Synthesis of two repeat units corresponding to the backbone of the pectic polysaccharide rhamnogalacturonan I

Article abstract of DOI:10.1016/j.carres.2007.10.030

A tetrasaccharide corresponding to a sequence of the rhamnogalacturonan I backbone has been synthesized. This synthesis relies on only two protected monosaccharides and proceeds through a common disaccharide intermediate. Synthesis of this tetrasaccharide

Full text of DOI:10.1016/j.carres.2007.10.030

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 179–188
Synthesis of two repeat units corresponding to the backbone of
the pectic polysaccharide rhamnogalacturonan I
Dominic Reiffarth and Kerry B. Reimer*
Department of Chemistry, University of Northern British Columbia, 3333 University Way, Prince George,
British Columbia, Canada V2N 4Z9
Received 3 October 2007; received in revised form 26 October 2007; accepted 29 October 2007
Available online 4 November 2007
Abstract—A tetrasaccharide corresponding to a sequence of the rhamnogalacturonan I backbone has been synthesized. This syn-
thesis relies on only two protected monosaccharides and proceeds through a common disaccharide intermediate. Synthesis of this
tetrasaccharide has been designed to allow for the addition of branching elements at the 4-positions of the rhamnosyl units, or
further chain elongation at the 2-position.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Rhamnogalacturonan I; Galacturonic acid; cis-Glycoside
1. Introduction
relates to its function.8 Currently, immunofluorescent
studies of RG-I investigating the role of RG-I in plant
tissue are usually limited to using antibodies that have
been raised against the galactan and arabinan side
chains (LM53 and LM69 antibodies, respectively). No
antibody is currently in use that demonstrates specific
binding to the RG-I backbone.
There are a few reports in the literature of chemical
synthesis of oligosaccharide fragments found within
the RG-I polysaccharide.10–12 We report here the syn-
thesis and characterization of a versatile tetrasaccharide
corresponding to two repeat units of the RG-I back-
bone. A block synthesis approach has been used which
allows for the coupling of two disaccharide units derived
from a single disaccharide to form the desired tetrasac-
charide 14. The 4-position of the rhamnosyl residue
has been orthogonally protected to allow for the possi-
ble substitution of RG-I side chains.
Pozsgay mentions the difficulties of late stage oxida-
tion in terms of achieving yields that are often not satis-
factory.13 Indeed, difficulties were experienced in our lab
with initial attempts at building an RG-I tetrasaccharide
using galactose, and thus a new synthetic strategy was
devised based on commercially available D-galacturonic
acid as one of the monosaccharide building blocks (com-
pounds 2 and 3); this avoided the difficulties of late stage
Molecular mechanisms in plant glycobiology are often
studied using immunofluorescent labeling techniques,
requiring the use of monoclonal antibodies.1–3 The bind-
ing specificity of the antibody is most highly defined
when the antibody has been raised against a well-charac-
terized epitope. Chemical synthesis is thus necessary to
ensure that the binding specificities of the monoclonal
antibodies that are generated are unambiguous.4,5
Rhamnogalacturonan I (RG-I) is a complex polysac-
charide found in plant tissues. The backbone of RG-I
consists of alternating repeat units of L-rhamnose and
D-galacturonic acid: [-a-L-Rhap-(1!4)-a-D-GalAp-
(1!2)-]. RG-I is a highly substituted polysaccharide,
with a high degree of substitution occurring at the C-4
position of the rhamnosyl residue,6 forming branched
chains. The chains consist mainly of galactose and arab-
inose,7 although substitution with glucuronic acid occa-
sionally occurs at O-3 of the galacturonic acid residue.
Many of the structural features of RG-I have been
identified, but little is known about how its structure
*
Corresponding author. Tel.: +1 250 960 6675; fax: +1 250 960
5845; e-mail: reimerk@unbc.ca
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.10.030

180
D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
oxidation at C-6 of galactose to galacturonic acid. The
resulting end product was easily isolated both in the
methyl ester form 16 and as the free carboxylic acid 17.
In principle, both 4 and 5 can lead to either disaccha-
ride acceptors (removal of the 2-O-acetate group) or
disaccharide donors (removal of the anomeric methyl
groups and subsequent activation); this flexibility pro-
vides considerable synthetic economy. Removal of the
2-O-acetate on the rhamnosyl portion of disaccharide
4 was accomplished by using methanolic HCl17 to give
acceptor 6 in a yield of 80%. We expected that reaction
of 5 under these conditions would result in the exchange
of the benzyl group as well as the loss of the 2-O-acetate.
Indeed, when 5 was let stand for an extended period
(77 h), a mixture of products was formed (4, 7%; 5,
5%; 6, 44%, and 7, 36%). We did not investigate this
avenue any further, although it is possible that even
longer reaction times would produce an even larger
amount of 6, providing a viable route to convert the
benzyl ester 3 into usable disaccharide product 6.
With disaccharides 4 and 5 in hand, we wished to
determine which of these would serve as the most effec-
tive donor in subsequent glycosylation reactions.
Accordingly, both 4 and 5 were, in three steps, trans-
formed into their glycosyl imidates 10 and 13, respec-
tively (see Scheme 2). Acetolysis of 4 and 5 afforded
the anomeric acetates 8 and 9 in 81% and 91%, respec-
tively.18 The reaction to form 8 was complete after
about 2 h, and further reaction times lead to no change
in yield. Conversely, if the acetolysis reaction to form 9
was left longer than about 2 h, a decrease in the yield
was observed; after 4–5 h the yields had dropped to
about 25%.
2. Results and discussion
This synthesis utilizes two protected monosaccharide
building blocks, an a-L-rhamnose derivative 1 and an
a-D-galacturonic acid derivative 2. Synthesis of mono-
saccharide 1 had been previously published.10 Monosac-
charide 2 was prepared from methyl (methyl 2-O-benzyl-
a-D-galactopyranosid)uronate14 by selective benzylation
at C-3. Addition of the benzyl group at C-3 was per-
formed by stannylene acetal formation under the condi-
tions similar to those cited by Nolting and co-workers.15
In addition to benzylation at C-3 (compound 2), benzyl-
ation was also observed at the C-6 position to give com-
pound 3. Production of this dibenzylated compound
could not be avoided, so we decided to carry both
monosaccharides forward to the disaccharide stage in
parallel syntheses. The strategy was to transform both
these disaccharides into glycosyl donors that could be
then used to prepare a tetrasaccharide. This would allow
us to investigate the differences in donor reactivity due
to the protection of the carboxylic acid as either the
benzyl ester or the methyl ester.
Glycosylation of 2 or 3 using 110 with catalytic
amounts of triflic acid afforded disaccharides 4 and 5
in yields of 78% and 80%, respectively (see Scheme 1).
NMR spectroscopy was used to determine whether the
newly formed glycosidic linkages for 4 and 5 were a or
Removal of the anomeric acetates from 8 and 9 using
hydrazine acetate19 did not proceed smoothly, with
yields in the 50% range. This difficulty was overcome
by using a procedure provided by Kartha et al. where
b. The one-bond 13C–1H coupling constants (1J13C,1H
)
for 4 and 5 at C-1 of the rhamnosyl residues, as deter-
mined by analysis of the HMQC (inverse) spectra, were
found to be 173.7 Hz and 173.3 Hz, respectively, indica-
tive of an a-linkage.16
˚
powdered 4 A molecular sieves were added to methano-
lic solutions of anomeric acetates.20 Accordingly,
anomeric acetates 8 and 9 were dissolved in methanol
O
O
O
SEt
OR
HO
OR
O
AcO
BzO
O
O
+
AllO
O
a
BnO
BnO
OBz
OAc
BnO
BnO
1
OMe
OMe
2: R= Me
Or
3: R= Bn
4: R= Me
5: R= Bn
OAll
b
O
O
OR
O
HO
BzO
O
BnO
BnO
OMe
6: R= Me
7: R= Bn
OAll
Scheme 1. Synthesis of the disaccharides and glycosyl acceptors. Reagents and conditions: (a) N-iodosuccinimide, triflic acid (cat.), in CH2Cl2 ꢀ40
ꢁC (78% 4 and 80% 5); (b) methanolic HCl in CH2Cl2 (80% 6 and 37% 7).

D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
181
O
OR2
O
O
R2=Me R2=Bn
R1
AcO
BzO
O
4
8
5
CH3
Ac
H
a
a
BnO
11
12
13
b
c
b
c
BnO
OR1
9
10
C(=NH)CCl3
OAll
Scheme 2. Synthesis of disaccharide glycosyl donors. Reagents and conditions: (a) 1% H2SO4 in AcOH and Ac2O (81% 8 and 91% 11); (b) powdered
molecular sieves in CH2Cl2/MeOH (89% 9 and 76% 12); (c) trichloroacetonitrile, DBU, in CH2Cl2 (81% 10 and 82% 13).
˚
and powdered 4 A molecular sieves were added to the
solution. The solutions were let stand at room tempera-
ture and the reactions proceeded slowly, but cleanly to
provide hemiacetals 10 and 11 in 81% and 82%, respec-
tively. Transformation of hemiacetals 9 and 12 into the
corresponding imidates 10 and 13 was accomplished
by the treatment with trichloroacetonitrile and DBU.21
The methyl ester 10 was isolated as the a-anomer,
whereas the benzyl ester 13 was isolated as an anomeric
mixture.
for 15, the sample failed both elemental and MS ana-
lyses, and is thus not included here. The low yield for
tetrasaccharide 14 is partially a result of multiple
chromatographic purification steps required to separate
the desired product from unreacted glycosyl acceptor 6.
No glycosylation attempts, despite the addition of more
equivalents of donor and promoter, were successful in
eliminating the presence of unreacted acceptor 6.
A number of methods were tried in an effort to
increase the conversion of glycosyl acceptor into tetra-
saccharide product. Activation of the imidate donors
10 and 13 using trimethylsilyl trifluoromethanesulfonate
(TMSOTf)22 or tert-butyldimethylsilyl trifluoromethane-
sulfonate (t-BDMSOTf)23 both proved unsuccessful.
Attempts to generate thioglycoside donors from the
1-O-acetate 8 using p-thiocresol and either boron tri-
fluoride etherate (BF3ÆEt2O) or tin(IV) chloride (SnCl4)
did not result in the formation of significant amounts
of thioglycoside. Attempts were also made to generate
an a-bromide donor in situ from hemiacetal 12 using tri-
phenyl phosphine (P(Ph)3) and carbon tetrabromide
(CBr4),24,25 followed by the addition of the disaccharide
Glycosylation of the disaccharide acceptor 6 to form
the tetrasaccharide structure proved to be problematic.
Glycosylation of 6, using silver triflate (AgOTf) as pro-
moter, with either the methyl ester 10 or the benzyl ester
13 imidates as donor, led to formation of the desired
tetrasaccharide product (see Scheme 3); however only
low yields were obtained (39% for 14 and an impure
sample for 15). The reaction mixture containing 15
was purified by chromatography multiple times using
various eluting solvents in an attempt to isolate pure tet-
rasaccharide but no attempts were successful. Although
the 1H, 13C, and COSY spectra could be clearly assigned
O
O
OR
O
OMe
O
AcO
BzO
HO
O
O
O
O
BnO
+
BnO
BnO
BzO
BnO
OC(=NH)CCl3
OMe
6
10: R= Me
13: R= Bn
OAll
OAll
O
O
OR
AcO
BzO
O
O
O
O
BnO
OMe
BnO
O
O
O
OAll
BnO
BzO
BnO
OMe
OAll
14: R= Me
15: R= Bn
Scheme 3. Synthesis of the fully protected tetrasaccharides. Reagents and conditions: AgOTf, molecular sieves in CH2Cl2 (39% 14 and undetermined
yield 13).

182
D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
acceptor 6; TLC analysis indicated that no reaction had
taken place.
O
OR2
O
O
R3O
R4O
This particular glycosylation is one that could benefit
by the use of solid support during solution phase synthe-
sis to circumvent some of the purification difficulties in
obtaining both tetrasaccharides 14 and 15.26
Complete NMR analysis was performed on tetrasac-
charide 14. Hydrogen and carbon assignments were per-
O
O
O
R1O
OR2
O
R1O
O
O
OR5
R1O
R4O
R1O
OMe
1
formed through a combined analysis of H NMR, 13C
OR5
NMR, COSY, and HMQC (inverse) spectra. All signals
corresponding to the ring protons and carbons could be
successfully accounted for, as well as all protecting
groups expected to be present.
R1
14 Bn
R2
R3
R4
R5
CH3
CH3
H
Ac
H
H
Bz
H
H
All
H
H
a, b, c
d
Four signals corresponding to C-1 of each of the four
monosaccharide residues could be seen in the 95 ppm to
100 ppm region of the 13C spectrum. HMQC (inverse)
analysis was used to determine the one-bond 13C–1H
coupling constants (1J13C,1H) for C-100 to H-100 on the
donor galacturonic acid residue to verify whether or
not the newly formed linkage was indeed in the desired
a-configuration. The observed coupling constant fell
within the expected range16 at 171.5 Hz, indicating that
the a-linkage had been successfully formed.
16
17
H
H
Scheme 4. Deprotection of tetrasaccharide 14. Reagents and condi-
tions: (a) (i) [(C6H5)3P]3RhCl, DABCO in EtOH/toluene/H2O; (ii)
HgO, HgCl2 in acetone/H2O; (b) NaOMe in MeOH (pH 11–12) (c) H2
(40 psi) over Pd(OAc)2; (d) (i) NaOHaq (pH 11–12); (ii) HClaq (pH 2–
3).
charide residues leading to branched segments found
within the RG-I polysaccharide structure.
A fully deprotected sample of tetrasaccharide was
obtained from 14 in four steps (see Scheme 4). The allyl
groups were removed by treatment with Wilkinson’s
catalyst ([(C6H5)3P]3RhCl), followed by the cleavage of
the resulting vinyl ether using a combination of mer-
cury(II) oxide and mercury(II) chloride.27 The yellow-
brown foam was treated with sodium methoxide to
remove the benzoate and the acetate groups. The crude
material was purified by chromatography before being
submitted to hydrogenation conditions.28 Removal of
the benzyl groups present on the partially deprotected
tetrasaccharide was successfully accomplished by plac-
ing the purified compound under H2(g) (40 psi) in the
presence of a palladium acetate catalyst, giving 16
(33% yield). 1H NMR, 13C NMR, HMQC (inverse),
and COSY analyses were performed on compound16;
the spectra thus obtained were consistent with the pro-
posed structure.
Tetrasaccharide 16 was dissolved in H2O and saponi-
fied by the addition of 1.0 M NaOH until pH 11–12, and
then acidified using 1.0 M HCl until pH 2–3. The prod-
uct was placed on a P2 gel column to remove any salts
present after saponification. The purified fractions were
pooled and lyophilized, producing the final target com-
pound 17 as a white solid (77% yield).
This synthesis has been planned to allow the fully
protected tetrasaccharide to be further elongated by
the removal of the 2-O-acetyl group of the non-reducing
terminus and thus generating a glycosyl acceptor, or
alternatively, removal of the anomeric methyl group
and the generation of a glycosyl donor. In addition,
the removal of the 4-O-allyl groups from the rhamnosyl
residues allows for the addition of branching monosac-
3. Experimental
3.1. General methods
1H NMR (300.13 MHz) and 13C NMR (75.03 MHz)
spectra were compiled with a Bruker AMX 300 spec-
trometer. Chemical shifts are relative to external tetra-
methyl silane (TMS). Electrospray ionization mass
spectra were measured on Water/Micromass LCT. The
samples were dissolved in MeOH. The solution concen-
tration was about 25–50 lM. They were infused to LCT
at a flow rate of 20 lL/min. Optical rotations were mea-
sured on an Autopol III polarimeter. Melting points
were performed on an Electrothermal Digital Melting
Point Apparatus (IA9100). All reactions were monitored
by thin-layer chromatography (TLC) using silica gel on
an aluminum support (Silicycle 250 lm, F-254 indica-
tor), with detection by charring with 5% sulfuric acid
(v/v) in EtOH. TLCs were performed with solvents of
appropriately adjusted polarity consisting of A, 1:1
hexane–EtOAc; B, 3:2 hexanes–EtOAc; C, 2:1 hexanes–
EtOAc; D, 3:1 hexanes–EtOAc; E, 2:1:1 hexanes–
EtOAc–CHCl3; F, 4:1 hexanes–EtOAc; G, 8:1
toluene–acetone; H, 3:1:1 hexanes–EtOAc–CHCl3; I,
2:1:1 hexanes–EtOAc–CH2Cl2; J, 4:1:1 hexanes–EtOAc–
CH2Cl2; K, 4:3:3 hexanes–EtOAc–CHCl3; L, 6:6:1
hexanes–EtOAc–MeOH; M, 12:2:1 EtOAc– hexanes–
MeOH; N, 16:2:1 EtOAc–hexanes–MeOH; O, 10:3:1
EtOAc–MeOH–AcOH; P, 20:20:1 EtOAc–MeOH–
AcOH; Q, 7:3 CH3CN–H2O. All chromatography was

D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
183
performed using low pressure liquid chromatography
columns packed with silica gel (Silicycle 230–400 mesh
(40–63 lm) 60 A).
mixture was washed sequentially with satd NaHCO3
and 10% (w/v) Na2S2O3. The organic layer was dried
over Na2SO4, filtered, evaporated to dryness, and
purified by column chromatography (eluting solvent
˚
22
3.2. Methyl (methyl 2,3-di-O-benzyl-a-D-galactopyran-
osid)uronate (2) and benzyl (methyl 2,3-di-O-benzyl-a-
D-galactopyranosid)uronate (3)
C), giving 4 (0.504 g, 78%). ½aꢁD +3.3 (c 0.6, CHCl3);
1H NMR (300.13 MHz, CDCl3): dH 8.05–7.95 (m, 2H,
aromatic), 7.61–7.24 (13H, aromatic), 5.78 (1H, OCH2-
CH@CH2), 5.61 (dd, 1H, J2,3 = 3.1 Hz, H-2 Rha), 5.49
(dd, 1H, J3,4 = 9.4 Hz, H-3 Rha), 5.20–5.04 (2H,
OCH2CH@CH2), 5.14 (d, J1,2 = 2.2 Hz, H-1 Rha),
4.90, 4.70 (2H, OCH2Ph), 4.84, 4.72 (2H, OCH2Ph),
4.71 (d, 1H, J1,2 = 3.5 Hz, H-1 GalA), 4.44 (1H,
H-4 GalA), 4.39 (1-H, H-5 GalA), 4.12 (2H,
OCH2CH@CH2), 4.09 (dd, 1H, J2,3 = 10.0 Hz, H-2
GalA), 3.95 (dd, 1H, J3,4 = 2.7 Hz, H-3 GalA), 3.83 (s,
3H, CO2CH3), 3.79 (dd, 1H, J5,6 = 6.2 Hz, H-5 Rha),
3.48 (dd, 1H, J4,5 = 9.5 Hz, H-4 Rha), 3.39 (s, 3H,
OCH3), 2.00 (s, 3H, OCOCH3), 1.35 (3H, H-6 Rha);
13C NMR (75.03 MHz, CDCl3): dC 169.5, 169.1
(C@O), 165.2 (C@O, benzoate), 138.4, 133.1, 130.4,
129.7, 128.8, 128.6, 128.5, 128.1, 127.8, 127.7 (C
aromatic), 134.9 (OCH2CH@CH2), 116.8 (OCH2-
Methyl (methyl 2-O-benzyl-a-D-galactopyranosid)uro-
nate14 (6.276 g, 20.08 mmol), Bu2SnO (6.000 g,
24.10 mmol), and toluene (240 mL) were heated at reflux
˚
for 5 h 40 min; water was removed by 4 A molecular
sieves placed in a Soxhlet extractor. The reaction mix-
ture was then heated in a 70 ꢁC oil bath and Bu4NBr
(7.8 g, 24 mmol) was added, followed by BnBr
(5.9 mL). The reaction was monitored by TLC (solvent
A) and stopped after 6.5 h and allowed to cool to rt;
MeOH was added and the mixture was evaporated to
dryness. The resulting syrup was purified by chromato-
graphy (eluting solvent B) to give 2 (5.376 g, 67%) and
22
3 (2.499 g, 26%). Anal. for 2: ½aꢁD +33.3 (c 0.6, CHCl3),
25
1
lit.29 ½aꢁD +33.3; H NMR (300.13 MHz, CDCl3): dH
7.41–7.26 (10H, aromatic), 4.83, 4.66 (2H, OCH2Ph),
4.81, 4.74 (2H, OCH2Ph), 4.76 (d, 1H, J2,1 = 3.3 Hz,
H-1), 4.39 (2H, H-4, H-5), 3.95 (dd, 1H, J3,4 = 3.1 Hz,
H-3), 3.89 (dd, 1H, J2,3 = 9.8 Hz, H-2), 3.82 (s, 3H,
CO2CH3), 3.41 (s, 3H, OCH3). Anal. Calcd for
C22H26O7: C, 65.66; H, 6.51. Found: C, 65.24; H, 6.40.
1
CH@CH2), 99.6 (C-1 GalA, J13C,1H = 170.8 Hz), 99.4
1
(C-1 Rha, J13C,1H = 173.7 Hz), 78.5 (C-4 Rha), 77.5
(C-3 GalA), 76.9 (C-4 GalA), 75.8 (C-2 GalA), 74.5
(CH2Ph), 73.6 (OCH2CH@CH2, CH2Ph), 72.0 (C-3
Rha), 70.5 (C-2 Rha), 70.3 (C-5, GalA), 68.4 (C-5
Rha), 56.3 (OCH3), 52.7 (CO2CH3), 21.0 (OCOCH3),
18.2 (C-6 Rha); ESI-MS found m/z 757.2839
[M+Na]+. Calcd for C40H46O13Na: 757.2836.
22
Anal. for 3: mp 101.2–102.0 ꢁC; ½aꢁD +24.4 (c 0.6,
1
CHCl3); H NMR (300.13 MHz, CDCl3): dH 7.45–7.26
(15H, aromatic), 5.27 (2H, CO2CH2Ph), 4.83, 4.66
(2H, OCH2Ph), 4.82, 4.72 (2H, OCH2Ph), 4.77 (d,
1H, J1,2 = 3.2 Hz, H-1), 4.41 (1H, H-5), 4.40–4.36 (1H,
H-4), 3.95 (dd, 1H, J3,4 = 3.0 Hz, H-3), 3.90 (dd, 1H,
J2,3 = 9.8 Hz, H-2), 3.83 (s, 3H, CO2CH3), 3.38 (s, 3H,
OCH3). Anal. Calcd for C28H30O7: C, 70.28; H, 6.32.
Found: C, 69.98; H, 6.50.
3.4. Benzyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-L-
rhamnopyranosyl)-(1!4)-(methyl 2,3-di-O-benzyl-a-
D-galactopyranosid)uronate (5)
A sample of compound 3 (0.432 g, 0.903 mmol) was
dissolved in freshly distilled CH2Cl2 (17.0 mL); 3 A
˚
molecular sieves were added, and the solution was
stirred under N2(g) in a pyr/N2(l) bath for 10 min (the
pyr/N2(l) bath (approximately ꢀ35 ꢁC to ꢀ40 ꢁC) was
maintained for the duration of the reaction). The thio-
glycoside donor 110 (0.499 g, 1.26 mmol, 1.4 equiv to
acceptor) was then added and the mixture was stirred
for an additional 10 min, followed by the addition of
NIS (0.304 g, 1.35 mmol, 1.07 equiv to donor) and stir-
ring for another 10 min 50 lL of TfOH (0.565 mmol,
0.45 equiv to donor) was added and the progress of
the reaction was followed by TLC (solvent C). The reac-
tion mixture turned a deep purple almost immediately
and was neutralized thereafter with Et3N, washed
sequentially with satd NaHCO3, and 10% (w/v)
Na2S2O3. The organic layer was dried over Na2SO4,
filtered, evaporated to dryness, and purified by column
chromatography (eluting solvent D), giving 5 (0.586 g,
3.3. Methyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-L-
rhamnopyranosyl)-(1!4)-(methyl 2,3-di-O-benzyl-a-
D-galactopyranosid)uronate (4)
A sample of compound 2 (0.356 g, 0.885 mmol) was dis-
˚
solved in freshly distilled CH2Cl2 (15.8 mL); 3 A molec-
ular sieves were added, and the solution was stirred
under N2(g) in a pyr/N2(l) bath for 10 min (the pyr/
N
2(l) bath (approximately ꢀ35 ꢁC to ꢀ40 ꢁC) was main-
tained for the duration of the reaction). The thioglyco-
side donor 110 (0.489 g, 1.24 mmol, 1.4 equiv to
acceptor) was then added and the mixture was stirred
for an additional 10 min. NIS (0.298 g, 1.32 mmol,
1.06 equiv to donor) was added and the mixture was
stirred for another 10 min followed by the addition of
49 lL of TfOH (0.554 mmol, 0.45 equiv to donor). The
reaction was monitored by TLC (solvent C) and halted
after 15 min by neutralization using Et3N. The reaction
22
80%). ½aꢁD +0.4 (c 0.5, CHCl3); 1H NMR
(300.13 MHz, CDCl3): dH 8.06–7.97 (2H, aromatic),

184
D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
7.63–7.19 (18H, aromatic), 5.80 (1H, OCH2CH@CH2),
5.65 (dd, 1H, J2,3 = 3.3 Hz, H-2 Rha), 5.54 (dd, 1H,
J3,4 = 9.5 Hz, H-3 Rha), 5.32, 5.14 (2H, CO2CH2Ph),
5.13 (2H, OCH2CH@CH2), 5.10 (d, 1H, J1,2 = 2.1 Hz,
H-1 Rha), 4.91, 4.74 (2H, OCH2Ph), 4.84, 4.68 (2H,
OCH2Ph), 4.72 (d, 1H, J1,2 = 3.5 Hz, H-1 GalA),
4.45–4.39 (2H, H-4, H-5, GalA), 4.23–4.06 (2H,
OCH2CH@CH2), 4.23–4.06 (1H, H-2, GalA), 3.98–
3.88 (1H, J3,4 = 2.8 Hz, H-3 GalA), 3.98–3.88 (1H,
J6,5 = 6.2 Hz, H-5 Rha), 3.50 (dd, 1H, J4,5 = 9.5 Hz,
H-4 Rha), 3.38 (s, 3H, OCH3), 2.01 (s, 3H, OCOCH3),
1.35 (3H, H-6 Rha); 13C NMR (75.03 MHz, CDCl3):
dC 169.5, 168.3 (C@O), 165.2 (C@O, benzoate), 138.5,
135.2, 133.1, 130.5, 129.7, 128.8, 128.7, 128.6, 128.5,
128.1, 127.9, 127.7 (C aromatic), 134.9 (OCH2-
134.8 (OCH2CH@CH2), 117.0 (OCH2CH@CH2), 102.0
(C-1 Rha, J13C,1H = 170.6 Hz), 99.4 (C-1 GalA,
1
1J13C,1H = 170.6 Hz), 78.9 (C-4 Rha), 77.7 (C-3 GalA),
77.1 (C-4 GalA), 75.9 (C-2 GalA), 74.3 (CH2Ph), 74.1
(C-3 Rha), 73.9 (CH2Ph), 73.1 (OCH2CH@CH2), 70.4
(C-5, GalA), 69.9 (C-2 Rha), 68.4 (C-5 Rha), 56.3
(OCH3), 52.7 (CO2CH3), 18.5 (C-6 Rha). Anal. Calcd
for C38H44O12: C, 65.88; H, 6.40. Found: C, 65.52; H,
6.32.
3.6. Benzyl (4-O-allyl-3-O-benzoyl-a-L-rhamnopyran-
osyl)-(1!4)-(methyl 2,3-di-O-benzyl-a-D-galactopyran-
osid)uronate (7)
A solution of 5 (0.500 g, 0.617 mmol) in CH2Cl2
(11.7 mL) was treated with 11.3 mL methanolic HCl
(prepared by the addition of 1.0 mL of acetyl chloride
to 35 mL distilled MeOH). The reaction was stirred
under N2(g) at rt for approximately 77.5 h, after which
time the mixture was worked up by diluting with CH2Cl2,
washing with satd NaHCO3, drying over Na2SO4, filter-
ing, and evaporating to dryness (TLC solvent B). The
resulting syrup was purified by chromatography (eluting
solvent B); two samples were collected, one of which
consisted of the compounds corresponding to the two
upper spots that appeared on TLC, and a second sample
that consisted of the compounds corresponding to the
two lower spots. Both of these samples were then indi-
vidually purified by chromatography again (eluting sol-
vent C) and yielded the following: 5, 0.027 g, 5%; 7,
0.174 g, 37%; 4, 0.033 g, 7%; 6, 0.189 g, 44%. Anal. for
CH@CH2), 116.8 (OCH2CH@CH2), 99.7 (C-1 Rha,
1
1J13C,1H = 173.3 Hz), 99.6 (C-1 GalA, J13C,1H
=
172.5 Hz), 78.6 (C-4 Rha), 77.7 (C-4 GalA), 77.5 (C-3
GalA), 75.9 (C-2 GalA), 74.6, 73.7 (CH2Ph), 73.6
(OCH2CH@CH2), 72.1 (C-3 Rha), 70.6 (C-2 Rha),
70.2 (C-5, GalA), 68.6 (C-5 Rha), 67.5 (CO2CH2Ph),
56.3 (OCH3), 21.0 (OCOCH3), 18.4 (C-6 Rha); ESI-
MS found m/z 833.3148 [M+Na]+. Calcd for
C40H46O13Na: 833.3149.
3.5. Methyl (4-O-allyl-3-O-benzoyl-a-L-rhamnopyran-
osyl)-(1!4)-(methyl 2,3-di-O-benzyl-a-D-galactopyran-
osid)uronate (6)
A sample of compound 4 (0.382 g, 0.520 mmol) was dis-
solved in freshly distilled CH2Cl2 (8.0 mL), to which
methanolic HCl (8.0 mL + 1.5 mL after approximately
49 h; prepared by the addition of 1.0 mL of acetyl chlo-
ride to 35 mL distilled MeOH) was added. The reaction
mixture was allowed to sit at rt for approximately 4
days, after which the reaction mixture was diluted with
CH2Cl2, washed with satd NaHCO3, filtered, dried over
Na2SO4, and evaporated to dryness (TLC solvent A).
The resulting syrup was purified by chromatography
22
1
7: ½aꢁD +6.9 (c 0.4, CHCl3); H NMR (300.13 MHz,
CDCl3): dH 8.14–8.07 (2H, aromatic), 7.65–7.22 (18H,
aromatic), 5.81 (1H, OCH2CH@CH2), 5.44 (dd, 1H,
J3,4 = 8.2 Hz, H-3 Rha), 5.30, 5.15 (2H, CO2CH2Ph),
5.15 (2H, OCH2CH@CH2), 5.13 (d, 1H J1,2 = 2.9 Hz,
H-1 Rha), 4.87, 4.70 (2H, OCH2Ph), 4.85, 4.68 (2H,
OCH2Ph), 4.75 (d, 1H, J1,2 = 3.4 Hz, H-1 GalA),
4.45–4.40 (2H, H-4, H-5, GalA), 4.38 (d, 1H, J3,2
=
22
(eluting solvent C) to give pure 6 (0.287 g, 80%). ½aꢁD
3.0 Hz, H-2 Rha), 4.28–4.03 (2H, OCH2CH@CH2),
4.07 (dd, 1H, J2,3 = 10.1 Hz, H-2 GalA), 3.95 (dd, 1H,
J3,4 = 2.7 Hz, H-3 GalA), 3.90 (dd, 1H, J5,6 = 6.2 Hz,
H-5 Rha), 3.54 (dd, 1H, J5,4 = 9.3 Hz, H-4 Rha), 3.39
(s, 3H, OCH3), 1.34 (3H, H-6 Rha); 13C NMR
(75.03 MHz, CDCl3): dC 168.2 (CO2CH3), 165.5
(C@O, benzoate), 138.4, 138.3, 135.3, 133.3, 129.9,
129.0 128.9, 128.8, 128.7, 128.1, 128.0, 128.0, 127.8,
127.2 (C aromatic), 134.8 (OCH2CH@CH2), 117.0
+13 (c 0.5, CHCl3);1H NMR (300.13 MHz, CDCl3):
dH 8.13–8.06 (m, 2H, aromatic), 7.64–7.21 (13H,
aromatic), 5.80 (1H, OCH2CH@CH2), 5.49 (dd, 1H,
J3,4 = 8.2 Hz, H-3 Rha), 5.13 (2H, OCH2CH@CH2),
5.13 (d, 1H, J1,2 = 2.8 Hz, H-1 Rha), 4.86, 4.69 (2H,
OCH2Ph), 4.84, 4.71 (2H, OCH2Ph), 4.75 (d, 1H,
J1,2 = 3.4 Hz, H-1 GalA), 4.48–4.44 (1H, H-4, GalA),
4.43–4.39 (1H, H-5, GalA), 4.32 (dd, 1H, J3,2
=
3.1 Hz, H-2 Rha), 4.24–4.05 (2H, OCH2CH@CH2),
4.05 (dd, 1H, J2,3 = 10.1 Hz, H-2 GalA), 3.97 (dd, 1H,
J3,4 = 2.7 Hz, H-3 GalA), 3.84 (s, 3H, CO2CH3), 3.77
(dd, 1H, J5,6 = 6.2 Hz, H-5 Rha), 3.51 (dd, 1H,
J4,5 = 9.3 Hz, H-4 Rha), 3.40 (s, 3H, OCH3), 1.33 (3H,
H-6 Rha); 13C NMR (75.03 MHz, CDCl3): dC 168.9
(CO2CH3), 165.5 (C@O, benzoate), 138.4, 138.2, 133.3,
130.4, 129.9, 128.7, 128.1, 128.0, 127.9 (C aromatic),
(OCH2CH@CH2), 102.5 (C-1 Rha, 1J13C,1H
=
1
170.4 Hz), 99.4 (C-1 GalA, J13C,1H = 170.9 Hz), 79.5
(C-4 Rha), 78.2 (C-4 GalA), 77.5 (C-3 GalA), 76.0
(C-2 GalA), 74.0 (CH2Ph), 74.2 (C-3 Rha), 74.0
(CH2Ph), 73.2 (OCH2CH@CH2), 70.3 (C-5, GalA),
69.8 (C-2 Rha), 68.6 (C-5 Rha), 67.6 (CO2CH2Ph),
56.3 (OCH3), 18.6 (C-6 Rha); ESI-MS found m/z
791.3041 [M+Na]+. Calcd for C44H48O12Na: 791.3043.

D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
185
3.7. Methyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-L-
3.9. Methyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-L-
rhamnopyranosyl)-(1!4)-(1-O-acetyl-2,3-di-O-benzyl-
rhamnopyranosyl)-(1!4)-2,3-di-O-benzyl-a-D-galacto-
a/b-D-galactopyranosid)uronate (8)
pyranosyluronate trichloroacetimidate (10)
A sample of compound 4 (1.860 g, 2.514 mmol) was
dissolved in AcOH (41.7 mL) and Ac2O (41.7 mL).
The reaction mixture was then cooled in an ice bath
for 10 min, after which H2SO4 (417 lL) was slowly
added. The reaction was followed by TLC (solvent E)
and showed completion after approximately 2 h
15 min. The reaction mixture was allowed to warm to
rt during the progress of the reaction. NaOAcÆ3H2O
(4.38 g) was then added and the mixture was stirred
until all NaOAcÆ3H2O had dissolved. The mixture
was then evaporated to dryness, taken up in EtOAc,
washed with H2O (extracted 4· with EtOAc), dried
over Na2SO4, filtered, evaporated to dryness, and
finally co-evaporated three times with EtOH. The
resulting syrup was purified by chromatography
(eluting solvent A) to obtain pure 8 (1.555 g, 81%).
NMR data are given only for the major component,
the a-anomer. 1H NMR (300.13 MHz, CDCl3): dH
2.12 (s, 3H, GalA –OCOCH3), 2.01 (s, 3 H, Rha
–OCOCH3); 13C NMR (75.03 MHz, CDCl3): dC
169.7, 169.5, 169.1, 168.8 (C@O, Rha OC(O)CH3,
GalA OC(O)CH3 and CO2CH3), 165.2 (C@O, benzo-
A sample of hemiacetal 9 (0.592 g, 0.821 mmol) was dis-
solved in freshly distilled CH2Cl2 (13.5 mL) and stirred
˚
under N2(g) in the presence of 4 A MS (0.590 g) at rt
for approximately 10 min, after which the mixture was
placed in an ice bath and stirred for an additional
15 min. Trichloroacetonitrile (837 lL, 8.35 mmol,
10.2 equiv) was then added, followed by DBU
(147 lL, 0.983 mmol, 1.20 equiv). The reaction was
followed by TLC (TLC solvent A) and stopped after
10 min. The mixture was immediately placed on a
vacuum column (eluting solvent F); fractions containing
10 were combined and evaporated to dryness. Impure 10
was then purified by chromatography twice; 10 was first
purified by chromatography using eluting solvent C
(0.498 g pure 10); impure fractions were collected and
purified by chromatography again using eluting solvent
G (0.079 g). The overall yield of 10 was 0.577 g (81%).
1H NMR (300.13 MHz, CDCl3): dH 8.63 (s, 1H,
C@NH), 8.05–7.97 (2H, aromatic), 7.63–7.20 (13H,
aromatic), 6.67 (d, 1H, J1,2 = 3.3 Hz, H-1 GalA), 5.79
(1H, OCH2CH@CH2), 5.61 (dd, 1H, J2,1 = 2.2 Hz,
J2,3 = 3.1 Hz, H-2 Rha), 5.48 (dd, 1H, J3,4 = 9.4 Hz,
H-3 Rha), 5.25–5.03 (3H, H-1 Rha, OCH2CH@CH2),
4.85–4.70 (4H, 2 OCH2Ph), 4.65–4.61 (1H, H-5, GalA),
4.57–4.52 (1H, H-4, GalA), 4.30 (dd, 1H, J2,3 = 10.0 Hz,
H-2 GalA), 4.22–4.05 (2H, OCH2CH@CH2), 4.05 (dd,
1H, J3,4 = 2.7 Hz, H-3 GalA), 3.85 (s, 3H, CO2CH3),
3.78 (dd, 1H, J5,6 = 6.2 Hz, H-5 Rha), 3.50 (dd, 1H,
J4,5 = 9.5 Hz, H-4 Rha), 2.04 (s, 3H, OCOCH3), 1.37
(3H, H-6 Rha); 13C NMR (75.03 MHz, CDCl3): dC
169.6, 168.4 (C@O), 165.3 (C@O, benzoate), 160.7
(OC(NH)CCl3), 138.4, 138.0, 133.2, 130.3, 129.7, 128.6,
128.5, 128.5, 128.1, 127.9, 127.8, 127.7 (C aromatic),
134.8 (OCH2CH@CH2), 116.9 (OCH2CH@CH2),
ate), 138.2–127.8 (C aromatic), 134.8 (OCH2CH@CH2),
1
116.9 (OCH2CH@CH2), 99.3 (C-1 Rha, J13C,1H
=
1
174.8 Hz), 90.6 (C-1 GalA, J13C,1H = 177.9 Hz), 78.5
(C-4 Rha), 77.2 (C-3 GalA), 75.8 (C-4 GalA), 74.9
(C-2 GalA), 74.1 (CH2Ph), 73.7 (OCH2CH@CH2),
73.4 (CH2Ph), 72.6 (C-5 GalA), 72.0 (C-3 Rha), 70.5
(C-2 Rha), 68.5 (C-5 Rha), 52.9 (CO2CH3), 21.2 (GalA
OCOCH3), 21.0 (Rha OCOCH3), 18.2 (C-6 Rha). Anal.
Calcd for C41H46O14: C, 64.56; H, 6.08. Found: C,
64.70; H, 5.79.
3.8. Methyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-
L-rhamnopyranosyl)-(1!4)-(2,3-di-O-benzyl-a/b-D-
galactopyranosid)uronate (9)
1
99.3 (C-1 Rha, J13C,1H = 174.7 Hz), 94.9 (C-1 GalA,
1J13C,1H = 180.4 Hz), 78.5 (C-4 Rha), 76.3 (C-3
GalA), 75.7 (C-4 GalA), 75.5 (C-2 GalA), 73.7
(OCH2CH@CH2), 73.7, 73.3 (CH2Ph), 72.8 (C-5,
GalA), 72.0 (C-3 Rha), 70.5 (C-2 Rha), 68.6 (C-5
Rha), 52.9 (CO2CH3), 21.0 (OCOCH3), 18.2 (C-6
Rha); ESI-MS found m/z 886.1777 [M+Na]+. Calcd
for C41H44NO13Cl3Na: 886.1776.
A sample of compound 8 (0.794 g, 1.04 mmol) was dis-
solved in distilled MeOH (25.8 mL) and CH2Cl2
˚
(14.7 mL). Powdered 4 A MS (0.529 g, 0.667 equiv by
weight to 8) were then added. The reaction was stirred
at rt for approximately 4 days, after which the solids
were removed by filtration (TLC solvent A). The filtrate
was evaporated to dryness and the resulting syrup was
purified by chromatography (eluting solvent A) to give
pure 9 (0.670 g, 90%). 13C NMR (75.03 MHz, CDCl3):
dC 134.8 (OCH2CH@CH2), 116.9 (OCH2CH@CH2),
3.10. Benzyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-L-
rhamnopyranosyl)-(1!4)-(1-O-acetyl-2,3-di-O-benzyl-
a/b-D-galactopyranosid)uronate (11)
1
99.9, 99.3 (C-1a/b Rha, J13C,1H = 173.4 Hz), 92.4, 92.3
A sample of 11 was prepared by dissolving dissacharide
5 (1.830 g, 2.257 mmol) in AcOH (37.3 mL) and Ac2O
(37.3 mL). The reaction mixture was then cooled in an
ice bath for 10 min, after which H2SO4 (370 lL) was
slowly added. The reaction was followed by TLC
1
(C-1a/b GalA, J13C,1H = 171.5 Hz, 172.3 Hz), 52.7
(CO2CH3), 21.0 (Rha OCOCH3), 18.4, 18.2 (C-6a/b
Rha); ESI-MS found m/z 743.2676 [M+Na]+. Calcd
for C39H44O13Na: 743.2680.

186
D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
(TLC solvent H) and showed completion after approxi-
mately 2 h 05 min. The reaction mixture was allowed to
warm to rt during the progress of the reaction. NaO-
AcÆ3H2O (3.911 g) was then added and the mixture
was stirred until all NaOAcÆ3H2O had dissolved. The
mixture was then evaporated to dryness, taken up in
EtOAc, washed with H2O (extracted 4· with EtOAc),
dried over Na2SO4, filtered, evaporated to dryness,
and finally co-evaporated three times with EtOH. The
resulting syrup was purified by chromatography (eluting
solvent C) to obtain pure 11 (1.730 g, 91%). NMR data
are given only for the major component, the a-anomer.
1H NMR (300.13 MHz, CDCl3): dH 2.12 (s, 3H, GalA
–OCOCH3), 2.01 (s, 3H, Rha –OCOCH3); 13C NMR
(75.03 MHz, CDCl3): dC 169.7, 169.5, 169.0, 167.6
(C@O, Rha OC(O)CH3, GalA OC(O)CH3 and
CO2CH2Ph), 165.2 (C@O, benzoate), 138.2–127.8 (C
aromatic), 134.8 (OCH2CH@CH2), 116.9 (OCH2-
(605 lL, 6.03 mmol, 10.0 equiv) was then added,
followed by DBU (108 lL, 0.725 mmol, 1.20 equiv).
The reaction was followed by TLC (TLC solvent I)
and stopped after 20 min. The mixture was immediately
placed on a vacuum column (eluting solvent F); frac-
tions containing 13 were combined and evaporated to
dryness. Impure 13 was then purified by chromato-
graphy twice (eluting solvent J, followed by chromato-
graphy with eluting solvent G), giving pure 13 (0.464
g, 82%). NMR data are given only for the major compo-
nent, the a-anomer. 1H NMR (300.13 MHz, CDCl3): dH
8.61 (s, C@NH); 13C NMR (75.03 MHz, CDCl3): dC
169.5, 167.5 (C@O), 165.2 (C@O, benzoate), 160.7
(OC(NH)CCl3), 138.4–127.7 (C aromatic), 134.8
(OCH2CH@CH2), 116.9 (OCH2CH@CH2), 99.6 (C-1
1
1
Rha, J13C,1H = 173.2 Hz), 95.0 (C-1 GalA, J13C,1H
=
180.1 Hz), 78.5 (C-4 Rha), 76.7 (C-4 GalA), 76.3 (C-3
GalA), 75.6 (C-2 GalA), 73.7 (OCH2CH@CH2,
CH2Ph), 73.4 (CH2Ph), 72.8 (C-5, GalA), 72.1 (C-3
Rha), 70.6 (C-2 Rha), 68.7 (C-5 Rha), 67.8
(CO2CH2Ph), 21.0 (OCOCH3), 18.4 (C-6 Rha); ESI-
MS found m/z 962.2087 [M+Na]+. Calcd for
C47H48NO13Cl3Na: 962.2089.
1
CH@CH2), 99.6 (C-1 Rha, J13C,1H = 173.9 Hz), 90.6
1
(C-1 GalA, J13C,1H = 177.5 Hz), 78.5 (C-4 Rha), 77.2
(C-3 GalA), 76.6 (C-4 GalA), 75.0 (C-2 GalA), 73.8
(OCH2CH@CH2), 73.5, 73.3 (CH2Ph), 72.5 (C-5 GalA),
72.1 (C-3 Rha), 70.5 (C-2 Rha), 68.7 (C-5 Rha), 67.8
(CO2CH2Ph), 21.3 (GalA OCOCH3), 21.0 (Rha
OCOCH3), 18.4 (C-6 Rha). Anal. Calcd for
C47H50O14: C, 67.29; H, 6.01. Found: C, 67.08; H, 6.05.
3.13. Methyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-L-
rhamnopyranosyl)-(1!4)-(methyl 2,3-di-O-benzyl-a-D-
galactopyranosyluronate)-(1!2)-(2-O-acetyl-4-O-allyl-
3-O-benzoyl-a-L-rhamnopyranosyl)-(1!4)-(methyl 2,3-
di-O-benzyl-a-D-galactopyranosid)uronate (14)
3.11. Benzyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-L-
rhamnopyranosyl)-(1!4)-(2,3-di-O-benzyl-a/b-D-galacto-
pyranosid)uronate (12)
Acceptor 6 (0.243 g, 0.351 mmol, 1.00 equiv) and donor
10 (0.426 g, 0.492 mmol, 1.40 equiv) were placed
together in a flask along with 4 A MS (ꢂ0.530 g).
A sample of compound 11 (0.277 g, 0.330 mmol) was
˚
dissolved in distilled MeOH (8.2 mL) and CH2Cl2
˚
(4.7 mL). Powdered 4 A MS (0.185 g, 0.667 equiv by
Freshly distilled CH2Cl2 (5.7 mL) was added; the flask
was then purged with N2(g) and allowed to stir at rt
and in the dark for 1 h, after which AgOTf was added
(0.0483 g, 0.382 equiv to donor). The reaction was fol-
lowed by TLC (TLC solvent K) and worked up after
17 h 20 min. Workup was performed by diluting the
reaction mixture with CH2Cl2, removing the solids by
filtration, washing the filtrate sequentially with H2O
and satd NaHCO3, and drying the organic layer over
Na2SO4. The drying agent was removed by filtration
and the filtrate evaporated to dryness to afford 14 as a
syrup. The crude syrup was purified by chromatography
using eluting solvent K; impure fractions were combined
and again purified by chromatography with the same
solvent. The crude mixture was purified by chromato-
graphy a total of four times to give pure 14 (0.190 g,
weight to 11) were then added. An additional 0.055 g
˚
of 4 A molecular sieves was added on day 3. The reac-
tion mixture was stirred at rt for approximately 4 days
in total, after which the solids were removed by filtra-
tion. The filtrate was evaporated to dryness and the
resulting syrup was purified by chromatography (eluting
solvent I) to give pure 12 (0.200 g, 76%). 13C NMR
(75.03 MHz, CDCl3): dC 134.9 (OCH2CH@CH2),
1
116.9 (OCH2CH@CH2), 99.5 (C-1a/b Rha, J13C,1H
=
172.6 Hz), 92.4, (C-1a/b GalA, J13C,1H = 173.2 Hz),
21.0 (Rha OCOCH3), 18.3 (C-6a/b Rha). Anal. Calcd
for C45H48O13: C, 67.83; H, 6.07. Found: C, 67.96; H,
6.16.
1
3.12. Benzyl (2-O-acetyl-4-O-allyl-3-O-benzoyl-a-L-
rhamnopyranosyl)-(1!4)-2,3-di-O-benzyl-a/b-D-galacto-
pyranosyluronate trichloroacetimidate (13)
22
39%). ½aꢁD +63.3 (c 0.2, CHCl3); 1H NMR (300.13
MHz, CDCl3): dH 8.03–7.94 (4H, aromatic), 7.64–7.21
(26H, aromatic), 5.86–5.68 (2H, OCH2CH@CH2), 5.55
(dd, 1H, J3,2 = 3.3 Hz, H-2000), 5.44 (1H, H-3000), 5.41
(1H, H-30), 5.31 (1H, H-10), 5.19–5.00 (4H, OCH2-
CH@CH2), 5.11 (1H, H-1000), 4.94, 4.75 (2H, OCHa2Ph),
4.85, 4.61 (2H, OCH2bPh), 4.78 (1H, H-100), 4.76 (1H,
A sample of hemiacetal 12 (0.480 g, 0.602 mmol) was
dissolved in freshly distilled CH2Cl2 (12.9 mL) and stir-
˚
red under N2(g) in the presence of 4 A MS (0.476 g) at rt
for
approximately 10 min.
Trichloroacetonitrile

D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
187
H-1), 4.72, 4.66 (2H, OCHd2Ph), 4.58, 4.42 (2H,
OCHc2Ph), 4.47 (2H, H-4, H-500), 4.40 (1H, H-20), 4.31
(1H, H-400), 4.20–4.10 (4H, OCH2CH@CH2), 4.14 (1H,
H-2), 3.94 (1H, H-3), 3.94 (1H, H-300), 3.85 (s, 3H,
CO2CH3), 3.84 (1H, H-200), 3.71 (1H, H-50), 3.70 (1H,
H-5000), 3.50 (1H, H-40), 3.46 (1H, H-4000), 3.83 (s, 3H,
CO2CH030), 3.30 (s, 3H, OCH3), 2.02 (s, 3H,
OCOCH0300), 1.36 (d, 3H, J6,5 = 6.1 Hz, H-60), 1.29 (d,
3H, J6,5 = 6.3 Hz, H-6000); 13C NMR (75.03 MHz,
CDCl3): dC 169.7, 169.2, 168.4 (C@O), 165.3, 165.2
(C@O, benzoate), 139.0–127.7 (C aromatic), 135.0,
134.8 (OCH2CH@CH2), 116.9, 116.7 (OCH2CH@CH2),
ples containing the target compound were pooled to
yield a yellow-brown foam.
This residue was taken up in freshly distilled MeOH
(12.0 mL) and 1.0 M NaOMe was added until litmus
paper indicated a pH between 11 and 12. TLC (TLC
solvent M) taken approximately 24 h after the start of
the reaction indicated that the reaction had gone to
completion. The reaction was neutralized by the
addition of Amberlystꢂ 15H+ resin beads, filtered, and
evaporated to dryness to afford a yellowish syrup. The
syrup was purified by chromatography using eluting
solvent N, giving a clear, colorless syrup.
1
1
99.4 (C-1, J13C,1H = 173.2 Hz), 99.0 (C-1000, J13C,1H
=
This syrup was dissolved in EtOAc–MeOH–AcOH
5:5:1 (11.0 mL), to which Pd(OAc)2 (0.209 g, 0.931
mmol) was added. The mixture was placed under
H2(g) at 40 psi for 1 h. TLC (solvent N) indicated no
starting material, with all material present on the base-
line. The mixture was worked up by filtering through
Celite (rinsed with EtOAc and MeOH), followed by
evaporation and drying under high vacuum; a white
powder (0.032 g, 33%) was collected. TLC showed
(TLC solvent O) one spot without any visible trace of
1
174.4 Hz), 98.4 (C-10, J13C,1H = 172.6 Hz), 96.7 (C-100,
1J13C,1H = 171.5 Hz), 78.5 (C-40, C-4000), 77.6 (C-3),
76.7 (C-300), 76.5 (C-2), 76.0 (C-4, C-400), 75.5 (C-200),
75.3 (C-20), 74.4 (OCH2aPh), 74.1 (OCH2bPh), 73.7, 73.5
(OCH2CH@CH2), 73.5 (C-30), 72.7 (OCH2cPh,
OCHd2Ph), 72.1 (C-3000), 70.6 (C-500, C-2000), 70.5 (C-5),
68.6 (C-50), 68.3 (C-5000), 56.3 (OCH3), 52.7 (CO2CH030),
52.1 (CO2CH3), 21.0 (OCOCH0300), 18.4 (C-60), 18.2
(C-6000). Anal. Calcd for C77H86O24: C, 66.27; H, 6.21.
Found: C, 66.60; H, 6.34.
22
impurities. ½aꢁD +59.7 (c 0.2, MeOH); 1H NMR
(300.13 MHz, CH3OD): dH 5.21 (1H, H-10), 5.14 (d,
1H, J1,2 = 1.5 Hz, H-1000), 5.11 (1H, H-5), 5.00 (d, 1H,
J1,2 = 3.7 Hz, H-1), 4.81 (d, 1H, J1,2 = 3.7 Hz, H-100),
4.52 (1H, H-500), 4.39–4.32 (2H, H-400, H-4), 4.11–4.06
(1H, H-20), 4.01–3.90 (3H, H-2000, H-3, H-300), 3.83–3.69
(3H, H-200, H-2, H-30), 3.81 (s, 3H, CO2CH3), 3.78 (s,
3.14. Methyl (a-L-rhamnopyranosyl)-(1!4)-(methyl a-D-
galactopyranosyluronate)-(1!2)-(a-L-rhamnopyranosyl)-
(1!4)-(methyl a-D-galactopyranosid)uronate (16)
A sample of 14 (0.190 g, 0.136 mmol) was dissolved in
EtOH–toluene–H2O 10:3:1 (28.0 mL) and the reaction
flask was purged with N2(g). Wilkinson’s catalyst
([(C6H5)3P]3RhCl; 0.0502 g, 0.0543 mmol, 0.4 equiv)
was added, followed by DABCO (0.0153 g, 0.136 mmol,
1.00 equiv). The reaction was set to reflux and moni-
tored by TLC (TLC solvent K). After approximately
22 h an additional 0.2 equiv of Wilkinson’s catalyst
was added, followed by 0.5 equiv of DABCO; the reac-
tion was stopped after 46 h. The mixture was evaporated
to dryness, taken up in CH2Cl2, and sequentially washed
with 0.1 M HCl, satd NaHCO3, and H2O. The organic
layer was dried over Na2SO4, filtered, and evaporated
to dryness. The resulting syrup was dissolved in 9:1 ace-
tone–H2O (20 mL). HgCl2 and HgO were then added
and the reaction mixture was allowed to stir at rt.
TLC (TLC solvent K) indicated that the reaction was
not complete, so additional portions of HgCl2
(0.0369 g) and HgO (0.0290 g) were added after 26 h
20 min and again after 45 h. No significant change was
observed, so the reaction mixture was filtered after
50 h 50 min through Celite and evaporated to dryness.
The residue was taken up in EtOAc and sequentially
washed with satd KI, satd Na2S2O3, and H2O. The
organic layer was dried over Na2SO4, filtered, and evap-
orated to dryness. The residue was purified by
chromatography (TLC and eluting solvent L) and sam-
3
H, CO2CH3), 3.63 (dd, 1H, J3,2 = 3.2 Hz,
J3,4 = 9.4 Hz, H-3000), 3.54–3.39 (2H, H-50, H-5000), 3.41
(s, 3H, OCH3), 3.38–3.29 (2H, H-4000, H-40), 1.35–1.20
(6H, H-60, H-6000); 13C NMR (75.03 MHz, CH3OD): dC
1
170.1, 169.7 (CO2CH3), 102.2 (C-1000, J13C,1H
=
1
171.5 Hz), 100.6 (C-100, J13C,1H = 171.0 Hz), 99.8
1
1
(C-10, J13C,1H = 171.5 Hz), 98.3 (C-1, J13C,1H = 171.5
Hz), 78.4 (C-400), 77.9 (C-4), 76.9 (C-20), 72.6 (C-4000),
72.5 (C-40), 70.9 (C-2000), 70.8 (C-3), 70.6 (C-5), 70.3
(C-3000), 70.1 (C-300), 69.9 (C-500), 69.8 (C-30), 69.4
(C-50), 69.1 (C-5000), 68.6 (C-200, C-2), 55.1 (OCH3), 55.1
(OCH3), 51.7, 51.6 (CO2CH3), 17.0 (C-6000), 16.9 (C-60).
Anal. Calcd for C27H44O21: C, 46.02; H, 6.29. Found:
C, 45.88; H, 6.39.
3.15. Methyl a-L-rhamnopyranosyl-(1!4)-a-D-galacto-
pyranosyluronate-(1!2)-a-L-rhamnopyranosyl-(1!4)-
(a-D-galactopyranosid)uronate (17)
Saponification of the methyl-esterified tetrasaccharide
was achieved by dissolving 16 (0.024 g, 0.034 mmol) in
deionized water (2.0 mL). NaOH (1.0 M) was added
until pH 11–12; TLC (TLC solvent P) was used to verify
the de-esterification and the presence of the highly polar
product. HCl (1.0 M) was then added until pH 2–3. The
reaction mixture was evaporated to dryness, dissolved in
a minimal amount of deionized water, and purified on a

188
D. Reiffarth, K. B. Reimer / Carbohydrate Research 343 (2008) 179–188
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P2 gel column with deionized water as the eluent (TLC
solvent Q). Fractions containing the product were
pooled and dried via lyophilization to give pure 17
1
(17.6 mg, 77%). H NMR (300.13 MHz, D2O): dH 5.11
(1H, H-10), 5.09 (1H, H-1000), 4.88 (d, 1H, J1,2
=
3.5 Hz, H-1), 4.72 (d, 1H, J1,2 = 3.7 Hz, H-100), 4.57
(1H, H-500), 4.31 (1H, H-400), 4.28 (1H, H-4), 4.16 (1H,
H-5), 4.01 (1H, H-300), 4.00 (1H, H-20), 3.95 (1H,
H-2000), 3.91 (dd, 1H, J3,4 = 2.7 Hz, H-3), 3.81 (1H, H-200),
3.80 (1H, H-2), 3.79 (1H, H-30), 3.68 (1H, H-300), 3.67
(1H, H-50), 3.66 (1H, H-5000), 3.30 (1H, H-40), 3.26 (s,
3H, OCH3), 3.30 (1H, H-40), 3.34 (1H, H-4000), 1.14
(3H, H-60), 1.13 (3H, H-6000);13C NMR (75.03 MHz,
D2O): dC 175.9 (COOH, COOH00), 102.2 (C-1000,
´
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1
1J13C,1H = 173.6 Hz), 100.9 (C-100, J13C,1H = 171.5 Hz),
1
1
100.1 (C-10, J13C,1H = 172.4 Hz), 98.9 (C-1, J13C,1H
=
171.5 Hz), 78.8 (C-4), 77.9 (C-400), 77.5 (C-20), 73.7
(C-4000), 73.4 (C-40), 72.7 (C-500), 72.0 (C-5), 71.9 (C-2000,
C-3), 71.8 (C-300), 71.5 (C-3000), 70.8 (C-30), 78.4 (C-400),
70.6 (C-50), 70.3 (C-5000), 69.4 (C-200), 69.3 (C-2), 56.8
(OCH3), 18.2, 18.1 (C-60, C-6000); ESI-MS found m/z
699.1958 [M+Na]+. Calcd for C25H40O21Na: 699.1960.
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Acknowledgment
We wish to thank the Natural Sciences and Engineering
Research Council of Canada for financial support of
this work.
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Supplementary data
1H and 13C NMR spectra of compounds 4, 5, 7, 9, 10, 13
and 17. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.carres.2007.10.030.
26. Ko, K.-S.; Jaipuri, F. A.; Pohl, N. L. J. Am. Chem. Soc.
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