Efficient synthesis of polylactosamine structures through regioselective glycosylations

Article abstract of DOI:10.1081/CAR-100108275

Di-, tri-and tetramers of β-(1→3)-linked N-acetyllactosamine residues have been synthesised as their methyl glycosides, to be used in ITC binding studies to various galectins. The synthetic strategy involves two types of regioselective glycosylations: cou

Full text of DOI:10.1081/CAR-100108275

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EFFICIENT SYNTHESIS OF POLYLACTOSAMINE
STRUCTURES THROUGH REGIOSELECTIVE
GLYCOSYLATIONS
Therese Buskas a , Peter Konradsson b & Stefan Oscarson a
a Department of Organic Chemistry, Arrhenius Laboratory , Stockholm University ,
Stockholm, S-106 91, Sweden
b Department of Chemistry , University of Linköping , Linköping, S-581 83, Sweden
Published online: 16 Aug 2006.
To cite this article: Therese Buskas , Peter Konradsson & Stefan Oscarson (2001) EFFICIENT SYNTHESIS OF
POLYLACTOSAMINE STRUCTURES THROUGH REGIOSELECTIVE GLYCOSYLATIONS , Journal of Carbohydrate Chemistry, 20:7-8,
569-583
To link to this article: http://dx.doi.org/10.1081/CAR-100108275
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J. CARBOHYDRATE CHEMISTRY, 20(7&8), 569–583 (2001)
EFFICIENT SYNTHESIS OF POLYLACTOSAMINE
STRUCTURES THROUGH REGIOSELECTIVE
GLYCOSYLATIONS1
Therese Buskas,1 Peter Konradsson,2 and Stefan Oscarson1
1Department of Organic Chemistry, Arrhenius Laboratory,
Stockholm University, S-106 91 Stockholm, Sweden
2Department of Chemistry, University of Linköping,
S-581 83 Linköping, Sweden
ABSTRACT
Di-, tri- and tetramers of ꢀ-(1→3)-linked N-acetyllactosamine residues have
been synthesised as their methyl glycosides, to be used in ITC binding studies
to various galectins. The synthetic strategy involves two types of regioselective
glycosylations: couplings of a galactosyl donor to 3,4-diol N-tetrachloroph-
thalimido glucose acceptors to give the lactosamine monomer building blocks,
and subsequent formation of the oligomers through consecutive couplings of
lactosamine donors to 2ꢁ,3ꢁ,4ꢁ-lactosamine acceptors, with high selectivity for
the desired products.
INTRODUCTION
Polylactosamines consist of ꢀ-(1→3)-linked N-acetyllactosamine chains at-
tached to sphingolipids and proteins. They are the backbones of keratan sulfates
and are present at the surface of mammalian cells with or without distal decorations
(sulfate groups, single monosaccharides, and numerous oligosaccharide determi-
nants).2 A few chemical syntheses of polylactosamine structures have already been
published, the tetramer by Alais and Veyrieres,3–5 Srivastava and Hindsgaul6 and
Shimizu et al.,7 the latter performed on solid phase, and the trimer by Nilsson and
Norberg8 as an intermediate in the synthesis of a trimeric Lewis x structure. Also
enzymatic syntheses have been described.9
569
Copyright © 2001 by Marcel Dekker, Inc.
www.dekker.com

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BUSKAS, KONRADSSON, AND OSCARSON
Figure 1.
Galectins, named after their preferential binding to ꢀ-galactose motifs, are
known to have a number of important biological functions,10,11 and to further in-
vestigate these, the study of their binding to polylactosamine structures was im-
portant. Lactosamine oligomers with varying length were essential for these stud-
ies. Consequently, the di- and trimer of ꢀ-(1→3)-linked N-acetyllactoseamine
residues (17 and 20, Figure 1) were synthesised. These will primarily be used in
isothermal titration calorimetry (ITC) studies of their binding to galectins, to ren-
der possible the determination of the size of the binding site and the thermody-
namics of the binding.
RESULTS AND DISCUSSION
Although a number of syntheses of 2-amino-2-deoxylactose from a disac-
charide precursor have been published,3,12–14 the alternative to start from monosac-
charides is still a relevant option. The “drawback” of having to perform an extra
glycosylation reaction is balanced by the advantage of the easy access to monosac-
charide precursors, protected in a suitable way for subsequent reactions. Especially
with phthalimido-protected derivatives the latter approach is feasible, since it has
been found that the coupling between galactosyl donors and, easily obtainable, 3,4-
diol N-phthalimido glucose acceptors is high-yielding and very regioselective for
the 4-position due to the bulkiness of the phthalimido group.15–17 This regioselec-
tivity is even more pronounced for tetrachlorophthalimido compounds.18
Regioselective benzylation of the tin-activated pentenyl glycoside 119 gave di-
rectly the 3,4-diol 2 in 69% yield (Scheme 1). This procedure was found to be more
convenient than the two-step one, involving 4,6-O-benzylidene formation and re-
ductive opening used earlier.20 The silver triflate-promoted coupling between 2 and
galactosyl donor 3 (obtained from the known corresponding thioethyl glycoside21
by bromine treatment) proceeded in high yield (87%) and with regiospecificity. The
1→4-linkage in the product 4 was proven by the downfield shift of the H-3 proton
in the benzoylated derivative 5. To allow for more flexibility in the synthetic strat-
egy, thioglycoside donors were also synthesised (Scheme 2). Hence, derivatives 10
and 12 were synthesised in a parallel fashion from the same galactosyl donor (3) and
the 3,4-diol thioglycoside acceptors 7 and 8,17 respectively. Once more the glyco-
sylations gave high regioselectivity and yield of products.
Since the primary use of the target compounds was to perform ITC-studies of

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571
Scheme 1. i: (Bu3Sn)2O, MeOH; ii: BnBr, QBr, 90 ꢀC ; iii: Br2, CH2Cl2; iv: AgOTf, CH2Cl2, 3Å
MS, ꢂ40ꢀC; v: BzCl, pyridine.
their binding to various animal lectins, and accordingly no conjugate formation
was necessary, it was decided to synthesise them as their methyl ꢀ-glycosides.
Therefore, the pentenyl glycoside 5 was transformed into the methyl glycoside 13
(93%) by treatment with 5 equiv of MeOH in the presence of NIS/TESOTf
(triethylsilyl triflate) (Scheme 3). If a larger excess of MeOH was used, the major
reaction was addition of MeOH to the double bond.
Another known type of regioselective glycosylation, preferentially used
in sialylation of galactose derivatives, was utilised to synthesise oligomers. In
2,3,4-triol galactose acceptors a high selectivity for the 3-hydroxyl group is usually
observed in glycosylations, sialylation on 2,4-protected derivatives being often
impossible.22 Already anticipating this route when chosing galactosyl donor 3,
the acetyl groups of compound 13 were now removed chemoselectively by treat-
ment with HCl in MeOH23 to give the 2ꢁ,3ꢁ,4ꢁ-triol 14 (82%). In the coupling
reaction to give the dimer it was most important to match the reactivity of the
reacting species, acceptor, donor and promoter, to get a high yield of product
(Table 1, Scheme 4). However, all the glycosylations were regioselective for the
Scheme 2. i: (Bu3SnO)2, MeOH; ii: BnBr (5 eq), TEABr, toluene, 90 ꢀC; iii: AgOTf, CH2Cl2, 3Å
MS, ꢂ40ꢀC; iv: BzCl, pyridine.

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BUSKAS, KONRADSSON, AND OSCARSON
Scheme 3. i: MeOH, NIS, TESOTf, CH2Cl2, 3Å MS; ii: 5% HCl/MeOH.
3-position. The use of an effective promoter combined with especially tetra-
chlorophthalimido donors gave mainly decomposition of the donor. If the promoter
was changed into a less active one or if a phthalimido donor was employed, good
yields of the desired ꢀ-(1→3)-linked products 15 and 16 were obtained.
Choosing the most high-yielding conditions (donor 10, promoter MeOTf)
from this dimer glycosylation study, syntheses of a trimer and a tetramer were at-
tempted (Scheme 5). Chemoselective removal of the three acetates from derivative
15, again proceeded smoothly to give an 88% yield of compound 18. Reasoning
Table 1. Glycosylation of Acceptor 14. General Conditions: 1.7 Equiv of the
Donor, 3Å MS, CH2Cl2, Ar. Bromide Donors were Prepared by Br2 Treatment of
the Corresponding Thioglycoside Just Prior to Coupling. aThe Donor Decompose

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573
Scheme 4.
that glycosylation at the 3ꢁ-position should even more decrease the already low nu-
cleophilicity of the 2ꢁ- and 4ꢁ-hydroxyl groups, pentaol 18 was tried as an accep-
tor in a glycosylation employing the conditions used above, and was found to give
the trimer 19 in a most satisfactory yield (61%). Continued use of this methodol-
ogy then effectively gave first the heptaol acceptor 21 (81%) and then the tetramer
22 (60%, 69% calculated on consumed acceptor).
Since the 3-position of the glucosamine residue, as discussed and shown, is
quite unreactive also, perhaps its protection was not necessary, i.e., the regioselec-
tive glycosylation could be performed on a 3,2ꢁ,3ꢁ,4ꢁ-tetraol acceptor using a 3-OH
donor. This approach would have the advantage of fewer reaction steps (no ben-
zoylation) and more easily performed deacetylation steps (nonchemoselective). To
test this hypothesis the pentenyl glycoside 4 was transformed into the methyl gly-
coside 23 using the same conditions as for the benzoylated analogue 5 above, and
in comparable yield (88%) (Scheme 6). Removal of the acetyl protecting groups
then gave the tetraol 24 in almost quantitative yield (97%). Coupling between ac-
ceptor 24 and the non-benzoylated donor 9 using methyl triflate as promoter pro-
Scheme 5. i: HCl/MeOH-CH2Cl2 1:1; ii: MeOTf, CH2Cl2, 3Å MS; iii: H2NNH2-H2O, MeCN-THF-
EtOH 2:1:1, 70 ꢀC; iv: Ac2O, pyridine; v: H2, Pd/C; vi: 1M NaOMe, MeOH.

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BUSKAS, KONRADSSON, AND OSCARSON
Scheme 6. i: MeOH, NIS, TESOTf, CH2Cl2, 3Å MS; ii: 5% HCl/MeOH; iii: 9, MeOTf, CH2Cl2,
3Å MS.
ceeded to give one major product, which by NMR was shown to be the expected
ꢀ-(1→3)-linked tetrasaccharide 25 (46%), proving this to be an attractive alterna-
tive pathway to lactosamine oligomers.
Deprotection of the oligomers 15 and 19 was accomplished using standard
conditions, hydrazine hydrate at elevated temperature followed by acetylation, hy-
drogenolysis and deacetylation with sodium methoxide (Scheme 5). Although
rather straightforward, the yield in the deprotection is acceptable but not high, as is
also the case with deprotections of similar compounds found in the literature.3–8
The yields are normally in the range of 40–50% corresponding to an 80–84% yield
in each step in a four-step deprotection scheme.
EXPERIMENTAL
General Methods. Organic solutions were dried over MgSO4 before con-
centrations, which were performed under reduced pressure at ꢃ40°C (water bath).
TLC was performed on silica gel 60 F254 (Merck) with detection by UV light
and/or charring with 8% sulfuric acid, ninhydrin or AMC (ammonium molybdate
10g, cerium(IV) sulfate 2 g, dissolved in aq 10% H2SO4 2 L). Silica gel
(0.0404–0.063 mm, Amicon) was used for column chromatography. NMR spectra
were recorded in CDCl3 at 25 °C (internal standard Me4Si,ꢄ ꢅ 0.00) unless other-
wise stated, using a Varian Mercury 300 at 300 MHz (1H) or 75 MHz (13C) or a
Varian Inova 400 at 400 MHz (1H) or 100 MHz (13C).
Pent-4-enyl 6-O-Benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-gluco-
pyranoside (2). Bis(tributyltin) oxide (14.5 mL, 28.54 mmol) was added to a so-
lution of 119 (10.5 g, 20.39 mmol) in dry MeOH (120 mL) stirred under argon.
After stirring at 75°C for 2 h the reaction mixture was concentrated and dried in
vacuo. The oily tin ether complex was dissolved in benzyl bromide (40 mL) and
tetrabutylammonium bromide (7.22 g, 22.4 mmol) was added. After stirring at
90°C for 20 h benzyl bromide was distilled from the reaction vessel. Flash chro-
matography (two columns: toluene-EtOAc 20:1→5:1 and toluene-EtOAc 5:1) of

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575
the oily residue furnished 2 (8.51 g, 14.07 mmol, 69%) having NMR data in agree-
ment with those reported elsewhere.20
Pent-4-enyl (2,3,4-Tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-
3-O-benzoyl-6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopyra-
noside (5). A solution of ethyl 2,3,4-tri-O-acetyl-6-O-benzyl-1-thio-ꢀ-D-galac-
topyranoside21 (6.59 g, 14.96 mmol) and bromine (2.3 mL, 44.89 mmol) in
CH2Cl 2 (100 mL) was stirred under argon for 30 min at room temperature. The
mixture was then concentrated and co-concentrated twice with toluene (Na-dried).
A solution of the residue and 2 (6.87 g, 11.26 mmol) in CH2Cl2 (150 mL) was
stirred with 3Å MS for 30 min at ꢂ60°C before AgOTf (7.30 g, 28.40 mmol) was
added. The reaction was quenched with triethylamine (6 mL) after 1 h and the mix-
ture was stirred for another 15 min, filtered through Celite and concentrated. Flash
chromatography of the residue (toluene-EtOAc 6:1) gave pent-4-enyl (2,3,4-tri-O-
acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-6-O-benzyl-2-deoxy-2-tetra-
chlorophthalimido-ꢀ-D-glucopyranoside (4) (9.51 g, 9.79 mmol, 87%). NMR
(CDCl3): 13C, ꢄ 21.1 (2C), 21.2 (CH3 acetyl), 29.1, 30.4 (CH2 pentenyl), 56.7,
67.4, 68.0, 68.2, 69.0, 69.1, 69.6, 71.0, 72.5, 73.6, 73.8, 74.4, 81.74 (C-2–6, 2ꢁ-6ꢁ,
OCH2 pentenyl, CH2 benzyl), 98.0 (C-1), 101.3 (C-1ꢁ), 115.0 (CH2 ꢅ pentenyl),
125.6–140.1 (aromatic C, CH ꢅ pentenyl), 162.4, 162.5 (CBO TCP), 169.4,
170.1, 170.3 (CBO acetyl);1H, ꢄ 1.53–1.64 (m, 2H, CH2 pentenyl), 1.89–1.94
(m, 2H, CH2 pentenyl), 1.97, 1.98, 2.06 (s, 9H, CH3 acetyl), 3.41–3.87 (m, 8H,
OCH2 pentenyl, H-4, 5, 5ꢁ, 6, and 6ꢁ), 4.17 (dd, 1H, H-2), 4.25–4.55 (5H, H-1ꢁ, 3,
CH2 benzyl), 4.71 (d, 1H, CH2 benzyl), 4.82–4.87 (m, 2H, CH2 ꢅ pentenyl), 4.94
(dd, 1H, H-3ꢁ), 5.09 (d, 1H, J1.2 8.42 Hz, H-1), 5.17 (dd, 1H, H-2ꢁ), 5.37 (d, 1H, H-
4ꢁ), 5.58–5.74 (m, 1H, CHꢅ pentenyl), 7.15–7.36 (m, 10H, aromatic H). Benzoyl
chloride (1.63 mL, 14.0 mmol) and pyridine (2.3 mL) were added to a stirred so-
lution of 4 (1.38 g, 1.40 mmol) in dry CH2Cl2 (20 mL) and the reaction mixture
was stirred at ambient temperature overnight and then concentrated. The residue
was dissolved in CH2Cl2 and the organic layer was washed with aq 1M HCl, satd
aq NaHCO3, and H2O, dried and concentrated. Flash chromatography (toluene-
EtOAc 8:1) of the residue gave 5 (1.35 g, 1.24 mmol, 88%). NMR (CDCl3): 13C, ꢄ
21.1 (2C), 21.3 (CH3 acetyl), 29.1, 30.4 (CH2 pentenyl), 56.1, 66.3, 67.2, 67.8,
69.5, 70.0, 71.6, 72.3, 73.5, 74.2, 75.1, 75.9 (C-2–6, 2ꢁ-6ꢁ, OCH2 pentenyl, CH2
benzyl), 98.2 (C-1), 100.7 (C-1ꢁ), 115.2 (CH2ꢅ pentenyl), 127.8–140.4 (aromatic
C, CH ꢅ pentenyl), 162.6, 162.8 (CBO TCP) 165.6 (CBO benzoyl), 169.2, 170.2
(2C) (CBO acetyl); 1H, ꢄ 1.56–1.64 (m, 2H, CH2 pentenyl), 1.85, 1.92, 1.94 (s,
9H, CH3 acetyl), 1.89–1.98 (m, 2H, CH2 pentenyl), 3.47–3.51 (m, 1H, OCH2 pen-
tenyl), 2.70 (dd, 1H), 2.91 (dd, 1H) 3.65–3.69 (m, 1H), 3.74–3.87 (m, 3H, OCH2
pentenyl, H-6), 4.07 (d, 1H, CH2 benzyl), 4.16 (dd, 1H, H-4), 4.25 (d, 1H, CH2
benzyl), 4.38 (dd, 1H, H-2), 4.45 (d, 1H, J1.2 8.06 Hz, H-1ꢁ), 4.54 (d, 1H, CH2 ben-
zyl), 4.74–4.89 (m, 4H, H-3ꢁ, CH2 benzyl, CH2ꢅ pentenyl), 4.94 (dd, 1H, H-2ꢁ),
5.22 (d, 1H, H-4ꢁ), 5.35 (d, 1H, J1.2 8.42 Hz, H-1), 5.64–4.74 (m, 1H, CHꢅ pen-
tenyl), 5.88 (dd, 1H, H-3) 7.14–8.17 (m, 15H, aromatic H). MS (MALDI-TOF):
(MꢆNa)ꢆ 1108.1.

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BUSKAS, KONRADSSON, AND OSCARSON
Ethyl (2,3,4-Tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-3-O-
benzoyl-6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-1-thio-ꢀ-D-glucopyra-
noside (10). Bis(tributyltin) oxide (3.53 mL, 6.93 mmol) was added to a solution
of 624 (3.09 g, 6.3 mmol) in dry MeOH (60 mL) stirred under argon. The reaction
was stirred at 75°C for 2 h, concentrated and dried in vacuo. The yellow oil was
dissolved in toluene (Na-dried) (70 mL) and benzyl bromide (3.75 mL, 31.5 mmol)
and tetrabutylammonium bromide (2.13 g, 6.62 mmol) were added. The reaction
was kept at 90°C for 21 h and then concentrated. Purification by flash chromatog-
raphy (toluene-EtOAc 4:1) furnished ethyl 6-O-benzyl-2-deoxy-2-tetrachloroph-
thalimido-1-thio-ꢀ-D-glucopyranoside (7) (2.45 g, 4.22 mmol, 67%). NMR
(CDCl3): 13C, ꢄ 15.3 (CH3CH2S), 24.5 (CH2S), 56.2, 70.6, 72.4, 73.7, 74.0, 78.3
(C-2–6, CH2 benzyl), 81.1 (C-1), 125.5–140.4 (aromatic C). Ethyl 2,3,4-tri-O-
acetyl-6-O-benzyl-1-thio-ꢀ-D-galactopyranoside21 (1.9 g, 4.34 mmol) was
converted into the corresponding bromosugar 3, by treatment with bromine (0.45
mL, 8.68 mmol). A mixture of 3, acceptor 7 (1.8 g, 3.1 mmol) and 3Å MS in
CH2Cl2 (40 mL) was stirred at ꢂ60°C for 30 min, whereafter AgOTf (2.23 g, 8.68
mmol) was added. After 45 min the reaction was quenched by addition of triethy-
lamine (1 mL) and the mixture was diluted with CH2Cl2 and filtered through Celite
and concentrated. Flash chromatography (toluene-EtOAc 6:1) gave ethyl (2,3,4-
tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-6-O-benzyl-2-deoxy-2-
tetrachlorophthalimido-1-thio-ꢀ-D-glucopyranoside (9) (2.61 g, 2.73 mmol, 88%).
NMR (CDCl3): 13C, ꢄ 15.3 (CH3CH2S), 20.9, 21.0, 21.1 (CH3 acetyl), 24.3
(CH2S), 56.0, 67.6, 68.2, 68.5, 69.3, 70.6, 71.2, 72.7, 73.8, 74.0, 78.6, 81.0 (C-2–6,
2ꢁ-6ꢁ, CH2 benzyl), 81.6 (C-1), 101.5 (C-1ꢁ), 125.9–140.3 (aromatic C), 162.9,
163.4 (CBO TCP), 169.3, 170.0, 170.2 (CBO acetyl). Benzoyl chloride (1.40
mL, 12.0 mmol) was added to a stirred solution of 9 (2.3 g, 2.40 mmol) in pyridine
(35 mL). After stirring at room temperature overnight the reaction mixture was
concentrated and worked up as described for 5. Flash chromatography (toluene-
EtOAc 4:1) of the crude product afforded 10 (2.17 g, 2.04 mmol, 85%). [ꢇ]D
ꢆ69°(c 1.1, CHCl3) NMR (CDCl3): 13C, ꢇ15.0 (CH3CH2S), 20.4, 20.5, 20.7 (CH3
acetyl), 24.2 (CH2S), 54.5, 65.6, 66.6, 67.4, 69.4, 71.0 (2C), 72.3, 72.9, 73.6, 74.9,
78.7 (C-2–6, 2ꢁ-6ꢁ, CH2 benzyl), 80.5 (C-1), 100.0 (C-1ꢁ), 125.0–140.1 (aromatic
C), 162.1, 162.6 (CBO TCP), 165.0 (CBO benzoyl), 168.6, 169.6 (CBO acetyl);
1H, ꢄ 1.24 (t, 3H, CH3CH2S), 1.83, 1.91, 1.94 (s, 9H, CH3 acetyl), 2.64–2.75 (m,
3H, CH2S), 2.89–2.92 (m, 1H), 3.30–3.34 (m, 1H), 3.70–3.79 (3H, H-5, H-6), 4.09
(d, 1H, CH2 benzyl), 4.19 (dd, 1H, H-4), 4.26 (d, 1H, CH2 benzyl), 4.46–4.55 (m,
3H, H-2, H-1ꢁ, CH2 benzyl), 4.76–4.79 (m, 2H, H-3ꢁ, CH2 benzyl), 4.95 (dd, 1H,
H-4ꢁ), 5.47 (d, 1H, J1.2 10.62 Hz, H-1), 5.95 (dd, 1H, H-3), 7.12–8.10 (m, 15H, aro-
matic H).
Anal. Calcd for C49H47O15NCl4: C, 55.33; H, 4.45%. Found: C, 55.19; H,
4.48%.
Ethyl (2,3,4-Tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-3-O-
benzoyl-6-O-benzyl-2-deoxy-2-phthalimido-1-thio-ꢀ-D-glucopyranoside (12).
A solution of ethyl 2,3,4-tri-O-acetyl-6-O-benzyl-1-thio-ꢀ-D-galactopyranoside21
(3.45 g, 7.83 mmol) and bromine (0.8 mL, 15.65 mmol) in CH2Cl2 (60 mL) was

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stirred for 30 min at room temperature and under argon. The mixture was then con-
centrated and co-evaporated twice from toluene (Na-dried). A solution of the
residue and ethyl 6-O-benzyl-2-deoxy-2-phthalimido-1-thio-ꢀ-D-glucopyran-
oside17 (8, 2.89 g, 6.53 mmol) in CH2Cl2 (75 mL) was stirred with 3Å MS for 30
min at ꢂ60°C before AgOTf (4.0 g, 15.67 mmol) was added. After 1 h, triethy-
lamine (3 mL) was added and the mixture was allowed to attain room temperature,
whereafter it was filtered through Celite and concentrated. Flash chromatography
of the residue (toluene-EtOAc 6:1) gave ethyl (2,3,4-tri-O-acetyl-6-O-benzyl-ꢀ-D-
galactopyranosyl)-(1→4)-6-O-benzyl-2-deoxy-2-phthalimido-1-thio-ꢀ-D-glu-
copyranoside (11) (3.70 g, 4.51 mmol, 69%). NMR (CDCl3): 13C, ꢄ 15.4
(CH3CH2S), 20.9 (2C), 21.1 (CH3 acetyl), 24.2 (CH2S), 55.5, 67.6, 67.7, 68.5,
69.3, 71.0, 71.2, 72.6, 73.8, 73.9, 78.6, 81.3 (C-2–6, 2ꢁ-6ꢁ, CH2 benzyl), 81.7
(C-1), 101.5 (C-1ꢁ), 123.4–138.3 (aromatic C), 167.8, 168.2 (CBO NPhth), 169.4,
170.0, 170.2 (CBO acetyl). Benzoyl chloride (2.09 mL, 18.04 mmol) was added
to a stirred solution of 11 (3.70 g, 4.51 mmol) in pyridine (100 mL). After stirring
in room temperature for 15 h the reaction was concentrated and then worked up as
described for compound 5. Flash chromatography (toluene-EtOAc 6:1) gave 12
(3.63 g, 3.92 mmol, 87%). NMR (CDCl3): 13C, ꢄ 15.6 (CH3CH2S), 20.9, 20.9, 21.1
(CH3 acetyl), 24.5 (CH2S), 54.2, 66.1, 67.1, 68.0, 69.9, 71.5, 72.8 (2C), 73.3, 74.0,
75.8, 79.2 (C-2–6, 2ꢁ-6ꢁ, CH2 benzyl), 81.3 (C-1), 100.57 (C-1ꢁ), 125.5–138.0
(aromatic C), 165.2 (CBO benzoyl), 167.5, 167.8 (CBO NPhth), 169.1, 170.1
1
(2C) (CBO acetyl); H, ꢄ 1.23 (t, 3H, CH3CH2S), 1.84, 1.91, 1.94 (s, 9H, CH3
acetyl), 2.61–2.72 (m, 3H, CH2S), 2.90 (dd, 1H), 3.30 (dd, 1H), 3.74–3.80 (m, 3H,
H-5, 6), 4.07 (d, 1H, CH2 benzyl), 4.18 (dd, 1H, H-4), 4.25 (d, 1H, CH2 benzyl),
4.47–4.55 (m, 3H, H-1 (J1.2 10.2 Hz), H-2, CH2 benzyl), 4.75–4.79 (m, 2H, H-3ꢁ,
CH2 benzyl), 4.96 (dd, 1H, H-2ꢁ), 5.22 (d, 1H, H-4ꢁ), 5.50 (d, 1H, J1.2 10.6 Hz,
H-1ꢁ), 6.03 (dd, 1H, H-3), 7.14–8.12 (m, 19H, aromatic H). MS (MALDI-TOF):
(MꢆNa)ꢆ 948.3; (MꢆK)ꢆ 964.3.
Methyl (6-O-Benzyl-ꢀ-D-galactopyranosyl)-(1→4)-3-O-benzoyl-6-O-
benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopyranoside (14). A so-
lution of 5 (2.23 g, 2.05 mmol) and dry MeOH (0.412 mL, 10.25 mmol) in CH2Cl2
(100 mL) was stirred under argon for 30 minutes, whereafter NIS (0.922 g, 4.10
mmol) and TESOTf (0.927 mL, 4.10 mmol) were added. After 15 min TLC indi-
cated complete reaction, and the mixture was diluted with CH2Cl2, filtered through
Celite, washed with satd aq Na2S2O3 and satd aq NaHCO3, dried and concentrated.
Flash chromatography (toluene-EtOAc 5:1) yielded methyl (2,3,4-tri-O-acetyl-6-
O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-3-O-benzoyl-6-O-benzyl-2-deoxy-2-
tetrachlorophthalimido-ꢀ-D-glucopyranoside (13) (1.97 g, 1.91 mmol, 93%).
NMR (CDCl3): 13C, ꢄ 20.8 (2C), 21.0 (CH3 acetyl), 55.7, 57.1, 67.9, 66.9, 67.4,
69.7, 71.2, 71.3, 71.9, 73.2, 73.9, 74.8, 75.5 (C-2–6, 2ꢁ-6ꢁ, CH2 benzyl, CH3O),
98.8 (C-1), 100.3 (C-1ꢁ), 125.3–140.1 (aromatic C), 162.3, 162.4 (CBO TCP),
165.3 (CBO benzoyl), 168.9, 169.9 (2C) (CBO acetyl); 1H, ꢄ 1.85, 1.91, 1.94 (s,
9H, CH3 acetyl), 2.66 (dd, 1H), 2.89 (dd, 1H), 3.26–3.41 (m, 1H), 3.47 (s, 3H,
CH3O), 3.68 (m, 1H, H-5), 3.80 (m, 2H, H-6), 4.08 (d, 1H, CH2 benzyl), 4.17 (dd,
1H, H-2), 4.25 (d, 1H, CH2 benzyl), 4.38 (dd, 1H, H-4), 4.44 (d, 1H, J1.2 7.87 Hz,

ORDER
REPRINTS
578
BUSKAS, KONRADSSON, AND OSCARSON
H-1ꢁ), 4.55 (dd, 1H, H-3ꢁ), 4.80 (d, 1H, CH2 benzyl), 4.94 (dd, 1H, H-2ꢁ), 5.22 (d,
1H, H-4ꢁ), 5.28 (d, 1H, J1.2 8.42 Hz, H-1), 5.89 (dd, 1H, H-3). Disaccharide 13
(1.74 g, 1.68 mmol) was dissolved in 5% HCl/MeOH (70 mL) and the mixture was
stirred for 15 h and then concentrated. Flash chromatography (toluene-EtOAc
1:1–1:3) of the residue afforded 14 (1.25 g, 1.38 mmol, 82%). [ꢇ]D ꢆ35° (c 1.0,
CHCl3) NMR: 13C (CDCl3), ꢄ 55.7, 57.3, 67.6, 68.3, 68.5, 72.3, 72.4, 72.9, 73.3
(2C), 73.7, 74.8, 76.9 (C-2–6, 2ꢁ-6ꢁ, CH2 benzyl, CH3O), 98.9 (C-1), 103.7 (C-1ꢁ),
127.3–140.0 (aromatic C), 162.4, 162.5 (CBO TCP), 166.3 (CBO benzoyl); 1H
(CD3OD ꢆ 2 drops of CDCl3), ꢄ 2.90 (dd, 1H), 3.09 (dd, 1H), 3.22–3.34 (m, 5H,
H-3ꢁ, CH3O), 3.39 (dd, 1H, H-2ꢁ), 3.65 (d, 1H, H-4ꢁ), 3.82 (m, 1H, H-5), 3.95 (dd,
1H, H-6), 4.12 (dd, 1H, H-6), 4.17–4.34 (m, 4H, CH2 benzyl, H-1ꢁ, 2), 4.65 (dd,
2H, CH2 benzyl), 5.30 (d, 1H, H-1), 5.85 (dd, 1H, H-3).
Anal. Calcd for C42H38O12NCl4: C, 56.64; H, 4.30%. Found: C, 56.44; H,
4.50%.
Methyl (2,3,4-Tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-
O-benzoyl-6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopy-
ranosyl)-(1→3)-(6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-O-benzoyl-
6-O-benzyl-2-deoxy-2-tetrachlorophthalimidoꢀ-D-glucopyranoside) (15).
Methyl triflate (0.28 mL, 1.5 mmol) was added to a stirred mixture of 14 (0.417 g,
0.46 mmol), 10 (0.685 g, 0.644 mmol) and 3Å MS in CH2Cl2. The reaction
mixture was stirred under argon for 18 h, quenched by addition of triethylamine (1
mL), filtered through Celite and concentrated. Flash chromatography of the residue
gave 15 (0.615 g, 0.322 mmol, 69%). NMR (CDCl3): 13C, ꢄ 20.9 (2C), 21.1 (CH3
acetyl), 55.8, 57.2, 66.1, 67.0, 67.5, 67.6, 68.3, 69.9, 71.1, 71.4, 71.5, 71.8, 72.4,
72.8, 73.4, 73.4, 73.8, 74.0, 74.4, 74.9, 75.6, 76.7, 77.5 (C-2–6, 2ꢁ-6ꢁ, 2ꢈ-6ꢈ, 2—6ꢉ,
CH3O, CH2 benzyl), 99.1, 99.2, 100.5, 103.3 (C-1–1ꢉ), 125.4–140.2 (aromatic C),
162.3, 163.7 (CBO TCP), 165.3, 165.7 (CBO benzoyl), 169.0, 170.0 (2C)
(CBO acetyl). An aliquot of 15 was acetylated (pyridine-acetic anhydride 3:2) to
1
give material having NMR (CDCl3): H, ꢄ 1.86, 1.90, 1.91, 1.97 (CH3 acetyl),
2.54–2.68 (m, 2H), 2.86–2.92 (m, 2H), 2.23–3.30 (m, 2H), 3.44 (s, 3H, CH3O),
3.55–3.61 (m, 2H, H-3ꢁ), 3.68–3.77 (m, 5H), 4.05–4,24 (m, 8H, H-4, 4ꢈ, 2ꢈ, 1ꢁ,
CH2 benzyl), 4.35 (dd, 1H, H-2), 4.46–4.62 (m, 4H, H-1ꢉ, 2ꢁ, CH2 benzyl),
4.75–4.82 (m, 3H, H-3ꢉ, CH2 benzyl), 4.97 (dd, 1H, H-2ꢉ), 5.13 (d, 1H, J1.2 8.42
Hz, H-1ꢈ), 5.20–5.22 (m, 2H, H-4ꢉ, 1) 5.34 (d, 1H, H-4ꢁ), 5.74 (dd, 1H, H-3), 5.94
(dd, 1H, H-3ꢈ), 7.12–7.83 (m, 30H, aromatic H). The signals for H-2ꢁ (4.61 ppm)
and H-4ꢁ (5.34 ppm) in the acetylated tetrasaccharide was shifted downfield, prov-
ing selective coupling at 3ꢁ-OH of the acceptor.
Methyl (2,3,4-Tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-
O-benzoyl-6-O-benzyl-2-deoxy-2-phthalimido-ꢀ-D-glucopyranosyl)-(1→3)-
(6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-O-benzoyl-6-O-benzyl-2-deoxy-
2-tetrachlorophthalimido-ꢀ-D-glucopyranoside) (16). A solution of 12 (0.90
g, 0.97 mmol) and bromine (0.1 mL, 1.95 mmol) in CH2Cl2 (10 mL) was stirred
under argon for 30 min. The mixture was then concentrated and co-concentrated

ORDER
REPRINTS
REGIOSELECTIVE GLYCOSYLATIONS
579
twice with toluene (Na-dried). A solution of the obtained glycosyl bromide and 14
(0.50 g, 0.55 mmol) in CH2Cl2-MeCN 3:1 (20 mL) was stirred with 3Å MS for 30
min at ꢂ40 °C before AgOTf (0.51 g, 1.98 mmol) was added. After stirring at
ꢂ40°C for 1 h the reaction was allowed to warm to ꢂ10 °C, and after an additional
4 h Et3N (1 mL) was added. The mixture was stirred for another 15 min then fil-
tered through Celite and concentrated. Flash chromatography (toluene-EtOAc 3:1)
of the residue yielded 594 mg of 16 (0.34 mmol, 62%). NMR (CDCl3): 13C, ꢄ 20.8,
20.9, 21.1 (CH3 acetyl), 55.1, 55.8, 57.2, 66.1, 67.0, 67.5, 68.2, 69.9, 71.0, 71.4,
71.5, 71.8, 72.5 (2C), 72.7 (2C), 73.3, 73.4, 73.6, 74.0, 74.6, 75.0, 75.9, 76.6, 82.3
(C-2–6, 2ꢁ-6ꢁ, 2ꢈ-6ꢈ, 2—6ꢉ, CH3O), 99.0, 99.2, 100.6, 103.4 (C-1–1ꢉ),
123.7–138.2 (aromatic C), 162.4, 162.6 (CBO TCP), 165.1, 165.7 (CBO ben-
zoyl), 167.6, 167.8 (CBO NPhth) 169.1, 170.1 (2C) (CBO acetyl). An aliquot of
16 was treated with benzoyl chloride (10 equiv) in pyridine and then acetylated
(pyridine-Ac2O 3:2) to give the corresponding 2ꢁ-O-acetylated-4ꢁ-O-benzoylated
1
tetrasaccharide with NMR (CDCl3): H, ꢄ 1.70, 1.84, 1.91, 1.95 (s, 12H, CH3
acetyl), 2.52–2-62 (m, 2H), 2.79–2.90 (m, 2H), 3.20–3.25, 3.35–3.43 (m, 5H,
H-5ꢁ, 5ꢉ, CH3O), 3.57 (m, 1H, H-5), 3.65–3.78 (m, 7H, H-3ꢁ), 3.99–4.25 (m, 8H,
H-4, 4ꢈ, 2, 1ꢁ), 4.37 (dd, 1H, H-2ꢈ), 4.46–4.52 (m, 4H, H-1ꢉ, CH2 benzyl),
4.72–4.86 (m, 4H, H-3ꢉ, 2ꢁ, CH2 benzyl), 4.96 (dd, 1H, H-2ꢉ), 5.20–5.24 (m, 2H,
H-4ꢉ, 1ꢈ (J1.2 8.24 Hz)), 4.36 (d, 1H, J1.2 8.24 Hz), 5.61 (d, 1H, H-4ꢁ), 5.79 (dd,
1H, H-3ꢈ), 5.92 (dd, 1H, H-3), 6.86–8.14 (m, 34H, aromatic H). The downfield
shift for H-2ꢁ (4.72–4.85 ppm) and H-4ꢁ (5.61 ppm) in the acylated tetrasaccharide
confirmed the (1→3)-linkage.
Methyl (ꢀ-D-Galactopyranosyl)-(1→4)-(2-acetamido-2-deoxy-ꢀ-D-glu-
copyranosyl)-(1→3)-(ꢀ-D-galactopyranosyl)-(1→4)-2-acetamido-2-deoxy-ꢀ-
D-glucopyranoside (17). Hydrazine hydrate (0.145 mL, 3 mmol) was added to a
solution of tetrasaccharide 15 (0.130 g, 0.068 mmol) in MeCN-THF-EtOH (2:1:1,
4 mL). After stirring at 70°C for 18 h, the mixture was concentrated and co-con-
centrated with EtOH. The residue was purified on a short silica gel column
(EtOAc-MeOH-H2O 7:2:1). The obtained material was dissolved in pyridine (3
mL) and acetic anhydride (1 mL), and the mixture was stirred overnight and con-
centrated. Flash chromatography (CHCl3-MeOH 30:1→10:1) followed by further
purification by size exclusion chromatography (LH-20 Sephadex, eluted with
MeOH) of the residue gave the fully acetylated tetrasaccharide (63 mg, 0.044
mmol, 65%), which was hydrogenolysed (120 psi) over Pd/C (10%) in a solution
of EtOAc-MeOH-H2O (7:2:1, 3 mL). After 12 h the mixture was filtered through
Celite and concentrated. The residue was dissolved in MeOH and a catalytic
amount of 1 M NaOMe in MeOH was added. The mixture was stirred for 5 h, then
neutralised with Dowex Hꢆ ion exchange resin, filtered and concentrated. Size ex-
clusion chromatography (Biogel P2 column, eluted with H2O containing 1% n-
BuOH) gave 17 (25 mg, 0.033 mmol, 49% from 15). [ꢇ]D ꢂ12° (c 1.1, H2O), [Lit.3
1
ꢂ10° (c 1.0, H2O)]. NMR (D2O): H (selected data) ꢄ 2.04, 2.05 (s, 6H, CH3
acetyl), 3.51 (s, 3H, CH3O), 4.48 (2d, 3H), 4.71 (2d, 1H). NMR data were in agree-
ment with those reported elsewhere.3,6,8

ORDER
REPRINTS
580
BUSKAS, KONRADSSON, AND OSCARSON
Methyl (ꢀ-D-galactopyranosyl)-(1→4)-(2-acetamido-2-deoxy-ꢀ-D-glu-
copyranosyl)-(1→3)-(ꢀ-D-galactopyranosyl)-(1→4)-(2-acetamido-2-deoxy-ꢀ-
D-glucopyranosyl)-(1→3)-(ꢀ-D-galactopyranosyl)-(1→4)-2-acetamido-2-de-
oxy-ꢀ-D-glucopyranoside (20). Tetrasaccharide 15 (71 mg, 0.037 mmol) was
dissolved in 5% HCl/MeOH-CH2Cl2 (1:1, 4 mL), and the solution stirred under
argon for 37 h, then concentrated and purified by flash chromatography (toluene-
EtOAc 2:3) to afford methyl (6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-O-ben-
zoyl-6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopyranosyl)-(1→3)-
(6-O-benzyl—D-galactopyranosyl)-(1→4)-(3-O-benzoyl-6-O-benzyl-2-deoxy-2-t
etrachlorophthalimido-ꢀ-D-glucopyranoside) (18) (58 mg, 0.033 mmol, 88%).
NMR (CDCl3): 13C, ꢄ 55.7 (2C), 57.3, 67.5, 67.7, 67.8, 68.2, 68.3, 68.5, 70.9, 72.2,
72.5, 72.8, 72.9, 73.4, 73.4, 73.7, 73.8, 74.4, 74.6, 76.6, 77.5, 77.7, 82.4 5
(C-2–6, 2ꢁ-6ꢁ, 2ꢈ-6ꢈ, 2—6ꢉ, CH3O), 99.0, 99.2, 103.3, 103.8 (C-1–1ꢉ), 127.2–140.3
(aromatic C), 162.7, 163.4 (CBO TCP), 165.7, 165.7 (CBO benzoyl). A mixture
of 18 (52 mg, 0.029 mmol), 10 (53 mg, 0.05 mmol), and 3Å MS in CH2Cl2 (2 mL)
was treated with methyl triflate (13 ꢊL, 0.116 mmol) under argon. After stirring for
48 h, Et3N (0.250 mL) was added and stirring was continued for 15 min. The mix-
ture was then diluted with CH2Cl2, filtered through Celite, and the solvent removed
under reduced pressure. Flash chromatography (toluene-EtOAc 3:1) of the residue
gave methyl (2,3,4-tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-O-
benzoyl-6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopyranosyl)-
(1→3)-(6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-O-benzoyl-6-O-benzyl-2-
deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopyranosyl)-(1→3)-(6-O-benzyl-ꢀ-D-ga
lactopyranosyl)-(1→4)-(3-O-benzoyl-6-O-benzyl-2-deoxy-2-tetrachlorophthal-
imido-ꢀ-D-glucopyranoside) (19) (49 mg, 0.018 mmol, 61%). NMR (CDCl3): 13C,
20.8, 21.1, 21.0 (CH3 acetyl), 55.7 (3C), 57.3, 66.0, 67.0, 67.4, 67.6, 68.1, 68.2,
69.8, 71.0, 71.4, 71.5, 71.8, 72.1, 72.3, 72.7, 73.3, 73.4, 73.6, 73.7, 73.9, 74.0, 74.3,
74.4, 74.9, 75.6, 76.3, 77.6, 82.4 (C-2–6, 2ꢁ-6ꢁ, 2ꢈ-6ꢈ, 2—6ꢉ, 2ꢈꢈ-6ꢈꢈ, 2ꢉꢈ-6ꢉꢈ,
CH3O, CH2 benzyl), 99.0, 99.1, 99.3, 100.5, 103.3 (2C) (C-1–1ꢉꢈ), 125.5–140.3
(aromatic C), 162.7, 163.3 (CBO TCP), 165.3, 165.6, 165.7 (CBO benzoyl),
170.0, 170.1 (CBO acetyl). An aliquot of 19 was acetylated (pyridine-Ac2O 3:2).
1H and COSY NMR experiments showed downfield shifts for H-2ꢁ, 2ꢉ (4.50–4.66
ppm), H-4ꢁ, 4ꢉ (5.29–5.34 ppm), whereas H-3ꢁ, 3ꢉ (3.5–3.7 ppm) which proved the
(1→3)-linkage. Hexasaccharide 19 (100 mg, 0.036 mmol) was deprotected as de-
scribed for compound 15 to give 16 mg (0.013 mmol, 39%) of 20. [ꢇ]D ꢂ8°(c 1.0,
H2O), [Lit.3 ꢂ6° (c 1.0, H2O)]. NMR (D2O): 1H, (selected data) ꢄ 2.03 (3s, 9H, CH3
acetyl), 3.50 (s, 3H, CH3O), 3.93 (d, 1H), 4.16 (d, 2H), 4.48 (2d, 4H), 4.71 (2d, 2H).
NMR data were in agreement with those reported elsewhere.3,6,8
Methyl (2,3,4-Tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-
O-benzoyl-6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopyra-
nosyl)-(1→3)-(6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-O-benzoyl-6-O-
benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopyranosyl)-(1→3)-(6-O-
benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(3-O-benzoyl-6-O-benzyl-2-deoxy-2-
tetrachlorophthalimido-ꢀ-D-glucopyranosyl)-(1→3)-O-(6-O-benzyl-ꢀ-D-

ORDER
REPRINTS
REGIOSELECTIVE GLYCOSYLATIONS
581
galactopyranosyl)-(1→4)-O-(3-O-benzoyl-6-O-benzyl-2-deoxy-2-tetra-
chlorophthalimido-ꢀ-D-glucopyranoside) (22). 5% HCl/MeOH (1 mL) was
added to a stirred solution of 19 (50 mg, 0.018 mmol) in CH2Cl2 (1 mL). After 42
h the mixture was concentrated and the residue subjected to flash chromatography
(toluene-EtOAc 1:1) to give 21 (39 mg, 0.015 mmol, 81%). NMR (selected data)
(CDCl3): 1H ꢄ 3.41 (s, 3H, CH3O), 5.21, 5.48, 5.58 (d, 3H, H-1, 1ꢈ, 1ꢈ), 5.86, 5.99
(dd, 3H, H-3, 3ꢈ, 3ꢈꢈ). Compound 21 (39 mg) and 10 (27 mg, 0.026 mmol) was dis-
solved in CH2Cl2 (1 mL) and stirred with 3Å MS for 30 min when methyl triflate
(9 ꢊL, 0.075 mmol) was added. After 24 h the reaction was quenched by addition
of Et3N (0.1 mL), and the mixture diluted with CH2Cl2, filtered through Celite and
concentrated. Flash chromatography (toluene-EtOAc 3:1→2:1) of the crude
product gave 22 (31 mg, 0.009 mmol, 60%). Further elution (toluene-EtOAc 1:1)
rendered 7 mg of unreacted 19 (0.002 mmol, 13%). 22: NMR (CDCl3): 13C, 20.9,
21.0, 21.1 (CH3 acetyl), 55.8 (4C), 57.2, 66.1, 67.0, 67.1, 67.5, 67.6, 67.7, 68.1,
68.3, 69.8, 71.0, 71.3, 71.5, 71.8, 72.0, 72.4, 72.8, 73.4 (2C), 73.6, 73.7, 74.0, 74.4,
74.9, 75.6, 76.3, 76.6, 76.7, 77.5, 82.4 (C-2–6, 2ꢁ-6ꢁ, 2ꢈ-6ꢈ, 2—6ꢉ, 2ꢈꢈ-6ꢈꢈ, 2ꢈꢈ-6ꢉꢈ,
2ꢉꢈꢁ-6ꢉꢈꢁ, 2ꢉꢈꢁꢁ-6ꢉꢈꢁꢁ, CH2 benzyl, CH3O), 99.0 (2C), 99.2 (2C), 100.5, 103.3 (3C) (C-
1–1 ꢁ) 128.3–140.1 (aromatic C), 162.7, 163.4 (CBO TCP), 165.3, 165.6 (2C),
165.7 (CBO benzoyl), 169.0, 170.0, 170.1 (CBO acetyl).
An aliquot of 22 was acetylated and used for NMR analysis to prove the se-
lectivity in the coupling. 1H and COSY NMR experiments showed the character-
istic downfield shifts for H-2ꢁ, 2ꢉ, 2ꢉꢈ, 4ꢁ, 4ꢉ and 4ꢉꢈ compared to H-3ꢁ, 3ꢉ, and
3ꢉꢈ, and it could be concluded that the coupling was selective for 3ꢉꢈ-OH.
Methyl (6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-6-O-benzyl-2-deoxy-
2-tetrachlorophthalimido-ꢀ-D-glucopyranoside (24). 120 mg (0.122 mmol) of
4 was treated with dry MeOH (25 ꢊL, 0.61 mmol), NIS (55 mg, 0.244 mmol) and
TESOTf (55 ꢊL, 0.244 mmol) in CH2Cl2 and then worked up as described for 13.
Flash chromatography (toluene-EtOAc 5:1) gave methyl (2,3,4-tri-O-acetyl-6-O-
benzyl-ꢀ-D-galactopyranosyl)-(1→4)-6-O-benzyl-2-deoxy-2-tetrachlorophthal-
imido-ꢀ-D-glucopyra-noside (23) (99 mg, 0.107 mmol, 88%). NMR (CDCl3): 13
C
ꢄ 20.9, 21.0, 21.1 (CH3 acetyl), 56.8, 57.0, 67.6, 68.2, 68.3, 69.3, 69.7, 71.2, 72.6,
73.7, 73.9, 74.6, 81.9 (C-2–6, C-2ꢁ-6ꢁ, CH2 benzyl, CH30), 99.1, 101.5 (C-1, C-1ꢁ),
125.5–138.2 (aromatic C), 169.3, 170.0, 170.1 (CBO acetyl). Disaccharide 23 (95
mg, 0.102 mmol) was dissolved in 5% HCl/MeOH and stirred at room temperature
for 36 h. The mixture was then concentrated and the residue subjected to flash
chromatography (toluene-EtOAc 1:2) to give tetraol 24 (79 mg, 0.099 mmol,
97%). NMR (CDCl3): 13C ꢄ 56.8, 56.9, 69.0, 69.2, 69.5, 69.6, 71.5, 73.7, 73.8, 73.9
(2C), 74.5, 82.9 (C-2–6, C-2ꢁ-6ꢁ, CH3O, CH2 benzyl), 99.0, 103.9 (C-1, C-1ꢁ),
127.5–140.1 (aromatic C), 163.1 (CBO TCP).
Methyl (2,3,4-Tri-O-acetyl-6-O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(6-
O-benzyl-2-deoxy-2-tetrachlorophthalimido-ꢀ-D-glucopyranosyl)-(1→3)-(6-
O-benzyl-ꢀ-D-galactopyranosyl)-(1→4)-(6-O-benzyl-2-deoxy-2-tetra-
chlorophthalimidoꢀ-D-glucopyranoside) (25). Methyl triflate (40 ꢊL, 0.35
mmol), was added to a stirred solution of thioglucoside 9 (117 mg, 0.122 mmol),

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BUSKAS, KONRADSSON, AND OSCARSON
24 (70 mg, 0.087 mmol) and 3Å MS in CH2Cl2 (3 mL). The reaction mixture was
stirred under argon for 24 h, quenched by addition of triethylamine (0.25 mL), fil-
tered through Celite and concentrated. Flash chromatography (toluene-EtOAc
4:1–3:1) gave 25 (68 mg, 40 mmol, 46%). NMR (CDCl3): 13C ꢄ 20.9, 21.0, 21.1,
56.6, 56.7, 57.0, 67.6, 68.2, 68.3, 68.5, 68.8, 69.3, 69.6, 69.7, 69.9, 70.3, 71.2, 72.4,
73.5, 73.6, 73.8, 73.9, 74.0, 74.4, 77.5, 81.9, 83.1, 84.7 (C-2–6, 2ꢁ-6ꢁ, 2ꢈ-6ꢈ, 2—C-
6ꢉ, CH3O, CH2 benzyl), 99.0, 99.3, 101.4, 104.0 (C-1–1ꢉ), 127.4–140.2 (aromatic
C), 163.5, 163.9 (CBO TCP), 169.2, 170.0, 170.1 (CBO acetyl). An aliquot of 25
was acetylated (pyridine:Ac2O 3:2) to give the corresponding 3,2ꢁ,4ꢁ,3ꢈ-O-acety-
lated tetrasaccharide with NMR (CDCl3): 1H ꢄ 1.77, 1.82, 1.90, 1.96, 1.99, 2.01,
2.04 (CH3 acetyl), 3.25 (m, 1H), 3.33–3.58 (m, 15H), 3.62–3.68 (m, 3H, H-2 Glc-
NTCP, H-4ꢋ2 GlcNTCP), 4.16 (dd, 1H, H-2 GlcNTCP), 4.20 (d, 1H, H-1ꢁ),
4.34–4.66 (m, 6H, H-1ꢉ, 2ꢁ), 4.77 (m, 2H), 4.91 (dd, 1H, H-3ꢉ), 5.02 (dd, 1H,
H-2ꢉ), 5.11 (d, 1H, H-1 GlcNTCP), 5.15 (d, 1H, H-1 GlcNTCP), 5.41–5-47
(m, 3H, H-3 GlcNTCP, H-4ꢁ, 4ꢉ), 5.66 (dd, 1H, H-3 GlcNTCP). The downfield
shift for H-3, 2ꢁ, 4ꢁ and 3ꢈ confirmed the regioselective coupling.
ACKNOWLEDGMENTS
This is a collaboration with Professor Fred Brewer at the Albert Einstein Col-
lege of Medicine, New York. Financial support from the Swedish Natural Science
Research Council is gratefully acknowledged.
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Received March 23, 2001
Accepted July 25, 2001

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