 Hz,1H),4.99 (bd, J ˆ 10.2 Hz,1H),4.95 (d, J ˆ 11.1 Hz,
1H),4.78 (d, J ˆ 11.1 Hz,1H),4.76 (s,2H),4.42 (d,
J ˆ 7.7 Hz,1H),4.27
(Methyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(methyl
2,3-di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl 2,3-di-O-
benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl 2,3-di-O-benzyl-a-
d-galactopyranosyluronate)-(1 ! 4)-(benzyl 2,3-di-O-benzyl-a-d-galacto-
pyranosyluronate)-(1 ! 4)-(methyl (benzyl 2,3-di-O-benzyl-b-d-galacto-
pyranosid)uronate) (26): 1H NMR: d ˆ 7.41 6.98 (m,85H),5.22 (d, J ˆ
(dd, J ˆ 9.8,6.4 Hz,1H),4.18 (dd, J ˆ 9.8,6.0 Hz,1H),4.12 (d, J ˆ 3.1 Hz,
1H),3.98 (dt, J ˆ 9.4,6.4 Hz,1H),3.78 (s,3H),3.74 (t, J ˆ 6.0 Hz,1H),3.70
(dd, J ˆ 9.4,7.7 Hz,1H),3.61 3.56 (m,2H),2.63 (bs,1H),2.19 (m,2H),
1.78 ppm (m,2H); 13C NMR: d ˆ 154.18,152.85,138.70,138.15,137.94,
128.54 (3C),128.37,128.14 (3C),127.97,127.89,127.67,115.90 (2C),114.92,
114.73 (2C),103.81,80.70,79.05,75.27,72.74,72.63,69.37,67.57,66.69,55.78,
30.33,29.09 ppm; elemental analysis calcd (%) for C 32H38O7: C 71.89,H
7.16; found: C 71.62,H 6.96.
3.4 Hz,1H),5.11 (d, J ˆ 3.1 Hz,2H),5.07 4.98 (m,4H),4.90 (d,
11.9 Hz,1H),4.86 (d,
J ˆ
J ˆ 11.9 Hz,1H),4.84 4.21 (m,41H),4.16 (m,
1H),3.98 (dd, J ˆ 10.3,3.4 Hz,1H),3.89 3.82 (m,4H),3.81 3.76 (m,
2H),3.68 (dd, J ˆ 10.2,2.6 Hz,1H),3.65 3.51 (m,4H),3.64 (s,3H),3.30
Benzyl
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-
(dd, J ˆ 9.8,2.6 Hz,1H),3.24 (s,3H),3.11 ppm (s,3H);
13C NMR: d ˆ
(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-
169.12,168.28,168.19,168.06,167.88,167.70,102.66,99.32,98.68,98.55
(2C),98.54,52.21,51.66,51.51 ppm.
1
(1 ! 4)-6-O-acetyl-2,3-di-O-benzyl-b-d-galactopyranoside (22): H NMR:
d ˆ 7.50 7.16 (m,35H),6.79 6.54 (m,8H),5.15 (d,
J ˆ 3.0 Hz,1H),5.08
(d, J ˆ 3.4 Hz,1H),5.02 (d, J ˆ 11.9 Hz,1H),4.98 (d, J ˆ 11.9 Hz,1H),
4.94 4.84 (m,4H),4.80 4.55 (m,11H),4.52 (d, J ˆ 7.3 Hz,1H),4.47 (dd,
J ˆ 9.4,5.1 Hz,1H),4.38 4.32 (m,3H),4.13 (dd, J ˆ 10.7,2.6 Hz,1H),4.08
(dd, J ˆ 10.7,3.4 Hz,1H),4.04 (t, J ˆ 8.5 Hz,1H),4.01 (d, J ˆ 3.0 Hz,1H),
3.91 (dd, J ˆ 9.8,2.6 Hz,1H),3.90 (dd, J ˆ 9.8,3.0 Hz,1H),3.77 (s,3H),
3.76 3.67 (m,3H),3.69 (s,3H),3.62 (t, J ˆ 6.4 Hz,1H),3.47 (dd, J ˆ 10.2,
(Benzyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl
2,3-di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(methyl 2,3-di-O-
benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(methyl 2,3-di-O-benzyl-a-
d-galactopyranosyluronate)-(1 ! 4)-(methyl 2,3-di-O-benzyl-a-d-galacto-
pyranosyluronate)-(1 ! 4)-(benzyl (benzyl 2,3-di-O-benzyl-b-d-galacto-
pyranosid)uronate) (27): 1H NMR: d ˆ 7.41 6.95 (m,85H),5.20 (d, J ˆ
3.0 Hz,1H),2.60 (s,1H),2.15 ppm (s,3H);
13C NMR: d ˆ 170.50,153.87,
3.4 Hz,1H),5.12 4.98 (m,7H),4.88,(d,
11.3 Hz,1H),4.82 4.28 (m,38H),4.23 (d,
J ˆ 11.3 Hz,1H),4.86 (d, J ˆ
J ˆ 12.4 Hz,1H),4.20 (d, J ˆ
153.78,152.71,152.41,138.62,138.52,138.45 (2C),138.31,138.26,137.53,
115.42 (2C),115.39 (2C),114.52 (2C),114.47 (2C),102.75,100.88,99.91,
80.81,78.66,78.39,78.02,75.91,75.80,75.60,75.02,74.74,73.62,73.26,72.87,
72.74,72.41,72.02,71.05,69.59,68.06,66.70,66.37,64.69,62.55,55.64 (2C),
13.2 Hz,1H),4.06 (m,1H),4.00 (dd,
3.91 (dd, J ˆ 10.7,2.6 Hz,1H),3.84 (dd, J ˆ 10.2,3.4 Hz,1H),3.79 3.72 (m,
J ˆ 10.2,3.5 Hz,1H),3.93 (bs,1H),
3H),3.66 3.47 (m,5H),3.39 (s,3H),3.38 (m,1H),3.36 (s,3H),3.29 ppm
Chem. Eur. J. 2003, 9,3821 3832
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
3829

R. Madsen,M. H. Clausen
FULL PAPER
(s,3H); 13C NMR: d ˆ 168.81,168.76,168.62,168.43,167.51,167.41,102.67,
99.65,98.67,98.62,98.40,98.34,51.78 (2C),51.70 ppm; ESI MS (2649.0):
m/z 2671.9 [M ‡ Na].
(Benzyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(methyl
2,3-di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(methyl 2,3-di-O-
benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(methyl 2,3-di-O-benzyl-a-
d-galactopyranosyluronate)-(1 ! 4)-(methyl 2,3-di-O-benzyl-a-d-galacto-
pyranosyluronate)-(1 ! 4)-(benzyl (benzyl 2,3-di-O-benzyl-b-d-galacto-
pyranosid)uronate) (32): 1H NMR: d ˆ 7.41 7.01 (m,80H),5.29 (d, J ˆ
3.2 Hz,1H),5.13 4.96 (m,7H),4.89 4.27 (m,40H),4.07 (bs,1H),3.97
3.90 (m,3H),3.85 (dd, J ˆ 10.5,3.2 Hz,1H),3.81 3.74 (m,3H),3.67 3.61
(m,2H),3.59 3.52 (m,3H),3.41 (s,3H),3.38 (m,1H),3.37 (s,3H),3.29 (s,
3H),3.12 ppm (s,3H); 13C NMR: d ˆ 168.74,168.69,168.54,168.39,168.36,
167.32,102.58,99.39,98.60,98.52,98.48,98.25,51.71,51.69,51.62,
Benzyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-
6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-
O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-
(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-(4-
methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-b-d-galac-
topyranoside (28): 1H NMR: d ˆ 7.40 6.97 (m,70H),6.69 6.29 (m,16H),
5.10 (d, J ˆ 3.2 Hz,1H),5.08 (d, J ˆ 3.7 Hz,1H),5.04 (d, J ˆ 3.7 Hz,1H),
5.03 (d, J ˆ 3.7 Hz,1H),4.96 4.79 (m,8H),4.77 4.27 (m,31H),4.21 (d,
J ˆ 9.0 Hz,1H),4.19 (d, J ˆ 9.0 Hz,1H),4.10 4.03 (m,3H),3.97 3.61 (m,
15H),3.69 (s,3H),3.68 (s,3H),3.67 (s,3H),3.66 (s,3H),3.55 3.40 (m,
51.51 ppm; ESI MS (2573.0): m/z: 2595.9 [M ‡ Na]; elemental analysis
calcd for C152H154O37: C 70.96,H 6.03; found: C 70.67,H 6.13.
4H),3.26 3.10 (m,3H),2.66 (bs,1H),2.49 ppm (bs,1H);
13C NMR: d ˆ
Benzyl
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-
154.05 (2C),153.91,153.87,152.79,152.60,152.53,152.35,115.50 (2C),
115.41 (4C),115.38 (2C),114.90 (4C),114.79 (2C),114.69 (2C),103.22,
100.53 (3C),100.06,99.63,56.02,55.99 (2C),55.95 ppm; ESI MS (2677.2):
m/z 2700.2 [M ‡ Na]; elemental analysis calcd (%) for C162H170O35: C
72.68,H 6.40; found: C 72.47,H 6.46.
(1 ! 4)-2,3-di-O-benzyl-6-O-pentafluorobenzoyl-a-d-galactopyranosyl-
(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-b-d-galactopyranoside
(33): [a]2D0 ˆ ‡25.6 (c ˆ 1.7,CHCl 3); 1H NMR: d ˆ 7.42 7.14 (m,35H),
6.81 6.58 (m,8H),5.06 (d, J ˆ 3.4 Hz,1H),4.99 (d, J ˆ 3.4 Hz,1H),4.97
(d, J ˆ 12.0 Hz,1H),4.90 4.63 (m,15H),4.54 4.42 (m,5H),4.28 (d,
1.8 Hz,1H),4.16 (d, J ˆ 2.6 Hz,1H),4.10 4.07 (m,2H),4.01 (t, J ˆ 8.5 Hz,
1H),3.92 (dd, J ˆ 9.9,3.4 Hz,1H),3.90 (dd, J ˆ 9.4,3.1 Hz,1H),3.81 (dd,
J ˆ 9.9,3.1 Hz,1H),3.76 (s,3H),3.74 (s,3H),3.73 3.65 (m,3H),3.44 (dd,
J ˆ
(Methyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-2,3-di-
O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-(methyl
(benzyl 2,3-di-O-benzyl-b-d-galactopyranosid)uronate) (29): 1H NMR:
d ˆ 7.41 6.88 (m,70H),6.69 6.26 (m,16H),5.16 (d, J ˆ 3.4 Hz,1H),5.08
(d, J ˆ 3.4 Hz,1H),5.06 (d, J ˆ 12.0 Hz,1H),5.03 (d, J ˆ 2.6 Hz,1H),4.99
(d, J ˆ 3.4 Hz,1H),4.96 (d, J ˆ 3.4 Hz,1H),4.94 4.12 (m,42H),4.03 3.96
J ˆ 9.9,2.9 Hz,1H),2.50 ppm (bs,1H);
13C NMR: d ˆ 157.90,154.06,
153.91,152.73,152.47,145.39 (bd,
JC,F ˆ 257 Hz,2C),143.15 (bd, JC,F ˆ
260 Hz,1C),138.66,138.57,138.51,138.29,138.19,138.15,137.80,137.61
(bd, JC,F ˆ 257 Hz,2C),115.45 (2C),115.43 (2C),114.75 (2C),114.53 (2C),
107.78 (dt, JC,F ˆ 15,3.5 Hz,1C),102.91,100.42,100.20,79.84,79.25,78.46,
77.35,76.86,75.90,75.08,74.98,74.59,74.31,73.02,72.96,72.77,72.43,72.12,
71.09,69.00,68.38,66.66,66.43,65.65,63.77,55.72,55.69 ppm; ESI MS
(1540.6): m/z: 1563.7 [M ‡ Na].
(m,3H),3.90 (dd, J ˆ 10.2,3.0 Hz,1H),3.86 (dd,
J ˆ 10.2,3.4 Hz,1H),
3.84 3.62 (m,7H),3.80 (s,3H),3.79 (s,3H),3.78 (s,3H),3.66 (s,3H),3.63
(s,3H),3.61 3.54 (m,3H),3.50 3.42 (m,2H),3.40 (dd,
J ˆ 7.7,5.1 Hz,
Benzyl
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-
1H),3.09 ppm (s,3H); 13C NMR: d ˆ 169.38,168.52,153.81,153.76,153.73,
153.70,152.60,152.46,152.34,152.00,115.27 (2C),115.23 (2C),115.11 (4C),
114.67 (4C),114.60 (2C),114.50 (2C),102.82,100.08,100.12,100.00,99.55,
(1 ! 4)-2,3-di-O-benzyl-6-O-pentafluorobenzoyl-a-d-galactopyranosyl-
(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-
(1 ! 4)-2,3-di-O-benzyl-6-O-pentafluorobenzoyl-a-d-galactopyranosyl-
(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-b-d-galactopyranoside
99.32,55.83,56.82,55.77,55.74,52.39,51.67 ppm; ESI MS (2733.1):
2756.1 [M ‡ Na]; elemental analysis calcd (%) for C164H170O37: C 72.07,H
6.27; found: C 71.85,H 6.27.
m/z
(34): 1H NMR: d ˆ 7.41 7.02 (m,55H),6.77 6.45 (12H),5.06 (d,
1.7 Hz,1H),5.02 (d, J ˆ 3.4 Hz,1H),4.96 (d, J ˆ 3.7 Hz,1H),4.95 (d,
J ˆ 11.4 Hz,1H),4.93 (d, J ˆ 3.4 Hz,1H),4.88 4.01 (m,40H),3.93 3.88
(m,2H),3.86 (dd, J ˆ 9.9,3.4 Hz,1H),3.83 3.76 (m,4H),3.74 (s,6H),
3.71 3.62 (m,3H),3.69 (s,3H),3.51 (dd, J ˆ 8.2,4.5 Hz,1H),3.42 (dd, J ˆ
J ˆ
(Benzyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-2,3-di-
O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-(benzyl
(benzyl 2,3-di-O-benzyl-b-d-galactopyranosid)uronate) (30): 1H NMR:
d ˆ 7.41 6.89 (m,80H),6.69 6.27 (m,16H),5.18 (d, J ˆ 3.0 Hz,1H),5.09
9.9,2.8 Hz,1H),2.45 ppm (bs,1H);
153.91,153.89,152.68,152.48,152.35,145.45 (bd,
(bd, JC,F ˆ 260 Hz,2C),137.69 (bd, JC,F ˆ 257 Hz,4C),115.47 (2C),115.38
(2C),115.27 (2C),114.79 (2C),114.71 (2C),114.54 (2C),107.56 (m,2C),
102.94,100.59,100.43,100.15,99.79,55.80 ppm (3C); elemental analysis
calcd (%) for C142H134F10O31: C 67.50,H 5.35; found: C 67.17,H 5.15.
13C NMR: d ˆ 157.99,157.66,154.09,
JC,F ˆ 257 Hz,4C),143.22
(d, J ˆ 3.4 Hz,1H),5.08 5.02 (m,2H),5.00 (d,
J ˆ 3.4 Hz,1H),4.93 (d,
J ˆ 3.4 Hz,1H),4.90 (d, J ˆ 3.4 Hz,1H),4.88 4.16 (m,43H),4.08 (t,
2.1 Hz,1H),4.03 (dd, J ˆ 10.2,2.1 Hz,1H),4.01 3.96 (m,2H),3.90 (dd,
J ˆ 10.7,3.4 Hz,1H),3.86 (dd, J ˆ 10.2,3.4 Hz,1H),3.83 3.64 (m,8H),
3.70 (s,3H),3.69 (s,3H),3.68 (s,3H),3.66 (s,3H),3.61 3.56 (m,2H),3.54
J ˆ
Benzyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-
6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-a-
d-galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-
galactopyranosyl-(1 ! 4)-2,3-di-O-benzyl-a-d-galactopyranosyl-(1 ! 4)-
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-b-d-galactopyranoside (35): 1H
(dd, J ˆ 10.7,3.0 Hz,1H),3.48 (dd,
J ˆ 9.8,3.0 Hz,1H),3.46 (m,1H),
3.41 ppm (dd, J ˆ 8.1,5.1 Hz,1H); 13C NMR: d ˆ 168.63,167.75,153.78,
153.73,153.69,153.66,152.55,152.33,152.30,151.97,115.23 (4C),115.07
(4C),114.62 (4C),114.55 (2C),114.46 (2C),102.69,100.14,100.03 (2C),
NMR: d ˆ 7.42 6.97 (m,70H),6.73 6.34 (m,12H),5.01 (d,
J ˆ 4.0 Hz,
1H),5.00 (d, J ˆ 3.6 Hz,1H),4.99 4.94 (m,4H),4.91 (d, J ˆ 11.0 Hz,1H),
4.89 (d, J ˆ 11.0 Hz,1H),4.83 4.43 (m,28H),4.27 3.99 (m,14H),3.95
3.74 (m,10H),3.72 (s,3H),3.71 3.52 (m,6H),3.69 (s,3H),3.67 (s,3H),
3.43 (dd, J ˆ 10.0,2.9 Hz,1H),3.40 (m,1H),3.32 3.24 (m,3H),3.08 (bs,
99.94,99.14,55.78,55.77,55.73,55.70 ppm; ESI MS (2885.2):
m/z: 2908.3
[M ‡ Na]; elemental analysis calcd (%) for C176H178O37: C 73.26,H 6.22;
found: C 72.98,H 6.14.
1H),2.81 ppm (m,1H);
13C NMR: d ˆ 153.97,153.74,153.72,152.46,
(Methyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl
2,3-di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl 2,3-di-O-
benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl 2,3-di-O-benzyl-a-
d-galactopyranosyluronate)-(1 ! 4)-(benzyl 2,3-di-O-benzyl-a-d-galacto-
pyranosyluronate)-(1 ! 4)-(methyl (benzyl 2,3-di-O-benzyl-b-d-galacto-
pyranosid)uronate) (31): 1H NMR: d ˆ 7.41 6.92 (m,90H),5.21 (d, J ˆ
3.7 Hz,1H),5.12 (d, J ˆ 3.1 Hz,1H),5.09 (d, J ˆ 3.7 Hz,1H),5.07 (d, J ˆ
3.1 Hz,1H),5.04 (d, J ˆ 12.1 Hz,1H),5.02 (d, J ˆ 3.2 Hz,1H),4.99 (d, J ˆ
12.1 Hz,1H),4.92 (d, J ˆ 12.1 Hz,1H),4.86 4.28 (m,42H),4.23 4.18 (m,
2H),4.14 (m,1H),4.02 (dd, J ˆ 10.0,3.2 Hz,1H),3.87 3.76 (m,6H),3.68
152.33,152.29,115.43 (2C),115.20 (4C),114.70 (2C),114.59 (2C),114.52
(2C),102.99,100.34,100.26,100.19,99.84,99.77,55.74 (2C),53.52 ppm; ESI
MS (2571.1): m/z: 2593.7 [M ‡ Na]; elemental analysis calcd (%) for
C155H164O34: C 72.41,H 6.43; found: C 72.16,H 6.31.
Benzyl (methyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-
2,3-di-O-benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-
(methyl 2,3-di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-2,3-di-O-
benzyl-6-O-(4-methoxyphenyl)-a-d-galactopyranosyl-(1 ! 4)-(methyl 2,3-
di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-2,3-di-O-benzyl-6-O-
(4-methoxyphenyl)-b-d-galactopyranoside (36): 1H NMR: d ˆ 7.42 6.86
(m,70H),6.80 6.28 (m,12H),5.15 (d, J ˆ 3.1 Hz,1H),5.10 (d, J ˆ 3.3 Hz,
1H),5.06 (d, J ˆ 3.1 Hz,1H),5.02 (d, J ˆ 3.1 Hz,1H),5.00 4.03 (m,46H),
3.99 (t, J ˆ 9.0 Hz,1H),3.88 (dd, J ˆ 10.5,3.3 Hz,1H),3.85 (dd, J ˆ 10.5,
3.3 Hz,1H),3.81 3.58 (m,9H),3.75 (s,3H),3.70 (s,3H),3.69 (s,3H),
(dd, J ˆ 10.0,2.6 Hz,1H),3.64 (s,3H),3.60 3.61 (m,4H),3.30 (dd,
J ˆ
10.0,3.1 Hz,1H),3.21 ppm (s,3H);
167.85,167.67,167.37,102.60,99.45,98.63,98.46 (2C),98.40,52.15,
13C NMR: d ˆ 168.99,168.13,167.99,
51.57 ppm; ESI MS (2725.1): m/z: 2748.1 [M ‡ Na]; elemental analysis
calcd (%) for C164H162O37: C 72.28,H 5.99; found: C 71.97,H 5.91.
3830
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9,3821 3832

Hexasaccharide Fragments of Pectin
3821 3832
3.45 3.35 (m,2H),3.33 (s,3H),3.14 (s,3H),3.12 ppm (s,3H);
13C NMR:
[1] a) C. Rolin,B. U. Nielsen,P.-E. Glahn in Polysaccharides - Structural
Diversity and Functional Versatility (Ed.: S. Dumitriu),Marcel
Dekker,New York, 1996,p 377; b) Progress in Biotechnology 14 -
Pectin and Pectinases (Eds.: J. Visser,A. G. J. Voragen),Elsevier,
Amsterdam, 1996; c) The Chemistry and Technology of Pectin (Ed.:
R. H. Walter),Academic Press,San Diego, 1991.
d ˆ 169.62,169.60,169.39,154.38,154.09,153.99,152.46,152.40,152.29,
115.63 (2C),115.50 (2C),115.46 (2C),115.05 (2C),114.91 (2C),114.75 (2C),
103.27,100.25,100.11,100.09,99.57 (2C),56.02 (2C),53.80,52.17,52.05,
51.92 ppm; elemental analysis calcd (%) for C158H164O37: C 71.48,H 6.23;
found: C 71.26,H 6.47.
[2] B. R. Thakur,R. K. Singh,A. K. Handa, Crit. Rev. Food Sci. Nut. 1997,
37,47.
[3] For recent examples,see: W. G. T. Willats,C. Orfila,G. Limberg,H. C.
Buchholt,G.-J. W. M. van Alebeek,A. G. J. Voragen,S. E. Marcus,
(Methyl 2,3,4-tri-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl
2,3-di-O-benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(methyl 2,3-di-O-
benzyl-a-d-galactopyranosyluronate)-(1 ! 4)-(benzyl 2,3-di-O-benzyl-a-
d-galactopyranosyluronate)-(1 ! 4)-(methyl 2,3-di-O-benzyl-a-d-galacto-
pyranosyluronate)-(1 ! 4)-(benzyl (benzyl 2,3-di-O-benzyl-b-d-galacto-
pyranosid)uronate) (37): 1H NMR: d ˆ 7.42 6.95 (m,85H),5.25 (d, J ˆ
3.2 Hz,1H),5.14 (d, J ˆ 12.4 Hz,1H),5.09 4.94 (m,7H),4.87 4.28 (m,
T. M. I. E. Christensen,J. D. Mikkelsen,B. S. Murray,J. P. Knox,
Biol. Chem. 2001, 276,19404; P. J. H. Daas,A. G. J. Voragen,H. A.
Schols, Biopolymers 2001, 58,195.
J.
[4] a) R. P. de Vries,J. Visser, Microbiol. Mol. Biol. Rev. 2001, 65,497;
b) S. R. Herron,J. A. E. Benen,R. D. Scavetta,J. Visser,F. Jurnak,
Proc. Natl. Acad. Sci. USA 2000, 97,8762; c) T. Sakai,T. Sakamoto,J.
Hallaert,E. J. Vandamme, Adv. Appl. Microbiol. 1993, 39,213.
[5] a) K. E. Mutenda,R. Kˆrner,T. M. I. E. Christensen,J. Mikkelsen,P.
Roepstorff, Carbohydr. Res. 2002, 337,1213; b) E. Bonnin,A. Le Goff,
42H),4.22 (d, J ˆ 12.7 Hz,1H),4.12 (m,1H),4.00 (dd,
J ˆ 10.2,3.2 Hz,
1H),3.92 (bs,1H),3.87 (dd, J ˆ 10.2,2.2 Hz,1H),3.82 3.75 (m,3H),3.63
(dd, J ˆ 10.2,2.6 Hz,1H),3.61 3.52 (m,3H),3.38 (s,3H),3.36 (dd,
J ˆ
10.2,2.9 Hz,1H),3.27 (s,3H),3.21 ppm (s,3H);
13C NMR: d ˆ 169.14,
168.80,168.53,168.02,167.52,167.41,102.64,99.66,98.65,98.52 (2C),98.40,
51.74,51.68,51.65 ppm; ESI MS (2649.0): m/z: 2671.7 [M ‡ Na]; elemental
analysis calcd (%) for C158H158O37: C 71.64,H 6.01; found: C 71.27,H 5.88.
R. Kˆrner,J. Vigouroux,P. Roepstorff,J.-F. Thibault,
Biochim.
Biophys. Acta 2002, 1596,83; c) G. Limberg,R. Kˆrner,H. C.
Buchholt,T. M. I. E. Christensen,P. Roepstorff,J. D. Mikkelsen,
Carbohydr. Res. 2000, 327,321; d) H. C. M. Kester,J. A. E. Benen,J.
Visser,M. E. Warren,R. Orlando,C. Bergmann,D. Magaud,D.
Anker,A. Doutheau, Biochem. J. 2000, 346,469; e) R. Kˆrner,G.
Limberg,T. M. I. E. Christensen,J. D. Mikkelsen,P. Roepstorff, Anal.
Chem. 1999, 71,1421.
(Methyl a-d-galactopyranosyluronate)-(1 ! 4)-(methyl a-d-galactopyra-
nosyluronate)-(1 ! 4)-(a-d-galactopyranosyluronic acid)-(1 ! 4)-(a-d-gal-
actopyranosyluronic acid)-(1 ! 4)-(a-d-galactopyranosyluronic acid)-
(1 ! 4)-(methyl D-galactopyranuronate) (1): 13C NMR (D2O): d ˆ 171.62,
170.94,170.08,100.67,99.95,99.85,96.08,92.76,79.12,78.87,78.30,78.19,
73.47,71.69,71.50,71.32,70.77,70.22,68.84,68.64,68.27,68.17,68.05,67.99,
53.22,53.14,53.04 ppm; ESI MS/MS: m/z: 1114.6,925.1,749.0,572.8; ESI
HRMS calcd for C39H56O37Na: m/z: 1139.2398; found: m/z: 1139.2382
[6] For recent developments,see: P. J. H. Daas,B. Boxma,A. M. C. P.
Hopman,A. G. J. Voragen,H. A. Schols, Biopolymers 2001, 58,1; G.
Limberg,R. Kˆrner,H. C. Buchholt,T. M. I. E. Christensen,P.
Roepstorff,J. D. Mikkelsen, Carbohydr. Res. 2000, 327,293; P. J. H.
‡
[M ‡ Na] .
(a-d-Galactopyranosyluronic acid)-(1 ! 4)-(a-d-galactopyranosyluronic
acid)-(1 ! 4)-(methyl a-d-galactopyranosyluronate)-(1 ! 4)-(methyl a-d-
galactopyranosyluronate)-(1 ! 4)-(methyl a-d-galactopyranosyluronate)-
(1 ! 4)-d-galactopyranuronic acid (2): 13C NMR (D2O): d ˆ 171.48,
171.38,101.08,100.30,96.90,82.49,79.67,79.52,79.21,78.75,78.70,72.79,
72.72,72.02,71.28,71.23,69.97,69.31,69.07,68.87,68.73,68.67,68.51,
Daas,A. G. J. Voragen,H. A. Schols,
120.
Carbohydr. Res. 2000, 326,
[7] W. G. T. Willats,G. Limberg,H. C. Buchholt,G.-J. van Alebeek,J.
Benen,T. M. I. E. Christensen,J. Visser,A. Voragen,J. D. Mikkelsen,
J. P. Knox, Carbohydr. Res. 2000, 327,309.
[8] For a recent review,see: B. G. Davis, J. Chem. Soc. Perkin Trans. 1
2000,2137.
53.60 ppm; ESI MS/MS: m/z: 1115.1,939.0,749.0,559.0,368.7; ESI HRMS
calcd for C39H56O37Na: m/z: 1139.2398; found: m/z: 1139.2406 [M ‡ Na] .
‡
(Methyl a-d-galactopyranosyluronate)-(1 ! 4)-(a-d-galactopyranosylur-
onic acid)-(1 ! 4)-(a-d-galactopyranosyluronic acid)-(1 ! 4)-(a-d-galacto-
pyranosyluronic acid)-(1 ! 4)-(a-d-galactopyranosyluronic acid)-(1 ! 4)-
(methyl d-galactopyranuronate) (3): 13C NMR (D2O): d ˆ 171.67,170.93,
170.05,99.93,96.59,92.63,78.83,78.41,78.12,73.45,71.66,71.44,71.30,
[9] M. H. Clausen,M. R. J˘rgensen,J. Thorsen,R. Madsen, J. Chem. Soc.
Perkin Trans. 1 2001,543.
[10] H. C. M. Kester,D. Magaud,C. Roy,D. Anker,A. Doutheau,V.
Shevchik,N. Hugouvieux-Cotte-Pattat,J. A. E. Benen,J. Visser,
J.
Biol. Chem. 1999, 274,37053.
70.25,70.06,68.90,68.74,68.16,68.03,53.19,53.14,53.03 ppm; ESI MS/MS:
m/z: 1100.9,910.9,734.9,558.9,382.8; ESI HRMS calcd for C 38H54O37Na:
m/z: 1125.2242; found: m/z: 1125.2218 [M ‡ Na] .
(a-d-Galactopyranosyluronic acid)-(1 ! 4)-(methyl a-d-galactopyranosy-
luronate)-(1 ! 4)-(methyl a-d-galactopyranosyluronate)-(1 ! 4)-(methyl
a-d-galactopyranosyluronate)-(1 ! 4)-(methyl a-d-galactopyranosyluro-
nate)-(1 ! 4)-d-galactopyranuronic acid (4): 13C NMR (D2O): d ˆ 170.96,
170.86,100.60,99.90,96.40,79.22,78.90,78.24,72.18,71.54,70.81,70.61,
¬
[11] a) L. Parenicova,J. A. E. Benen,H. C. M. Kester,J. Visser, Biochem.
¬
J. 2000, 345,637; b) L. Parenicova ,H. C. M. Kester,J. A. E. Benen,J.
Visser, FEBSLett. 2000, 467,333; c) J. A. E. Benen,H. C. M. Kester,
L. Parenicova,J. Visser, Biochemistry 2000, 39,15563; d) C. Roy,H.
Kester,J. Visser,V. Shevchik,N. Hugouvieux-Cotte-Pattat,J. Robert-
Baudouy,J. Benen, J. Bacteriol. 1999, 181,3705; e) V. E. Shevchik,
H. C. M. Kester,J. A. E. Benen,J. Visser,J. Robert-Baudouy,N.
Hugouvieux-Cotte-Pattat, J. Bacteriol. 1999, 181,1652; f) L. Paren-
‡
¬
¬
icova,J. A. E. Benen,H. C. M. Kester,J. Visser, Eur. J. Biochem. 1998,
69.31,68.04,53.14 ppm; ESI MS/MS:
m/z: 1128.9,952.9,762.9,572.8,
251,72; g) E. M. W. Chen,A. J. Mort, Carbohydr. Polym. 1996, 29,
129.
[12] Larger non-esterified oligogalacturonates can be prepared by syn-
thesis or isolated pure by chromatography,see: Y. Nakahara,T.
Ogawa, Carbohydr. Res. 1990, 205,147; A. T. Hotchkiss,Jr.,S. L.
Lecrinier,K. B. Hicks, Carbohydr. Res. 2001, 334,135.
382.8 ppm; ESI HRMS calcd for C40H58O37Na: m/z: 1153.2555; found: m/z:
1153.2551 [M ‡ Na] .
‡
(Methyl a-d-galactopyranosyluronate)-(1 ! 4)-(a-d-galactopyranosylur-
onic acid)-(1 ! 4)-(methyl a-d-galactopyranosyluronate)-(1 ! 4)-(a-d-gal-
actopyranosyluronic acid)-(1 ! 4)-(methyl a-d-galactopyranosyluronate)-
(1 ! 4)-d-galactopyranuronic acid (5): 13C NMR (D2O): d ˆ 171.67,171.02,
99.90,94.45,92.61,82.00,78.77,78.68,78.16,72.14,71.45,70.77,70.25,68.92,
68.59,68.53,68.22,53.13,53.05 ppm; ESI MS/MS: m/z: 1115.1,939.1,749.0,
572.9,382.9; ESI HRMS calcd for C 39H56O37Na: m/z: 1139.2398; found:
[13] For previous syntheses of selectively methyl-esterified di- and
trigalacturonates,see: S. Kramer,B. Notting,A.-J. Ott,C. Vogel,
Carbohydr. Chem. 2000, 19,891; D. Magaud,C. Grandjean,A.
Doutheau,D. Anker,V. Shevchik,N. Cotte-Pattat,J. Robert-
Baudouy, Carbohydr. Res. 1998, 314,189; see Reference [10].
[14] B. Fraser-Reid,U. E. Udodong,Z. Wu,H. Ottosson,J. R. Merritt,
C. S. Rao,C. Roberts,R. Madsen, Synlett 1992,927.
J.
‡
m/z: 1139.2435 [M ‡ Na] .
[15] B. Fraser-Reid,Z. Wu,U. E. Udodong,H. Ottosson,
J. Org. Chem.
Acknowledgement
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[16] P. Konradsson,B. Fraser-Reid, J. Chem. Soc. Chem. Commun. 1989,
1124.
We thank Dr. Kudzai E. Mutenda,Department of Biochemistry and
Molecular Biology,University of Southern Denmark for conducting
analyses by mass spectrometry. Financial support from the Danish
Research Agency Center Contract Program is gratefully acknowledged.
[17] K. C. Nicolaou,R. E. Dolle,D. P. Papabatjis,J. L. Randall,
J. Am.
Chem. Soc. 1984, 106,4189. For a recent review on synthesis of
glycosyl fluorides,see: M. Yokoyama, Carbohydr. Res. 2000, 327,5.
Chem. Eur. J. 2003, 9,3821 3832
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
3831

R. Madsen,M. H. Clausen
FULL PAPER
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¬
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Received: December 4,2002 [F4636]
3832
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9,3821 3832