Synthesis of glycopeptide sequences of repeating units of the mucins MUC 2 and MUC 3 containing oligosaccharide side-chains with core 1, core 2, core 3, core 4 and core 6 structure

Article abstract of DOI:10.1039/a701742a

An efficient synthesis of glycosylamino acid building blocks containing core 1, core 2, core 3, core 4 or core 6 mucin core oligosaccharide structures linked O-glycosidically to threonine has been developed. These building blocks 6, 10, 16, 24 and 30 can be used directly for coupling reactions in a glycopeptide synthesis. In a multiple-column solid-phase synthesis, they have been used to prepare different series of glycopeptides. Decapeptide sequences have been synthesized from repeating units of the mucins MUC 2 and MUC 3 in which different threonine residues are each systematically glycosylated with an oligosaccharide of core 1, core 2, core 3, core 4 or core 6 structure. Glycopeptides are substrates for the study of the biosynthesis of the saccharide side-chains of mucins.

Full text of DOI:10.1039/a701742a

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Synthesis of glycopeptide sequences of repeating units of the mucins
MUC 2 and MUC 3 containing oligosaccharide side-chains with
core 1, core 2, core 3, core 4 and core 6 structure
Nathalie Mathieux,a Hans Paulsen,a Morten Meldalb and Klaus Bock b
a
Institut für Organische Chemie, Universität Hamburg, Martin-Luther-King-Platz 6,
D-20146 Hamburg, Germany
b Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10,
DK-2500 Copenhagen Valby, Denmark
An efficient synthesis of glycosylamino acid building blocks containing core 1, core 2, core 3, core 4 or
core 6 mucin core oligosaccharide structures linked O-glycosidically to threonine has been developed.
These building blocks 6, 10, 16, 24 and 30 can be used directly for coupling reactions in a glycopeptide
synthesis. In a multiple-column solid-phase synthesis, they have been used to prepare different series
of glycopeptides. D ecapeptide sequences have been synthesized from repeating units of the mucins
M U C 2 and M U C 3 in which different threonine residues are each systematically glycosylated with an
oligosaccharide of core 1, core 2, core 3, core 4 or core 6 structure. G lycopeptides are substrates for the
study of the biosynthesis of the saccharide side-chains of mucins.
these glycopeptides proceeds as follows: first the 2-acetamido-
Introduction
2-deoxy--galactose is transferred from uridine 5Ј-diphosphate
(UDP)-GalNAc to the hydroxy group of threonine or serine by
the polypeptide-GalNAc-transferase and then further chain
elongation takes place stepwise with catalysis of the corre-
sponding stereoselective glycosyl transferases.5 For isolation
and precise characterisation of these enzymes the appropriate
synthetic glycopeptides with defined core structures are needed
as substrates and reference substances. A number of glycopep-
tides with core 5 and core 7 structures containing a further α-
glycosidically linked GalNAc to GalNAc have already been
synthesized.6 In the present publication, the synthesis of a series
of glycopeptides of varying amino acid sequence that contain
the core 1, core 2, core 3, core 4 and core 6 structure is
described. The compounds are substrates for a large number of
enzymes.
To synthesize glycopeptides, glycosylamino acid building
blocks are required which already contain the oligosaccharide
chain and threonine or serine in a form that enables direct inser-
tion of the building block in a multiple-column solid-phase
glycopeptide synthesis.7,8 Syntheses of some of this type of
building block have already been described.9,10 In the present
publication, an improved, more efficient method is described
which permits preparation of all five building blocks from a
single starting material. This method also provides larger
amounts, making it possible to synthesize series of glycopep-
tides with different types of linkage.
Mucins are high relative-molecular-mass glycoproteins that are
heavily glycosylated.1 They may be membrane-associated on the
surface of epithelial cells or secreted in gel form, and play an
important role in the respiratory and intestinal tracts. The pro-
tein backbone has a high content not only of proline but also
of threonine and serine residues, which have hydroxy groups
linked O-glycosidically to saccharide side-chains.1,2 The protein
backbone of mucins of known structure consists of repeating
units of approximately 10 to 20 amino acids which are joined to
form larger molecules of up to about 20 tandem units. Even
larger aggregates (400–1000 kDa) can be formed via disulfide
bridges. The saccharide chains linked to the threonine and
serine residues can vary in length, leading to a significant
heterogeneity of the molecules. The carbohydrate content is
high, constituting 50–85% of the total relative molecular
mass.1,3
A characteristic feature of all mucins is, however, the linking
region of the protein backbone with the saccharide side-chain.
The first saccharide unit is always α-glycosidic linked 2-
acetamido-2-deoxy--galactose connected to the hydroxy
group of threonine or serine. This saccharide forms the inner
part of the characteristic core oligosaccharides (core 1–7) which
have the following structures:1,3
Core 1: Galβ(1→3)-GalNAcα-Thr(Ser)
GlcNAc
↓β(1→6)
Core 2: Galβ(1→3)-GalNAcα-Thr(Ser)
Core 3: GlcNAcβ(1→3)-GalNAcα-Thr(Ser)
Results and discussion
The starting product for all syntheses was the glycosylamino
acid derivative 1 easily available from compound 25. The
saccharide residue in the glycoside 1 contained a free OH group
which could be coupled to afford disaccharide 3 by reaction
with the trichloroacetimidate group of galactose 211 in the
presence of catalytic amounts of trimethylsilyl trifluoro-
methanesulfonate (TMSOTf). The benzylidene group was
cleaved from compound 3 with acetic acid (80%) to give the diol
4. Conversion of the azido group of the saccharide 4 into an
acetamido group was most easily accomplished with activated
zinc in acetic anhydride, acetic acid and tetrahydrofuran (THF )
GlcNAc
↓β(1→6)
Core 4: GlcNAcβ(1→3)-GalNAcα-Thr(Ser)
Core 5: GalNAcα(1→3)-GalNAcα-Thr(Ser)
Core 6: GlcNAcβ(1→6)-GalNAcα-Thr(Ser)
Core 7: GalNAcα(1→6)-GalNAcα-Thr(Ser)
Attached to the core structures are other saccharide chains
of varying structure and length. In cancer cells, the carbo-
hydrate side-chains are incomplete, and the core 1 structure or
just the GalNAc unit may be exposed.3,4 The biosynthesis of
J. Chem. Soc., Perkin Trans. 1, 1997
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Scheme 1 Reagents: i, TMSOTf; ii, Zn, Ac2O, AcOH, THF; iii, Ac2O, pyridine (Pyr); iv, AcOH (80%); v, TFA (95%)
by reduction and acetylation.12 Subsequent O-acetylation of
the 4-OH and 6-OH groups afforded compound 5, from which
the tert-butyl group was cleaved with trifluoroacetic acid (TFA)
to give the acid 6. With compound 6 the desired core 1 building
block was available for glycopeptide synthesis (Scheme 1).
The diol 4 obtained as the intermediate product was also the
starting product for the synthesis of the core 2 building block
10. It was important to protect the 2-amino group for coupling
with glucosamine donors. It was also necessary to ensure the
reaction to the β-glycoside and the possibility of removing the
protecting group under mild conditions, thus avoiding degrad-
ation reactions. The best results were obtained with donor 7,
where the 2-amino group was protected with the trichloro-
ethoxycarbonyl (Teoc) residue.13 Reaction of the diol 3
proceeded regioselectively with β-glycosylation to give the tri-
saccharide 8. Treatment of compound 8 with activated zinc in
acetic anhydride, acetic acid and THF then resulted in a simul-
taneous reduction of the azido group and cleavage of the Teoc
group, with in situ N-acetylation of both amino groups formed.
Subsequent O-acetylation afforded compound 9, providing the
desired core 2 building block 10 ready for peptide synthesis
after cleavage of the tert-butyl ester, making this building block
available for a glycopeptide synthesis.
Synthesis of the core 3 and core 4 building block was possible
in a similar reaction sequence. The benzylidene compound 1
could be treated with the glycosyl donor 7 in the presence of the
TMSOTf promoter to give the disaccharide 11 in 82% yield.
Selective hydrolysis of the benzylidene group in compound 11
2360
J. Chem. Soc., Perkin Trans. 1, 1997

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afforded the diol 12 which could, in turn, be glycosylated regio-
selectively with the donor 7 at the 6-OH group to give the tri-
saccharide 14 in 88% yield. This could be converted in one step
by reduction with activated zinc and subsequent N-acetylation
into the N-acetylated compound 15. Hydrolysis of the ester
group of the amino acid in compound 15 afforded the free
carboxylic acid 16, thus providing the core 4 building block for
glycopeptide syntheses.
From the diol 11 the core 3 building block 13 was accessible
via a reaction sequence similar to that of compounds 4→6. We
had, however, already synthesized the core 3 building block by
another pathway. In this case the glycosyl donor 1711 was
allowed to react with the methyl glycoside 189,14 to give the
disaccharide 19 (Scheme 2). After cleavage of the phthalimido
Scheme 3 Reagents: i, (CH3)2CH(OCH3)2, PTSA; ii, TMSOTf; iii,
AcOH, Ac2O, Pyr; iv, Zn, Ac2O, AcOH, THF; v, TFA (95%)
acetic anhydride and the Teoc group was cleaved to give, after
N-acetylation, compound 29. Cleavage of the ester then pro-
vided the core 6 building block 30 ready for glycopeptide
synthesis.
With the synthesized building blocks multiple-column solid-
phase glycopeptide syntheses were performed. Two partial
sequences of the repeating units of the two human intestinal
mucins MUC 2 and MUC 3 which seemed promising for
enzymic studies were selected as the target peptide sequence
from MUC 2: Thr-Thr-Thr-Val-Thr-Pro-Thr-Pro-Thr-Gly and
from MUC 3: Thr-Glu-Thr-Thr-Ser-His-Ser-Thr-Pro-Gly.
These sequences have been synthesized with glycosylation on
Thr at various positions.
The multiple-column solid-phase synthesis (MCPS) was
carried out in a manual 20-column multiple synthesizer as pre-
viously described.7 With this synthesizer 20 different glyco-
peptides can be prepared in a parallel fashion.15 The Wang
resin 16 was selected as support material. The building blocks
of compounds 6 (core 1), 10 (core 2), 16 (core 4) and 30
(core 6) with free carboxy groups were activated for coupl-
ing with O-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (TBTU). The non glycosylated amino
acids were introduced as the N-fluoren-9-ylmethoxycarbonyl
pentafluorophenyl (Fmoc-Pfp) esters and, as was the case with
building block 24, coupled, with addition of 3,4-dihydro-3-
hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) in order to fol-
low the progress of the coupling reaction visually by the disap-
pearance of the yellow colour. Thr and Ser with free OH groups
were introduced as the But ethers, the Glu as the But ester and
His as the tert-butoxycarbonyl (Boc) compound. After removal
of the synthesized glycopeptides from the resin with TFA, the
compounds were treated with catalytic amounts of NaOMe in
methanol at pH 8.5 to remove the acetyl groups of the sacchar-
ide part. After preparative reversed-phase HPLC separation, all
compounds were isolated in their pure form. They were charac-
terised by 1D- and 2D-1H NMR experiments and by FAB mass
spectra.
Scheme 2 Reagents: i, TMSOTf; ii, hydrazine; iii, Ac2O, Pyr; iv,
H2SO4, Ac2O; v, TiBr4; vi, AgOTf; vii, Zn, Ac2O, AcOH, THF
group and N-acetylation to afford compound 20, the methyl
glycoside was cleaved by acetolysis to give the acetate 21, which
was subsequently converted with titanium tetrabromide into
the glycosyl bromide 22. The glycosylation of Fmoc-Thr-OPfp
with the disaccharide donor 22 then afforded the α-glycoside
23. Even in the labile pentafluorophenyl ester 23 reduction of
the azido group to give compound 24 was possible with acti-
vated zinc in acetic anhydride. Since this pathway provided an
adequate amount of the core 3 building block 24, it was used
for the other corresponding glycopeptide syntheses.
For synthesis of the core 6 building block the triol 25 was
converted with 2,2-dimethoxypropane into the isopropylidene
compound 26. Treatment of the acceptor 26 with the glycosyl
donor 7 then gave the disaccharide 27 (Scheme 3). After cleav-
age of the isopropylidene group and O-acetylation to afford the
saccharide 28, the azido group could be reduced with zinc in
Thus it was possible to synthesize from the MUC 2 sequence
the glycopeptides 31–36 with the monosaccharide (Tn-antigen
structure), compounds 37–42 with the core 1 structure (T-
antigen), compounds 43–48 with the core 2 structure (Table 1),
as well as compounds 60–65 with the core 3 structure, com-
J. Chem. Soc., Perkin Trans. 1, 1997
2361

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Table 1 Synthesized glycopeptides with Tn, Core 1 and Core 2
structure. FAB-MS data.
pounds 88–90 with the core 6 structure (Table 2). Thus a variety
of glycopeptides with MUC 2 and MUC 3 sequence carrying
all the different core structures are available by MCPS. Manual
MCPS was found to be a very efficient method for the prepar-
ation of a large number of glycopeptides using only a small
excess of the precious glycosylated amino acid building blocks.
Two types of carboxy-protection schemes were used and
both were efficient; however, the stability of the But group under
basic as well as weakly acidic conditions such as aq. acetic acid
allows for a larger range of protecting-group manipulation
when compared with the Pfp ester, leading to a large number of
building blocks from a single precursor. On the other hand
Fmoc-Thr/Ser-OPfp esters are sometimes more easily avail-
able.
This library of mucin-related compounds will be used for the
characterisation of the specificity of the glycosyl transferases
involved in the biosynthesis of the oligosaccharide side-chains
of mucins. They can be used in the study of the following
enzymes: polypeptide-GalNAc transferase, core 1-β3-Gal
transferase, core 2-β6-GlcNAc transferase, core 3-β3-GlcNAc
transferase, core 4-β4-GlcNAc transferase, core 6-β6-GlcNAc
transferase, α3-sialyl transferase, α6-sialyl transferase, as well as
transferases involved in elongation processes and also for
sulfotransferases.
No.
Sequence
M ϩ 1/Da
Mcalc/Da
MUC 2
ଙ
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
TTTVTPTPTG
1178.6
1178.9
1179.1
1179.0
1179.1
1382.3
1341.8
1341.4
1341.4
1341.4
1341.7
1706.6
1544.8
1544.7
1544.8
1544.8
1544.6
2113.3
1910.0
1910.1
1177.6
1177.6
1177.6
1177.6
1177.6
1380.7
1340.4
1340.4
1340.4
1340.4
1340.4
1705.8
1543.6
1543.6
1543.6
1543.6
1543.6
2112.2
1909.0
1909.0
ଙ
TTTVTPTPTG
ଙ
TTTVTPTPTG
ଙ
TTTVTPTPTG
ଙ
TTTVTPTPTG
ଙ
ଙ
TTTVTPTPTG
#
TTTVTPTPTG
#
TTTVTPTPTG
#
TTTVTPTPTG
#
TTTVTPTPTG
#
TTTVTPTPTG
#
#
TTTVTPTPTG
᭺
TTTVTPTPTG
᭺
TTTVTPTPTG
᭺
TTTVTPTPTG
Experimental
᭺
TTTVTPTPTG
᭺
Materials and methods
TTTVTPTPTG
All solvents were distilled at the appropriate pressure. Light
petroleum refers to the fraction distilled in the range 60–70 ЊC.
Dimethylformamide (DMF ) was analysed for free amines by
addition of Dhbt-OH prior to use. Reagents for peptide syn-
thesis were purchased as follows: Dhbt-OH and TBTU from
Fluka; Wang resin from Bachem; Fmoc amino acid Pfp esters
from NovaBiochem. 1H NMR spectra were recorded on a
Bruker AMX 400 spectrometer; δ-values are in ppm and
J-values are in Hz (±0.3 Hz). Column chromatography was
performed on Silica Gel (ICN Biochemical, 12–26 µm; 60 Å)
with 1.5–6 bar pressure.† HPLC was performed on a Merck/
Hitachi HPLC system with LiChrospher reversed-phase RP-18
columns (250 × 25 mm; 7 µm; flow rate 10 cm3 minϪ1 for pre-
parative separation) with buffer A (0.1% TFA in water) and
buffer B (0.1% TFA in acetonitrile). FAB mass spectra were
recorded on a double-focused VG-Analytical 70-250 S mass
spectrometer with m-nitrobenzyl alcohol matrix. Optical rota-
tions were recorded on a Perkin-Elmer Polarimeter 241, and
᭺
᭺
TTTVTPTPTG
᭺
#
TTTVTPTPTG
#
᭺
TTTVTPTPTG
MUC 3
ଙ
51
52
53
54
55
56
57
58
59
TETTSHSTPG
1220.8
1221.3
1221.1
1383.6
1383.8
1383.6
1586.8
1586.8
1586.7
1219.5
1219.5
1219.5
1382.4
1382.4
1382.4
1585.6
1585.6
1585.6
ଙ
TETTSHSTPG
ଙ
TETTSHSTPG
#
TETTSHSTPG
#
TETTSHSTPG
#
TETTSHSTPG
᭺
TETTSHSTPG
᭺
TETTSHSTPG
᭺
[α]D -values are given in units of 10Ϫ1 deg cm2 gϪ1
.
TETTSHSTPG
N á-(Fluoren-9-ylmethoxycarbonyl)-O-(2-azido-4,6-O-benzyl-
idene-2-deoxy-á-D-galactopyranosyl)-L-threonine tert-butyl ester
1
N-(Fluoren-9-ylmethoxycarbonyl)-O-(3,4,6-tri-O-acetyl-2-
azido-2-deoxy-α--galactopyranosyl)--threonine
tert-butyl
ester 17 (9.5 g, 13.3 mmol) was dissolved in methanol (200 cm3).
A solution of sodium methoxide (1  in methanol; 1.5 cm3) was
carefully added in order to prevent cleavage of the Fmoc group.
After 1 h (TLC chloroform–methanol, 10:1), the mixture was
neutralised by addition of ion exchanger resin Amberlite CG-
50I, filtered, and evaporated to give free triol 25. A solution of
compound 25 (7.75 g, 13.30 mmol), α,α-dimethoxytoluene
(3.95 cm3, 26.60 mmol) and toluene-4-sulfonic acid (PTSA)
(630 mg) in nitromethane (130 cm3) was stirred at room tem-
perature. After 1 h (TLC toluene–acetone, 10:1), the mixture
was neutralized with triethylamine and co-concentrated with
toluene. The residue was chromatographed on a silica gel col-
umn with light petroleum–ethyl acetate (3:1) as eluent to give
the title compound 1 (5.8 g, 65%), [α]D ϩ110 (c 1.07, CHCl3);
pounds 68–73 with the core 4 structure and compounds 76–81
with the core 6 structure (Table 2). Compounds 49, 50, 66, 67,
74 and 75 are glycopeptides with two different saccharide side-
chains. From the MUC 3 sequence the following compounds
were obtained: 51–53 with a monosaccharide structure, com-
pounds 54–56 with the core 1 structure, compounds 57–59 with
the core 2 structure (Table 1), compounds 82–84 with the core 3
structure, compounds 85–87 with the core 4 structure and com-
† 1 bar = 105 Pa.
2362
J. Chem. Soc., Perkin Trans. 1, 1997

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Table 2 Synthesized glycopeptides with Core 3, Core 4 and Core 6 structure. FAB-MS data.
No.
Sequence
M ϩ 1/Da
Mcalc/Da
No.
Sequence
M ϩ 1/Da
Mcalc/Da
MUC 2
ଙ
#
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
TTTVTPTPTG
1381.8
1382.0
1382.0
1382.2
1382.2
1788.0
1584.2
1584.8
1584.8
1585.0
1585.2
1585.2
1584.8
2194.2
1991.3
1991.4
1380.7
1380.7
1380.7
1380.7
1380.7
1786.8
1583.7
1583.7
1583.7
1583.7
1583.7
1583.7
1583.7
2193.0
1989.9
1989.9
76
77
78
79
80
81
TTTVTPTPTG
1382.0
1382.0
1382.1
1382.0
1382.2
1787.8
1380.7
1380.7
1380.7
1380.7
1380.7
1786.8
ଙ
#
TTTVTPTPTG
TTTVTPTPTG
ଙ
#
TTTVTPTPTG
TTTVTPTPTG
ଙ
#
TTTVTPTPTG
ଙ
TTTVTPTPTG
#
TTTVTPTPTG
TTTVTPTPTG
ଙ
ଙ
#
#
TTTVTPTPTG
TTTVTPTPTG
ଙ
ꢀ
TTTVTPTPTG
MUC 3
ꢀ
ଙ
ଙ
TTTVTPTPTG
82
83
84
85
86
87
88
89
90
TETTSHSTPG
1423.3
1423.8
1423.5
1627.1
1626.8
1627.0
1424.1
1423.8
1424.1
1422.6
1422.6
1422.6
1625.7
1625.7
1625.7
1422.6
1422.6
1422.6
᭺
ଙ
TTTVTPTPTG
TETTSHSTPG
᭺
ଙ
TTTVTPTPTG
TETTSHSTPG
᭺
᭺
TTTVTPTPTG
TETTSHSTPG
᭺
᭺
TTTVTPTPTG
᭺
TETTSHSTPG
᭺
TTTVTPTPTG
TETTSHSTPG
᭺
᭺
#
TETTSHSTPG
#
TTTVTPTPTG
᭺
#
TTTVTPTPTG
TETTSHSTPG
#
᭺
#
TTTVTPTPTG
TETTSHSTPG
δH (CDCl3; MeSi4) 7.82–7.25 (13 H, m, ArH), 5.75 (1 H, d,
JN H ,CH α 9.1, NH), 5.57 (1 H, s, PhCH), 5.10 (1 H, d, J1,2 3.6, 1-
H), 4.43 (2 H, m, Fmoc CH2), 4.34 (1 H, dd, JCH α,CH β 2.5,
JCH β,CH γ 7.6, Thr CHβ), 4.30–4.22 (4 H, m, 4-H, 6-H2 and Thr
CHα), 4.16 (1 H, ddd, J4,5 3.6, J5,6a 7.1, J5,6b 7.2, 5-H), 4.07 (1 H,
dd, J2,3 10.7, 3-H), 3.78 (1 H, s, Fmoc CH), 3.57 (1 H, dd, 2-H),
2.51 (1 H, d, OH), 1.51 (9 H, s, But) and 1.31 (3 H, d, Thr CHγ)
(Found: C, 64.3; H, 6.1; N, 8.2. C34H40N4O9 requires C, 64.4;
H, 6.0; N, 8.3%).
H, dd, 6Ј-Ha), 4.12 (1 H, dd, 6Ј-Hb), 4.08 (1 H, d, Thr CHα),
4.05 (1 H, dd, J2,3 11.2, 3-H), 3.94 (1 H, m, 5Ј-H), 3.80 (1 H,
dd, 2-H), 3.73 (1 H, s, Fmoc CH), 2.16, 2.06, 2.03 and 1.99
(12 H, 4 s, 4 × COCH3), 1.50 (9 H, s, But) and 1.33 (3 H,
d, Thr CHγ) (Found: C, 60.0; H, 6.0; N, 5.3. C50H58N4O18
requires C, 59.9; H, 5.8; N, 5.6%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-[O-(2Ј,3Ј,4Ј,6Ј-tetra-O-
acetyl-â-D-galactopyranosyl)-(1→3)-2-azido-2-deoxy-á-D-
galactopyranosyl]-L-threonine tert-butyl ester 4
N á-(Fluoren-9-ylmethoxycarbonyl)-O-[O-(2Ј,3Ј,4Ј,6Ј-tetra-O-
acetyl-â-D-galactopyranosyl)-(1→3)-2-azido-4,6-O-benzylidene-
2-deoxy-á-D-galactopyranosyl]-L-threonine tert-butyl ester 3
A mixture of compound 1 (2.77 g, 4.12 mmol), the imidate 211
(2.76 g, 5.60 mmol) and activated powdered 4 Å molecular
sieves in dry 1,2-dichloroethane (60 cm3) was stirred at 0 ЊC
under nitrogen. After 1 h, TMSOTf (80 mm3, 0.44 mmol) was
added. The mixture was stirred for 45 min at 0 ЊC (TLC toluene–
acetone, 10:1), neutralized with triethylamine, filtered, and then
concentrated. The residue was chromatographed on a silica gel
column and eluted with light petroleum–ethyl acetate (2:1) to
give title compound 3 (2.8 g, 68%), [α]D ϩ88.5 (c 1.03,
CHCl3); δH (CDCl3; MeSi4) 7.80–7.28 (13 H, m, ArH), 5.74 (1
H, d, JN H ,CH α 9.2, NH), 5.56 (1 H, s, PhCH), 5.41 (1 H, d,
J3Ј,4Ј 3.1, 4Ј-H), 5.31 (1 H, dd, J1Ј,2Ј 8.1, J2Ј,3Ј 11.2, 2Ј-H), 5.13
(1 H, d, J1,2 3.6, 1-H), 5.04 (1 H, dd, 3Ј-H), 4.81 (1 H, d, 1Ј-
H), 4.50 (1 H, ddd, J4,5 1.0, J5,6a 6.1, J5,6b 6.6, 5-H), 4.46 (1 H,
dd, JCH α,CH β 1.6, JCH β,CHγ 6.6, Thr CHβ), 4.41 (1 H, d, J3,4
2.5, 4-H), 4.35–4.23 (4 H, m, 6-H2 and Fmoc CH2), 4.21 (1
A mixture of compound 3 (844 mg, 0.84 mmol), acetic acid (12
cm3) and water (3 cm3) was stirred at 80 ЊC for 2 h (TLC ethyl
acetate–light petroleum, 3:1) then was cooled and co-
concentrated with toluene. The residue was chromatographed
on a silica gel column with ethyl acetate–light petroleum (3:1)
as eluent to give title compound 4 (667 mg, 87%), [α]D ϩ66.0 (c
1, CHCl3); δH (CDCl3; MeSi4) 7.81–7.28 (8 H, m, ArH), 5.70 (1
H, d, JN H ,CH α 9.7, NH), 5.41 (1 H, d, J3Ј,4Ј 2.5, 4Ј-H), 5.30 (1 H,
dd, J1Ј,2Ј 7.6, J2Ј,3Ј 10.2, 2Ј-H), 5.08 (1 H, d, J1,2 3.6, 1-H), 5.05 (1
H, dd, 3Ј-H), 4.75 (1 H, d, 1Ј-H), 4.47 (1 H, dd, JCH α,CH β 1.5,
JCH β,CH γ 6.1, Thr CHβ), 4.45 (1 H, s, Fmoc CH), 4.30 (1 H, d,
Thr CHα), 4.27 (2 H, ddd, JCH ,CH = JCH ,CH = 7.1, JCH
11.2, Fmoc CH2), 4.21 (1 H, d, J3,4 2.5, 4-H), 4.18 (1 H, dd,
J5Ј,6Јa 4.1, J6Јa,6Јb 11.7, 6Ј-Ha), 4.11 (1 H, dd, J5Ј,6Јb 5.2, 6Ј-Hb),
4.03 (1 H, dd, J2,3 10.7, 3-H), 3.98–3.89 (3 H, m, 5Ј-H and
6-H2), 3.80 (1 H, m, 5-H), 3.58 (1 H, dd, 2-H), 2.21, 2.10, 2.05
and 2.00 (12 H, 4 s, 4 × COCH3), 1.51 (9 H, s, But) and 1.26 (3
H, d, Thr CHγ) (Found: C, 56.8; H, 6.1; N, 6.0. C43H54N4O18
requires C, 56.5; H, 6.0; N, 6.1%).
2a
2b
2a,CH 2b
J. Chem. Soc., Perkin Trans. 1, 1997
2363

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N á-(Fluoren-9-ylmethoxycarbonyl)-O-[O-(2Ј,3Ј,4Ј,6Ј-tetra-O-
acetyl-â-D-galactopyranosyl)-(1→3)-2-acetamido-4,6-di-O-
acetyl-2-deoxy-á-D-galactopyranosyl]-L-threonine tert-butyl
ester 5
3-H), 3.95 (1 H, dd, 2Љ-H), 3.87 (1 H, m, 5-H), 3.56 (1 H, dd,
2-H), 2.17, 2.11, 2.03 and 2.01 (12 H, 4 s, 4 × COCH3), 1.51 (9
H, s, But) and 1.32 (3 H, d, Thr CHγ) (Found: C, 55.9; H, 5.1; N,
4.7. C73H78Cl3N5O27 requires C, 56.1; H, 5.0; N, 4.5%).
Compound 4 (745 mg, 0.74 mmol) was dissolved in THF–acetic
anhydride–acetic acid (3:2:1; 14 cm3). Zinc powder, activated
in 2% aq. copper sulfate, was added and the mixture was stirred
at room temp. for 20 min (TLC ethyl acetate–light petroleum,
5:1). The solid was removed by filtration, and the solution was
co-concentrated with toluene. To a solution of the residue in
dry pyridine (10 cm3) was added acetic anhydride (5 cm3). After
being stirred at room temp. for 2 h the solution was co-
concentrated with toluene. The residue was chromatographed
with ethyl acetate–light petroleum (3:1) as eluent to give title
compound 5 (606 mg, 82%); δH (CDCl3; MeSi4) 7.82–7.28 (8 H,
m, ArH), 5.89 (1 H, d, J2,N H 9.1, NH), 5.45 (1 H, d, JN H ,CH α 9.7,
NH Thr), 5.36 (2 H, d, J3,4 3.0, 4- and 4Ј-H), 5.09 (1 H, dd, J1Ј,2Ј
7.6, J2Ј,3Ј 10.7, 2Ј-H), 4.93 (1 H, dd, 3Ј-H), 4.84 (1 H, d, J1,2 3.6,
1-H), 4.54 (4 H, m, 1Ј-H and 2-H, Fmoc CH2), 4.26 (2 H, m, 5-
H and 6-Ha), 4.19–4.10 (5 H, m, 6-Hb, 6Ј-H2 and Thr CHβ), 3.99
(1 H, dd, JCH α,CH β 2.1, Thr CHα), 3.87 (1 H, t, 5Ј-H), 3.78 (1 H,
dd, J2,3 10.2, 3-H), 2.16, 2.13, 2.06, 2.05, 2.04, 2.03 and 1.97 (21
H, 7 s, 7 × COCH3), 1.46 (9 H, s, But) and 1.30 (3 H, d, JCH β,CH γ
6.6, Thr CHγ) (Found: C, 58.7; H, 6.4; N, 2.9. C49H62N2O20
requires C, 58.9; H, 6.3; N, 2.8%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-(2Ј,3Ј,4Ј,6Ј-tetra-O-
acetyl-â-D-galactopyranosyl)-(1Ј→3)-O-[2Љ-acetamido-
3Љ,4Љ,6Љ-tri-O-benzoyl-2Љ-deoxy-â-D-glucopyranosyl-(1Љ→6)]-
2-acetamido-4-O-acetyl-2-deoxy-á-D-galactopyranosyl}-L-
threonine tert-butyl ester 9
To a mixture of compound 8 (1.81 g, 1.15 mmol) in THF–acetic
anhydride–acetic acid (3:2:1, 30 cm3) was added zinc powder,
activated in 2% aq. copper sulfate. The mixture was stirred at
room temp. for 30 min (TLC toluene–acetone, 1:1), and was
then filtered, and co-concentrated with toluene. To a solution
of the residue in dry pyridine (20 cm3) was added acetic
anhydride (10 cm3). After being stirred at room temp. for 10 h
(TLC toluene–acetone, 1:1), the solution was co-concentrated
with toluene. The residue was chromatographed with toluene–
acetone (2:1) as eluent to give title compound 9 (1.24 g, 72%),
[α]D ϩ18.4 (c 1, CHCl3); δH (CDCl3; MeSi4) 8.04–7.17 (23 H, m,
ArH), 5.97 (1 H, d, JCH α,N H 9.7, NH Thr), 5.77 (1 H, d, J2Љ,N H
9.2, NHЉ), 5.72 (1 H, dd, J2Љ,3Љ 10.2, J3Љ,4Љ 9.7, 3Љ-H), 5.60 (1 H, d,
J2,N H 9.7, NH), 5.56 (1 H, dd, 4Љ-H), 5.34 (1 H, d, J3Ј,4Ј 2.5,
4Ј-H), 5.33 (1 H, d, J3,4 3.0, 4-H), 5.09 (1 H, dd, J1Ј,2Ј 7.6, J2Ј,3Ј
10.2, 2Ј-H), 4.94 (1 H, dd, 3Ј-H), 4.85 (1 H, d, J1,2 3.6, 1-H), 4.82
(1 H, d, J1Љ,2Љ 7.6, 1Љ-H), 4.58 (1 H, m, Fmoc CH), 4.51 (1 H, d,
1Ј-H), 4.50 (1 H, dd, JCH α,CH β 2.4, Thr CHα), 4.43 (2 H, m, Fmoc
CH2), 4.24 (1 H, dd, JCH β,CH γ 6.1, Thr CHβ), 4.20 (1 H, dd,
6Ј-Ha), 4.14–4.05 (7 H, m, 2- and 2Љ-H, 6Ј-Hb, and 6Љ- and
6-H2), 3.95 (1 H, dd, 5-H), 3.84 (1 H, dd, J2,3 10.7, 3-H), 3.80 (1
H, m, 5Љ-H), 3.56 (1 H, m, 5Ј-H), 2.14, 2.08, 2.07, 2.02, 1.99,
1.98 and 1.85 (21 H, 7 s, 7 × COCH3), 1.47 (9 H, s, But) and 1.30
(3 H, d, Thr CHγ) (Found: C, 61.6; H, 5.5; N, 2.8. C76H85N3O28
requires C, 61.3; H, 5.8; N, 2.8%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-[O-(2Ј,3Ј,4Ј,6Ј-tetra-O-
acetyl-â-D-galactopyranosyl)-(1Ј→3)-2-acetamido-4,6-di-O-
acetyl-2-deoxy-á-D-galactopyranosyl]-L-threonine 6
A solution of compound 5 (800 mg, 0.8 mmol) in TFA–water
(95:5; 10 cm3) was stirred at room temp. for 1 h (TLC ethyl
acetate–light petroleum, 3:1). The solution was then co-
concentrated with toluene to give title compound 6 (732 mg,
97%); δH (CDCl3, TFA 1%) 7.81–7.29 (8 H, m, ArH), 7.02 (1 H,
d, J2,N H 10.2, NH), 6.79 (1 H, d, JN H ,CH α 9.7, NH Thr), 5.45 (1
H, d, J1,2 3.1, 1-H), 5.39 (1 H, d, J3Ј,4Ј 3.0, 4Ј-H), 5.16 (1 H, d,
J3,4 3.0, 4-H), 5.09 (1 H, dd, J1Ј,2Ј 7.6, J2Ј,3Ј 10.1, 2Ј-H), 4.93 (1 H,
dd, 3Ј-H), 4.65 (2 H, m, Fmoc CH2), 4.54 (1 H, d, 1Ј-H), 4.36 (1
H, dd, JCH α,CH β 4.1, JCH β,CH γ 6.6, Thr CHβ), 4.28–4.15 (4 H, m,
3-H, 6- and 6Ј-Ha, Thr CHα), 4.16–4.01 (3 H, m, Fmoc CH,
6- and 6Ј-Hb), 3.90 (1 H, dd, 2-H), 3.86 (1 H, t, 5-H), 3.82 (1 H,
t, 5Ј-H), 2.17, 2.16, 2.10, 2.08, 2.05, 2.00 and 2.00 (21 H, 7 s,
7 × COCH3) and 1.09 (3 H, d, Thr CHγ) (Found: C, 57.14;
H, 5.98; N, 3.03. C45H54N2O20 requires C, 57.32; H, 5.77; N,
2.97%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-(2Ј,3Ј,4Ј,6Ј-tetra-O-
acetyl-â-D-galactopyranosyl)-(1Ј→3)-O-[2Љ-acetamido-
3Љ,4Љ,6Љ-tri-O-benzoyl-2Љ-deoxy-â-D-glucopyranosyl-(1Љ→6)]-
2-acetamido-2-deoxy-á-D-galactopyranosyl}-L-threonine 10
A solution of compound 9 (900 mg, 0.60 mmol) in TFA–water
(95:5; 10 cm3) was stirred at room temp. After 1 h (TLC
chloroform–methanol, 20:1), the solution was co-concentrated
with toluene to give title compound 10 (813 mg, 94%);
δH (CDCl3; MeSi4) 8.01–7.16 (23 H, m, ArH), 6.90 (1 H, d,
JCH α,NH 9.7, NH Thr), 6.86 (1 H, d, J2,N H 8.7, NH), 6.43 (1 H, d,
J2Љ,N H 8.6, NHЉ), 5.68 (1 H, dd, J2Љ,3Љ 10.1, J3Љ,4Љ 9.7, 3Љ-H), 5.66 (1
H, dd, 4Љ-H), 5.42 (1 H, d, J3,4 3.0, 4-H), 5.38 (1 H, d, J3Ј,4Ј 2.6,
4Ј-H), 5.09 (1 H, dd, J1Ј,2Ј 7.6, J2Ј,3Ј 10.2, 2Ј-H), 4.93 (1 H, dd, 3Ј-
H), 4.79 (1 H, d, J1Љ,2Љ 8.1, 1Љ-H), 4.66 (1 H, dd, Fmoc CH), 4.56
(1 H, d, 1Ј-H), 4.55 (1 H, d, J1,2 3.6, 1-H), 4.54 (1 H, dd, JCH α,CH β
2.6, Thr CHα), 4.44 (2 H, m, Fmoc CH2), 4.35 (1 H, dd, JCH β,CH γ
6.6, Thr CHβ), 4.25 (1 H, dd, 2Љ-H), 4.22 (2 H, m, 6Љ-H2), 4.18–
4.02 (5 H, m, 2-H, and 6Ј- and 6-H2), 3.91 (1 H, dd, J2,3 11.2, 3-
H), 3.82 (2 H, m, 5- and 5Ј-H), 3.51 (1 H, m, 5Љ-H), 2.17, 2.13,
2.11, 2.06, 2.04, 2.03 and 1.98 (21 H, 7 s, 7 × COCH3) and 1.19
(3 H, d, Thr CHγ) (Found: C, 60.6; H, 5.7; N, 2.7. C72H77N3O28
requires C, 60.4; H, 5.4; N, 2.9%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-(2Ј,3Ј,4Ј,6Ј-tetra-O-
acetyl-â-D-galactopyranosyl)-(1Ј→3)-O-[3Љ,4Љ,6Љ-tri-O-benzoyl-
2Љ-deoxy-2Љ-(2,2,2-trichloroethoxycarbonylamino)-â-D-gluco-
pyranosyl-(1Љ→6)]-2-azido-2-deoxy-á-D-galactopyranosyl}-
L-threonine tert-butyl ester 8
A mixture of disaccharide 4 (1.54 g, 1.68 mmol), imidate 713
(1.77 g, 2.18 mmol) and activated powdered 4 Å molecular
sieves in dry 1,2-dichloroethane (50 cm3) was stirred at 0 ЊC
under nitrogen. TMSOTf (40 mm3, 0.22 mmol) was added.
After 1 h (TLC ethyl acetate–light petroleum, 1:1) the mixture
was neutralized by addition of triethylamine, filtered and con-
centrated. The residue was chromatographed with ethyl
acetate–light petroleum (1:2) as eluent to give title compound 8
(2.0 g, 76%), [α]D ϩ28.5 (c 1, CHCl3); δH (CDCl3; MeSi4) 8.04–
7.29 (23 H, m, ArH), 5.78 (1 H, dd, J2Љ,3Љ 10.2, J3Љ,4Љ 9.1, 3Љ-H),
5.66 (1 H, d, JN H ,CH α 9.7, NH Thr), 5.64 (1 H, dd, 4Љ-H), 5.56 (1
H, d, J2Љ,N H 8.6, NHЉ), 5.40 (1 H, d, J3Ј,4Ј 3.0, 4Ј-H), 5.29 (1 H,
dd, J1Ј,2Ј 7.1, J2Ј,3Ј 10.2, 2Ј-H), 5.04 (1 H, dd, 3Ј-H), 5.01 (1 H, d,
J1,2 3.6, 1-H), 4.90 (1 H, d, J1Љ,2Љ 8.1, 1Љ-H), 4.77 (1 H, d,
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-[3Ј,4Ј,6Ј-tri-O-
benzoyl-2Ј-deoxy-2Ј-(2,2,2-trichloroethoxycarbonylamino)-â-
D-glucopyranosyl]-(1Ј→3)-2-azido-4,6-O-benzylidene-2-deoxy-
á-D-galactopyranosyl}-L-threonine tert-butyl ester 11
A mixture of compound 1 (1.84 g, 2.74 mmol), imidate 713
(2.60 g, 3.28 mmol) and activated powdered 4 Å molecular
sieves in dry 1,2-dichloroethane (40 cm3) was stirred at 0 ЊC
under nitrogen. After 1 h, a solution of TMSOTf in dry
dichloroethane (1 cm3; 0.50 mmol) was added. The mixture
was stirred for 45 min at 0 ЊC (TLC light petroleum–ethyl acet-
ate, 1:1) then was neutralized with triethylamine, filtered and
JCH
12.2, CH Teoc), 4.74 (1 H, d, 1Ј-H), 4.63 (1 H, dd,
2a,CH2b
Fmoc CH), 4.51 (1 H, d, CH Teoc), 4.47 (2 H, m, Fmoc CH2),
4.44 (1 H, dd, JCH α,CH β 2.8, JCH β,CH γ 6.1, Thr CHβ), 4.28 (3 H, m,
6-H2, Thr CHα), 4.19 (1 H, dd, 5Љ-H), 4.12 (1 H, d, J3,4 3.0, 4-H),
4.10–4.02 (5 H, m, 5Ј-H, 6Ј- and 6Љ-H2), 3.98 (1 H, dd, J2,3 10.2,
2364
J. Chem. Soc., Perkin Trans. 1, 1997

View Article Online
concentrated. The residue was chromatographed on a silica
gel column with light petroleum–ethyl acetate (5:2) as eluent
to give title compound 11 (2.97 g, 82%), [α]D ϩ62.6 (c 1,
CHCl3); δH (CDCl3; MeSi4) 8.07–7.24 (28 H, m, ArH), 5.94 (1
H, t, J2Ј,3Ј 10.2, 3Ј-H), 5.74 (1 H, d, JN H ,CH α 9.7, NH Thr),
5.62 (1 H, t, J3Ј,4Ј 9.7, 4Ј-H), 5.43 (1 H, s, PhCH), 5.26 (1 H,
d, JN H ,2Ј 9.7, NH Teoc), 5.19 (1 H, d, J1Ј,2Ј 7.6, 1Ј-H), 5.10 (1
H, d, J1,2 3.6, 1-H), 4.73 (1 H, dd, Fmoc CH), 4.65 (1 H, d,
JCH a,CH b 12.2, CH Teoc), 4.48 (2 H, dd, Fmoc CH2), 4.46 (1
H, m, 6-Ha), 4.45 (1 H, m, 5-H), 4.38 (1 H, dd, JCH α,CH β 2.1,
JCH β,CH γ 6.6, Thr CHβ), 4.35 (1 H, m, 6-Hb), 4.32 (1 H, dd,
Thr CHα), 4.27 (1 H, d, J3,4 3.1, 4-H), 4.10 (1 H, m, 5Ј-H),
4.06 (1 H, dd, J2,3 11.7, 3-H), 3.91 (1 H, dd, 2Ј-H), 3.85 (1
H, dd, 2-H), 3.77 (2 H, m, 6Ј-H2), 1.49 (9 H, s, But) and
1.27 (3 H, d, Thr CHγ) (Found: C, 60.2; H, 5.0; N, 5.1.
C66H64Cl3N5O18 requires C, 60.0; H, 4.9; N, 5.3%).
3.92–3.84 (4 H, m, 2Ј-, 2Љ-H and 6-H2), 3.89 (1 H, dd, 2-H), 1.50
(9 H, s, But) and 1.26 (3 H, d, JCH β,CH γ 7.1, Thr CHγ) (Found:
C, 56.5; H, 4.3; N, 4.6. C89H84Cl6N6O27 requires C, 56.8; H,
4.5; N, 4.5%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-(2Ј-acetamido-
3Ј,4Ј,6Ј-tri-O-benzoyl-2Ј-deoxy-â-D-glucopyranosyl)-(1Ј→3)-
O-[(2Љ-acetamido-3Љ,4Љ,6Љ-tri-O-benzoyl-2Љ-deoxy-â-D-gluco-
pyranosyl)-(1Љ→6)]-2-acetamido-4-O-acetyl-2-deoxy-á-D-
galactopyranosyl}-L-threonine tert-butyl ester 15
Compound 14 (1.0 g, 0.53 mmol) was dissolved in THF–acetic
anhydride–acetic acid (3:2:1; 10 cm3). Zinc powder, activated
in 2% aq. copper sulfate, was added and the mixture was stirred
for 1 h at room temp. (TLC toluene–acetone, 1:2). The residue
was acetylated as described for compound 9 and chromato-
graphed on a silica gel column with elution with toluene–
acetone (3:2) to give title compound 15 (530 mg, 60%), [α]D
ϩ13.8 (c 1, CHCl3); δH (CDCl3; MeSi4) 8.03–7.22 (38 H, m,
ArH), 6.48 (1 H, d, JCH α,N H 8.6, NH Thr), 6.03 (1 H, d, J2Ј,N H
7.6, NHЈ), 5.93 (1 H, d, J2Љ,N H 8.6, NHЉ), 5.79 (2 H, dd, J2Ј,3Ј
10.2, J3Ј,4Ј 9.3, 3Ј- and 3Љ-H), 5.74 (1 H, d, J2,N H 9.1, NH),
5.59 (1 H, dd, 4Љ-H), 5.54 (1 H, dd, 4Ј-H), 5.45 (1 H, d, J1,2
3.1, 1-H), 5.12 (1 H, d, J1Ј,2Ј 7.6, 1Ј-H), 4.88 (1 H, d, J1Љ,2Љ 8.1,
1Љ-H), 4.62 (1 H, dd, Fmoc CH), 4.52 (2 H, m, 6Ј- and 6Љ-
Ha), 4.48 (1 H, dd, JCH α,CH β 2.1, Thr CHα), 4.43 (2 H, m, 6Ј-
and 6Љ-Hb), 4.24 (3 H, m, Fmoc CH2, 5-H), 4.18 (1 H, dd,
JCH β,CH γ 6.1, Thr CHβ), 4.10–3.98 (5 H, m, 4-H, 6-Ha, 5Ј-, 2Љ-
and 5Љ-H), 3.92 (1 H, dd, J2,3 10.7, 2-H), 3.89 (1 H, dd, 6-Hb),
3.82 (1 H, dd, 2Ј-H), 3.55 (1 H, dd, 3-H), 2.06, 2.04, 1.90 and
1.84 (12 H, 4 s, 4 × COCH3), 1.47 (9 H, s, But) and 1.22 (3
H, d, Thr CHγ) (Found: C, 65.1; H, 5.8; N, 3.6. C91H92N4O27
requires C, 65.3; H, 5.5; N, 3.4%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-[3Ј,4Ј,6Ј-tri-O-
benzoyl-2Ј-deoxy-2Ј-(2,2,2-trichloroethoxycarbonylamino)-â-
D-glucopyranosyl]-(1Ј→3)-2-azido-2-deoxy-á-D-galacto-
pyranosyl}-L-threonine tert-butyl ester 12
A solution of compound 11 (2.97 g, 2.25 mmol) in a mixture
of acetic acid (16 cm3) and water (4 cm3) was stirred at 80 ЊC.
After 1 h (TLC ethyl acetate–light petroleum, 1:1), the solu-
tion was cooled, and co-concentrated with toluene. The resi-
due was chromatographed on a silica gel column eluted with
ethyl acetate–light petroleum (1:1) to give title compound 12
(2.28 g, 82%), [α]D ϩ39.4 (c 1, CHCl3); δH (CDCl3; MeSi4)
8.07–7.31 (23 H, m, ArH), 5.89 (1 H, dd, J2Ј,3Ј 10.2, J3Ј,4Ј 9.7,
3Ј-H), 5.69 (1 H, d, JN H ,CH α 9.1, NH Thr), 5.58 (1 H, dd, J4Ј,5Ј
10.2, 4Ј-H), 5.41 (1 H, d, JN H ,2Ј 9.6, NH Teoc), 5.17 (1 H, d,
J1Ј,2Ј 8.1, 1Ј-H), 5.05 (1 H, d, J1,2 3.6, 1-H), 4.74 (1 H, dd,
Fmoc CH), 4.69 (1 H, d, JCH a,CH b 12.2, CH Teoc), 4.54 (1 H,
d, CH Teoc), 4.46–4.33 (3 H, m, Fmoc CH2, Thr CHβ), 4.28
(1 H, dd, JCH α,CH β 2.6, Thr CHα), 4.24 (1 H, d, J3,4 3.1, 4-H),
4.15–4.11 (3 H, m, 5Ј-H and 6Ј-H2), 4.06 (1 H, dd, J2,3 10.7,
3-H), 3.89 (1 H, dd, 2Ј-H), 3.81 (1 H, m, 5-H), 3.74 (1 H, m,
6-Ha), 3.67 (1 H, dd, 2-H), 3.59 (1 H, m, 6-Hb), 2.85 (1 H, s,
4-OH), 2.39 (1 H, s, 6-OH), 1.50 (9 H, s, But) and 1.28 (3 H,
d, JCH β,CH γ 6.6, Thr CHγ) (Found: C, 57.3; H, 5.1; N, 5.6.
C59H60Cl3N5O18 requires C, 57.5; H, 4.9; N, 5.7%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-(2Ј-acetamido-
3Ј,4Ј,6Ј-tri-O-benzoyl-2Ј-deoxy-â-D-glucopyranosyl)-(1Ј→3)-
O-[(2Љ-acetamido-3Љ,4Љ,6Љ-tri-O-benzoyl-2Љ-deoxy-â-D-gluco-
pyranosyl)-(1Љ→6)]-2-acetamido-4-O-acetyl-2-deoxy-á-D-
galactopyranosyl}-L-threonine 16
A solution of compound 15 (450 mg, 0.27 mmol) in TFA–water
(95:5; 6 cm3) was stirred at room temp. for 1 h (TLC
chloroform–methanol, 19:1). The solution was then co-
concentrated with toluene to give title compound 16 (422 mg,
96%); δH (CDCl3; TFA 1%) 7.99–7.14 (38 H, m, ArH), 6.98 (1 H,
d, JCH α,NH 9.7, NH Thr), 6.94 (1 H, d, J2Ј,N H 9.2, NHЈ), 6.72 (1 H,
d, J2,N H 9.2, NH), 6.61 (1 H, d, J2Љ,N H 9.1, NHЉ), 5.72 (2 H, dd,
J2Ј,3Ј = J2Љ,3Љ = 10.2, J3Ј,4Ј = J3Љ,4Љ = 9.7, 3Ј- and 3Љ-H), 5.68 (1 H,
dd, 4Љ-H), 5.66 (1 H, dd, 4Ј-H), 5.50 (1 H, d, J3,4 3.1, 4-H), 5.03
(1 H, d, J1,2 3.6, 1-H), 4.85 (1 H, d, J1Ј,2Ј 8.1, 1Ј-H), 4.82 (1 H, d,
J1Љ,2Љ 7.6, 1Љ-H), 4.67 (1 H, dd, Fmoc CH), 4.65–4.54 (2 H, m,
6Ј-H2), 4.52 (2 H, m, 5Ј- and 5Љ-H), 4.46 (2 H, dd, Fmoc CH2),
4.29–4.21 (4 H, m, 2- and 2Ј-H, 6Љ-Ha and Thr CHβ), 4.15 (1 H,
dd, 2Љ-H), 4.11 (1 H, dd, 6Љ-Hb), 4.03 (1 H, dd, 6-Ha), 3.99 (1 H,
dd, 6-Hb), 3.96 (1 H, dd, J2,3 10.7, 3-H), 3.92 (1 H, dd, JCH α,CH β
2.7, Thr CHα), 3.49 (1 H, m, 5-H), 2.06, 2.04, 2.01 and 1.91 (12
H, 4 s, 4 × COCH3) and 1.12 (3 H, d, JCH β,CH γ 6.1, Thr CHγ)
(Found: C, 65.0; H, 5.0; N, 3.6. C87H84N4O27 requires C, 64.6;
H, 5.2; N, 3.5%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-[3Ј,4Ј,6Ј-tri-O-
benzoyl-2Ј-deoxy-2Ј-(2,2,2-trichloroethoxycarbonylamino)-â-
D-glucopyranosyl]-(1Ј→3)-O-[3Љ,4Љ,6Љ-tri-O-benzoyl-2Љ-deoxy-
2Љ-(2,2,2-trichloroethoxycarbonylamino)-â-D-glucopyranosyl]-
(1Љ→6)-2-azido-2-deoxy-á-D-galactopyranosyl}-L-threonine tert-
butyl ester 14
A mixture of disaccharide 12 (2.20 g, 1.78 mmol), imidate 713
(1.74 g, 2.14 mmol) and activated powdered 4 Å molecular
sieves in dry 1,2-dichloroethane (40 cm3) was stirred at 0 ЊC
under nitrogen. After 1 h, TMSOTf (58 mm3, 0.32 mmol) was
added. After being stirred at 0 ЊC for 30 min (TLC light
petroleum–ethyl acetate, 1:1), the solution was neutralized by
addition of triethylamine, filtered, and concentrated. The resi-
due was chromatographed on a silica gel column with light
petroleum–ethyl acetate (2:1) as eluent to give title compound
14 (2.95 g, 88%), [α]D ϩ15.3 (c 1, CHCl3); δH (CDCl3; MeSi4)
8.04–7.28 (38 H, m, ArH), 5.83 (1 H, dd, J2Ј,3Ј 10.1, J3Ј,4Ј 9.7,
3Ј-H), 5.78 (1 H, dd, J2Љ,3Љ 10.2, J3Љ,4Љ 9.7, 3Љ-H), 5.69 (1 H, d,
J2Ј,N H 9.6, NHЈ), 5.66 (1 H, dd, 4Ј-H), 5.59 (1 H, dd, 4Љ-H),
5.37 (1 H, d, J2Љ,N H 8.1, NHЉ), 5.14 (1 H, d, J1Ј,2Ј 8.1, 1Ј-H),
4.98 (1 H, d, J1,2 3.5, 1-H), 4.87 (1 H, d, J1Љ,2Љ 8.1, 1Љ-H), 4.82
(1 H, d, JCH α,N H 9.7, NH Thr), 4.70 (2 H, d, JCH a,CH b 12.2, CH
Teoc), 4.64 (1 H, dd, Fmoc CH), 4.53 (2 H, d, CH Teoc),
4.48 (1 H, dd, JCH α,CH β 2.5, Thr CHα), 4.42 (2 H, dd, Fmoc
CH2), 4.31–4.24 (3 H, m, 5Љ-H, 6Ј- and 6Љ-Ha and Thr CHβ),
4.16 (1 H, d, J3,4 3.0, 4-H), 4.11 (2 H, m, 6Ј- and 6Љ-Hb), 4.05 (1
H, m, 5Ј-H), 4.03 (1 H, dd, J2,3 10.7, 3-H), 3.94 (1 H, m, 5-H),
Methyl O-(3Ј,4Ј,6Ј-tri-O-acetyl-2Ј-deoxy-2Ј-phthalimido-â-D-
glucopyranosyl)-(1Ј→3)-2-azido-4,6-O-benzylidene-2-deoxy-
â-D-galactopyranoside 19
A mixture of compound 189,14 (310 mg, 1 mmol), imidate 17
(700 mg, 1.2 mmol) and activated powdered 4 Å molecular
sieves in dry 1,2-dichlorethane (10 cm3) was stirred at Ϫ20 ЊC
under nitrogen. A solution of TMSOTf in dry toluene (0.5 ;
0.3 cm3) was added. After 2 h (TLC toluene–ethyl acetate, 2:1),
the solution was neutralized with triethylamine, filtered and
concentrated. The residue was chromatographed on a silica gel
column and eluted with toluene–ethyl acetate (2:1) to give title
compound 19 (610 mg, 84%), [α]D Ϫ2.2 (c 1, CHCl3); δH (CDCl3;
J. Chem. Soc., Perkin Trans. 1, 1997
2365

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MeSi4) 7.88–7.32 (9 H, m, ArH), 5.80 (1 H, dd, J2Ј,3Ј 10.5, J3Ј,4Ј
9.0, 3Ј-H), 5.60 (1 H, d, J1Ј,2Ј 8.2, 1Ј-H), 5.54 (1 H, s, PhCH),
5.19 (1 H, dd, J4Ј,5Ј 9.5, 4Ј-H), 4.42 (1 H, dd, 2Ј-H), 4.38 (1 H,
dd, J5Ј,6Јa 2.5, J6Јa,6Јb 12.0, 6Ј-Ha), 4.30 (1 H, dd, J5,6a 1.5, J6a,6b
12.5, 6-Ha), 4.24 (1 H, dd, J3,4 3.5, J4,5 1.0, 4-H), 4.17 (1 H, dd,
J5Ј,6Јb 4.5, 6Ј-Hb), 4.09 (1 H, d, J1,2 8.0, 1-H), 4.05 (1 H, dd, J5,6b
2.0, 6-Hb), 3.86 (1 H, m, 5Ј-H), 3.59 (1 H, dd, 2-H), 3.47 (3 H, s,
OMe), 3.35 (1 H, dd, J2,3 10.5, 3-H), 3.33 (1 H, m, 5-H) and
2.05, 2.03 and 1.88 (9 H, 3 s, 3 × COCH3) (Found: C, 56.4; H,
5.2. C34H36N4O14 requires C, 56.4; H, 5.0%).
H, dd, J5,6b 7.6, 6-Hb), 3.94 (1 H, dd, 2-H), 3.90 (1 H, dd, 2Ј-H),
3.71 (1 H, m, 5Ј-H) and 2.15, 2.12, 2.07, 2.04, 2.03 and 1.93 (18
H, 6 s, 6 × COCH3).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-[O-(2Ј-acetamido-3Ј,4Ј,6Ј-
tri-O-acetyl-2Ј-deoxy-â-D-glucopyranosyl)-(1Ј→3)-4,6-di-O-
acetyl-2-azido-2-deoxy-á-D-galactopyranosyl]-L-threonine
pentafluorophenyl ester 23
A mixture of Fmoc-Thr-OPfp (188 mg, 0.37 mmol), activated
powdered 4 Å molecular sieves and silver trifluoromethanesul-
fonate (226 mg, 0.88 mmol) in dry dichloromethane (10 cm3)
was stirred at room temp. under nitrogen. After 1 h, the mixture
was cooled at Ϫ20 ЊC and a solution of the bromide 22 (300 mg,
0.44 mmol) in dry dichloromethane was added. The mixture
was stirred for 14 h at Ϫ20 ЊC (TLC toluene–acetone, 2:1) and
was then filtered. The solution was washed successively with aq.
NaHCO3 and water, dried (Na2SO4), and concentrated. The resi-
due was chromatographed on a silica gel column with toluene–
acetone (3:1) as eluent to give title compound 23 (197 mg, 48%),
[α]D ϩ25.2 (c 1, CHCl3); δH (CDCl3) 7.74–7.12 (8 H, m, ArH),
5.88 (1 H, d, JCH α,N H 9.1, NH), 5.53 (1 H, d, J2Ј,N H 8.7, NHЈ),
5.47 (1 H, d, J3,4 2.5, 4-H), 5.28 (1 H, dd, J2Ј,3Ј 9.7, J3Ј,4Ј 10.2, 3Ј-
H), 5.10 (1 H, d, J1,2 4.6, 1-H), 5.09 (1 H, dd, J4Ј,5Ј 9.2, 4Ј-H),
4.92 (1 H, d, J1Ј,2Ј 8.1, 1Ј-H), 4.75 (1 H, dd, JCH α,CH β 2.0, Thr
Methyl O-(2Ј-acetamido-3Ј,4Ј,6Ј-tri-O-acetyl-2Ј-deoxy-â-D-
glucopyranosyl)-(1Ј→3)-2-azido-4,6-O-benzylidene-2-deoxy-
â-D-galactopyranoside 20
A solution of disaccharide 19 (600 mg, 0.8 mmol) in a mix-
ture of hydrazine hydrate (6 cm3) and ethanol (13 cm3) was
stirred at 70 ЊC for 15 min (TLC toluene–ethyl acetate, 2:1).
The solution was then co-concentrated with ethanol. To a solu-
tion of the residue in dry pyridine (18 cm3) was added acetic
anhydride (9 cm3). After being stirred at 90 ЊC for 20 min (TLC
toluene–acetone, 1:1), the solution was co-concentrated with
toluene. The residue was chromatographed on a silica gel col-
umn with toluene–acetone (2:1) as eluent to give title compound
20 (471 mg, 86%), [α]D ϩ21.0 (c 1, CHCl3); δH (CDCl3) 7.55–7.22
(5 H, m, Ph), 6.60 (1 H, s, NH), 5.54 (1 H, s, PhCH), 5.41 (1 H,
dd, J2Ј,3Ј 10.2, J3Ј,4Ј 9.2, 3Ј-H), 5.06 (1 H, d, J1Ј,2Ј 8.6, 1Ј-H), 5.00 (1
H, dd, J4Ј,5Ј 9.6, 4Ј-H), 4.29 (1 H, dd, J3,4 3.5, J4,5 1.0, 4-H), 4.25
(1 H, dd, J5Ј,6Јa 2.6, J6Јa,6Јb 12.2, 6Ј-Ha), 4.23 (1 H, m, 6-Ha), 4.20
(1 H, d, J1,2 8.1, 1-H), 4.15 (1 H, dd, J5Ј,6Јb 4.6, 6Ј-Hb), 4.08 (1 H,
dd, J5,6a 1.5, J6a,6b 12.7, 6-Hb), 3.75 (1 H, m, 5Ј-H), 3.69 (1 H, dd,
J2,3 10.7, 2-H), 3.66 (1 H, dd, 2Ј-H), 3.56 (3 H, s, OMe), 3.52
(1 H, dd, 3-H), 3.43 (1 H, m, 5-H) and 2.05, 2.04, 2.01 and 1.93
(12 H, 4 s, 4 × COCH3) (Found: C, 53.1; H, 5.6. C28H36N4O13
requires C, 52.8; H, 5.7%).
CHα), 4.58 (1 H, dd, JCH ,CH 7.1, JCH
10.7, Fmoc
2a
2a,CH 2b
a
CH2 ), 4.54 (1 H, dd, JCH β,CH γ 6.1, Thr CHβ), 4.45 (1 H, dd,
b
JCH ,CH 7.1, Fmoc CH2 ), 4.29 (2 H, m, Fmoc CH, 6Ј-Ha), 4.17
2b
(3 H, m, 6-H2 and 6Ј-Hb), 4.03 (1 H, dd, J2,3 10.1, 3-H), 3.99 (1
H, m, 5-H), 3.80 (1 H, dd, 2Ј-H), 3.76 (1 H, dd, 2-H), 3.69 (1 H,
m, 5Ј-H), 2.13, 2.10, 2.06, 2.03, 2.02 and 1.90 (18 H, 6 s,
6 × COCH3) and 1.42 (3 H, d, Thr CHγ) (Found: C, 53.0; H,
4.8. C49H50F5N5O19 requires C, 53.1; H, 4.6%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-[O-(2Ј-acetamido-
3Ј,4Ј,6Ј-tri-O-acetyl-2Ј-deoxy-â-D-glucopyranosyl)-(1Ј→3)-
2-acetamido-4,6-di-O-acetyl-2-deoxy-á-D-galactopyranosyl]-L-
threonine pentafluorophenyl ester 24
O-(2Ј-Acetamido-3Ј,4Ј,6Ј-tri-O-acetyl-2Ј-deoxy-â-D-gluco-
pyranosyl)-(1Ј→3)-1,4,6-tri-O-acetyl-2-azido-2-deoxy-á-D-
galactopyranose 21
Compound 23 (292 mg, 0.26 mmol) was dissolved in THF–
acetic anhydride–acetic acid (3:2:1; 10 cm3). Zinc powder, acti-
vated in 2% aq. copper sulfate, was added and the mixture was
stirred at room temp. for 10 min (TLC toluene–acetone, 1:1).
The mixture was filtered and co-concentrated with toluene. The
residue was chromatographed on a silica gel column eluted with
toluene–acetone (2:1) to give title compound 24 (213 mg, 73%),
[α]D ϩ51.0 (c 1, CHCl3); δH (CDCl3) 7.78–7.29 (8 H, m, ArH),
6.18 (1 H, d, JCH α,NH 9.1, NH), 6.08 (1 H, d, J2,N H 8.6, NH), 5.81
(1 H, d, J2Ј,N H Ј 8.1, NHЈ), 5.37 (1 H, dd, J2Ј,3Ј 9.1, J3Ј,4Ј 10.7,
3Ј-H), 5.34 (1 H, d, J3,4 2.6, 4-H), 5.05 (1 H, dd, J4Ј,5Ј 9.7, 4Ј-H),
5.03 (1 H, d, J1,2 4.1, 1-H), 4.92 (1 H, d, J1Ј,2Ј 8.1, 1Ј-H), 4.70 (1
A solution of methyl glycoside 20 (1.25 g, 1.82 mmol) in acetic
anhydride (12 cm3) was stirred at 0 ЊC. A cold solution of acetic
anhydride–sulfuric acid (50:1; 12 cm3) was added. After 15 h
at 0 ЊC (TLC ethyl acetate–light petroleum, 4:1), the solution
was diluted with cold dichloromethane and washed succes-
sively with aq. NaHCO3 and water, dried (Na2SO4), and co-
concentrated with toluene to give the 1-O-acetate 21 (1.17 g,
97%); δH (CDCl3) α-anomer 6.25 (1 H, d, J1,2 3.6, 1-H), 5.62 (1 H,
d, J2Ј,N H 8.6, NH), 5.50 (1 H, dd, J3,4 3.5, J4,5 1.0, 4-H), 5.34 (1 H,
dd, J2Ј,3Ј 10.6, J3Ј,4Ј 9.1, 3Ј-H), 5.10 (1 H, dd, J4Ј,5Ј 9.7, 4Ј-H), 4.99
(1 H, d, J1Ј,2Ј 8.1, 1Ј-H), 4.32 (1 H, dd, J5Ј,6Јa 2.6, J6Јa,6Јb 12.2, 6Ј-
Ha), 4.19–3.97 (3 H, m, 6-H2 and 6Ј-Hb), 3.93 (1 H, dd, J2,3 10.2,
2-H), 3.78 (1 H, dd, 2Ј-H), 3.73 (1 H, m, 5Ј-H), 3.67 (1 H, dd, 3-
H), 3.57 (1 H, m, 5-H) and 2.16, 2.12, 2.10, 2.07, 2.03, 2.02 and
1.95 (21 H, 7 s, 7 × COCH3).
H, dd, JCH α,CH β 1.6, Thr CHα), 4.57 (2 H, ddd, JCH ,CH
=
2a
JCH ,CH = 6.1, JCH
11.8, Fmoc CH2), 4.46–4.34 (3 H,
2b
2a,CH 2b
m, Thr CHβ, 2- and 5Ј-H), 4.27 (1 H, t, Fmoc CH), 4.13 (2 H,
m, 6Ј-H2), 4.07–3.99 (2 H, m, 6-H2), 3.90 (1 H, dd, J2,3 9.7, 3-H),
3.70 (1 H, m, 5-H), 3.54 (1 H, dd, 2Ј-H), 2.11, 2.10, 2.08, 2.06,
1.92, 1.85 and 1.77 (21 H, 7 s, 7 × COCH3) and 1.40 (3 H, d,
JCH β,CH γ 6.1, Thr CHγ) (Found: C, 54.7; H, 5.0. C51H54F5N3O20
requires C, 54.5; H, 4.8%).
O-(2Ј-Acetamido-3Ј,4Ј,6Ј-tri-O-acetyl-2Ј-deoxy-â-D-gluco-
pyranosyl)-(1Ј→3)-4,6-di-O-acetyl-2-azido-2-deoxy-á-D-
galactopyranosyl bromide 22
A solution of compound 21 (140 mg, 0.21 mmol) and titanium
tetrabromide (118 mg, 0.3 mmol) in dry dichloromethane–ethyl
acetate (10:1; 2.2 cm3) was stirred at room temp. for 14 h. The
solution was diluted with dry toluene (5 cm3), and dry sodium
acetate was added until the mixture became colourless. The
mixture was filtered and concentrated to give the bromide 22
(130 mg, 91%); δH (CDCl3) 6.46 (1 H, d, J1,2 4.1, 1-H), 5.56 (1 H,
d, J2Ј,N H 6.6, NH), 5.54 (1 H, d, J3,4 3.6, 4-H), 5.23 (1 H, dd, J2Ј,3Ј
10.7, J3Ј,4Ј 9.7, 3Ј-H), 5.10 (1 H, dd, J4Ј,5Ј 10.2, 4Ј-H), 4.86 (1 H,
d, J1Ј,2Ј 8.1, 1Ј-H), 4.39 (1 H, m, 5-H), 4.31 (1 H, dd, J5Ј,6Јa 2.0,
J6Јa,6Јb 12.2, 6Ј-Ha), 4.20 (1 H, dd, J5,6a 4.6, J6a,6b 11.7, 6-Ha), 4.14
(1 H, dd, J5Ј,6Јb 4.6, 6Ј-Hb), 4.13 (1 H, dd, J2,3 10.2, 3-H), 4.03 (1
N á-(Fluoren-9-ylmethoxycarbonyl)-O-(2-azido-2-deoxy-3,4-O-
isopropylidene-â-D-galactopyranosyl)-L-threonine tert-butyl
ester 26
A solution of compound 25 (800 mg, 1.37 mmol) prepared as
previously described 11 with compound 1 and PTSA (11 mg) in
2,2-dimethoxypropane (14 cm3) was stirred at room temp. for
12 h (TLC ethyl acetate–light petroleum, 1:1). The solution was
then neutralized by addition of triethylamine, and co-
concentrated with toluene. A solution of the residue in
methanol–water (10:1; 11 cm3) was stirred at 80 ЊC. After 1 h
(TLC ethyl acetate–light petroleum, 1:1) the solution was co-
concentrated with toluene. The residue was chromatographed
2366
J. Chem. Soc., Perkin Trans. 1, 1997

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on a silica gel column with ethyl acetate–light petroleum (1:2)
as eluent to give title compound 26 (590 mg, 69%), [α]D ϩ99.5 (c
1, CHCl3); δH (CDCl3) 7.79–7.13 (8 H, m, ArH), 5.63 (1 H, d,
JN H ,CH α 9.1, NH), 5.02 (1 H, d, J1,2 3.6, 1-H), 4.44 (1 H, dd,
JCH ,CH 3.0, JCH ,CH 10.7, Fmoc CH), 4.41 (1 H, dd, J2,3 8.1,
N á-(Fluoren-9-ylmethoxycarbonyl)-O-[O-(2Ј-acetamido-
3Ј,4Ј,6Ј-tri-O-benzoyl-2Ј-deoxy-â-D-glucopyranosyl)-(1Ј→6)-2-
acetamido-3,4-di-O-acetyl-2-deoxy-á-D-galactopyranosyl]-L-
threonine tert-butyl ester 29
To a solution of compound 28 (450 mg, 0.34 mmol) in THF–
acetic anhydride–acetic acid (3:2:1; 10 cm3) was added zinc
powder, activated in 2% aq. copper sulfate. The mixture was
stirred at room temp. for 30 min (TLC toluene–acetone, 2:1)
and was then filtered and co-concentrated with toluene. The
residue was chromatographed on a silica gel column eluted with
toluene–acetone (2:1) to give title compound 29 (305 mg, 73%),
[α]D ϩ18.6 (c 1, CHCl3); δH (CDCl3) 8.02–7.28 (23 H, m, ArH),
6.08 (1 H, d, JN H ,CH α 10.2, NHThr), 5.83 (1 H, d, JN H ,2 9.6, NH),
5.81 (1 H, d, JN H Ј,2Ј 10.2, NHЈ), 5.76 (1 H, dd, J2Ј,3Ј
11.2, J3Ј,4Ј 8.7, 3Ј-H), 5.56 (1 H, dd, 4Ј-H), 5.35 (1 H, d, J3,4 2.6,
4-H), 5.08 (1 H, dd, J2,3 10.7, 3-H), 5.01 (1 H, d, J1Ј,2Ј 8.1, 1Ј-H),
4.88 (1 H, d, J1,2 2.5, 1-H), 4.54 (3 H, m, Thr-CHα, 2- and
6Ј-Ha), 4.46 (2 H, m, 6Ј-Hb and Fmoc-CH), 4.25 (1 H, m, 5-H),
4.23 (2 H, m, Fmoc CH2), 4.20 (1 H, dd, JCH α,CH β 2.0, JCH β,CH γ
6.6, Thr CHβ), 4.08 (1 H, m, 5Ј-H), 3.90 (1 H, dd, 6-Ha), 3.86 (1
H, dd, 2Ј-H), 3.65 (1 H, dd, 6-Hb), 2.10, 1.99, 1.98 and 1.87 (12
H, 4 s, 4 × COCH3), 1.47 (9 H, s, But) and 1.32 (3 H, d, Thr
CHγ) (Found: C, 64.2; H, 5.9; N, 3.5. C64H69N3O20 requires C,
64.0; H, 5.8; N, 3.5%).
2a
2b
J3,4 2.9, 3-H), 4.39 (1 H, dd, JCH α,CH β 2.0, JCH β,CH γ 6.6, Thr CHβ),
a
4.33 (1 H, dd, JCH
7.6, Fmoc CH2 ), 4.29 (1 H, dd, Thr
2a,CH 2
b
b
CHα), 4.26 (1 H, dd, Fmoc CH2 ), 4.24 (1 H, dd, 4-H), 4.15 (1
H, m, 5-H), 3.93 (1 H, dd, J5,6a 6.6, J6a,6b 11.7, 6-Ha), 3.84 (1 H,
dd, J5,6b 4.1, 6-Hb), 3.41 (1 H, dd, 2-H), 2.48 (1 H, d, OH), 1.52
(3 H, s, CH3), 1.49 (9 H, s, But), 1.47 (3 H, s, CH3) and 1.33 (3
H, d, Thr CHγ) (Found: C, 61.7; H, 6.7; N, 9.1. C32H40N4O9
requires C, 61.5; H, 6.5; N, 9.0%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-[3Ј,4Ј,6Ј-tri-O-
benzoyl-2Ј-deoxy-2Ј-(2,2,2-trichloroethoxycarbonylamino)-â-D-
glucopyranosyl]-(1Ј→6)-2-azido-2-deoxy-3,4-O-isopropyl-
idene-á-D-galactopyranosyl}-L-threonine tert-butyl ester 27
A mixture of compound 26 (660 mg, 1.08 mmol), imidate 7
(1.03 g, 1.26 mmol) and activated powdered 4 Å molecular
sieves in dry 1,2-dichloroethane (15 cm3) was stirred at room
temp. under nitrogen. After 1 h, TMSOTf (36 mm3, 0.19 mmol)
was added. After being stirred at room temp. for 25 min (TLC
light petroleum–ethyl acetate, 2:1), the solution was neutralized
by addition of triethylamine, filtered, and concentrated. The
residue was chromatographed on a silica gel column eluted with
light petroleum–ethyl acetate (3:1) to give title compound 27
(1.2 g, 81%), [α]D ϩ30.4 (c 1, CHCl3); δH (CDCl3) 8.02–7.25
(23 H, m, ArH), 5.79 (1 H, dd, J2Ј,3Ј 10.7, J3Ј,4Ј 9.7, 3Ј-H), 5.62
(1 H, d, JN H ,2Ј 9.1, NH Teoc), 5.60 (1 H, dd, 4Ј-H), 4.97 (1 H,
d, J1,2 3.1, 1-H), 4.92 (1 H, d, J1Ј,2Ј 8.1, 1Ј-H), 4.81 (1 H, d,
JN H ,CH α 9.0, NH Thr), 4.59 (1 H, dd, JCH ,CH 4.1, JCH ,CH
N á-(Fluoren-9-ylmethoxycarbonyl)-O-[O-(2Ј-acetamido-3Ј,4Ј,6Ј-
tri-O-benzoyl-2-deoxy-â-D-glucopyranosyl)-(1→6)-2-acetamido-
3,4-di-O-acetyl-2-deoxy-á-D-galactopyranosyl]-L-threonine 30
A solution of ester 29 (453 mg, 0.38 mmol) in TFA–water
(95:5; 4 cm3) was stirred at room temp. for 1 h (TLC dichloro-
methane–methanol, 20:1). The solution was then co-concen-
trated with toluene to give title compound 30 (414 mg, 96%);
δH (CDCl3) 8.01–7.12 (23 H, m, ArH), 6.29 (1 H, d, JN H ,CH α 9.6,
NH Thr), 6.12 (1 H, d, JN H ,2Ј 8.7, NHЈ), 6.07 (1 H, d, JN H ,2 9.1,
NH), 5.91 (1 H, dd, J2Ј,3Ј 10.7, J3Ј,4Ј 9.8, 3Ј-H), 5.58 (1 H, dd, J4Ј,5Ј
10.2, 4Ј-H), 5.35 (1 H, d, J3,4 2.6, 4-H), 5.12 (1 H, dd, J2,3 9.2, 3-
H), 5.09 (1 H, d, J1Ј,2Ј 7.6, 1Ј-H), 5.00 (1 H, d, J1,2 3.1, 1-H), 4.56
(1 H, dd, Fmoc CH), 4.49 (1 H, dd, 2-H), 4.48–4.36 (4 H, m,
Thr CHα, Thr CHβ, Fmoc CH2), 4.22 (2 H, m, 5- and 5Ј-H),
4.15 (1 H, m, 6Ј-Ha), 4.07 (1 H, m, 6Ј-Hb), 3.90 (1 H, dd, 2Ј-H),
3.87 (1 H, dd, 6-Ha), 3.62 (1 H, dd, 6-Hb), 2.08, 1.99, 1.93 and
1.89 (12 H, 4 s, 4 × COCH3) and 1.31 (3 H, d, JCH β,CH γ 6.6, Thr
CHγ) (Found: C, 63.1; H, 5.3; N, 3.5. C60H61N3O20 requires C,
63.0; H, 5.4; N, 3.7%).
2a
2b
12.2, Fmoc CH), 4.53 (1 H, d, JCH a,CH b 12.2, CHa Teoc), 4.51
(1 H, d, CHb Teoc), 4.46 (1 H, d, J3,4 3.0, 4-H), 4.39 (1 H,
dd, JCH α,CH β 2.0, JCH β,CH γ 6.6, Thr CHβ), 4.34–4.19 (4 H, m,
3-H, Thr CHα and Fmoc CH2), 4.10 (4 H, m, 6- and 6Ј-H2),
3.92 (1 H, dd, 2Ј-H), 3.90 (1 H, m, 5Ј-H), 3.88 (1 H, m,
5-H), 3.36 (1 H, dd, J2,3 8.1, 2-H), 1.51 (9 H, s, But), 1.47 (6
H, s, 2 × CH3) and 1.34 (3 H, d, Thr CHγ) (Found: C, 58.2;
H, 5.2; N, 5.6. C62H64Cl3N5O18 requires C, 58.5; H, 5.1; N,
5.4%).
N á-(Fluoren-9-ylmethoxycarbonyl)-O-{O-[3Ј,4Ј,6Ј-tri-O-
benzoyl-2Ј-deoxy-2Ј-(2,2,2-trichloroethoxycarbonylamino)-â-D-
glucopyranosyl]-(1Ј→6)-3,4-di-O-acetyl-2-azido-2-deoxy-á-
D-galactopyranosyl}-L-threonine tert-butyl ester 28
Synthesis of glycopeptides 31–90 on a manual 20-column peptide
synthesizer
A solution of disaccharide 27 (680 mg, 0.52 mmol) in a mixture
of acetic acid (16 cm3) and water (4 cm3) was stirred at 80 ЊC.
After 30 min (TLC light petroleum–ethyl acetate, 1:1), the solu-
tion was co-concentrated with toluene. To a solution of the
residue in dry pyridine (3 cm3) was added anhydride acetic
(1.5 cm3). After 1 h (TLC light petroleum–ethyl acetate, 2:1),
the solution was co-concentrated with toluene. The residue
was chromatographed on a silica gel column and eluted with
light petroleum–ethyl acetate (2:1) to give title compound 28
(612 mg, 87%), [α]D ϩ28.6 (c 1, CHCl3); δH (CDCl3) 8.02–7.28
(23 H, m, ArH), 5.84 (1 H, dd, J2Ј,3Ј 10.7, J3Ј,4Ј 9.7, 3Ј-H), 5.65
(1 H, d, JN H ,CH α 9.7, NH Thr), 5.58 (1 H, dd, 4Ј-H), 5.47 (1
H, d, J3,4 3.0, 4-H), 5.43 (1 H, d, JN H ,2Ј 9.1, NH Teoc), 5.35 (1
H, dd, J2,3 11.2, 3-H), 5.09 (1 H, d, J1,2 3.1, 1-H), 4.92 (1 H,
d, J1Ј,2Ј 7.6, 1Ј-H), 4.60 (1 H, dd, JCH ,CH 3.0, JCH ,CH 12.2,
The Wang resin (6.0 g) was placed in a glass reactor and swelled
in dichloromethane (15 cm3; 10 min). After washing of the resin
with dichloromethane, a mixture of Fmoc-Gly-OH (2.4 g, 8.1
mmol), 1-(mesitylenesulfonyl)-3-nitro-1,2,4-triazole (2.4 g, 8.1
mmol) and methylimidazole (504 mm3, 5.4 mmol) in dichloro-
methane (15 cm3) was added. After 2 h, the resin was washed
successively with dichloromethane and DMF (3×) and the
unchanged amino groups were acetylated with Ac2O–DMF
(1:7; 15 cm3). The resin was washed successively with dichloro-
methane and DMF (6×) and then dried.
The derivatized resin was weighed out and packed in the 20
columns of the manual synthesizer.15 All reagents were removed
by washing of the resins with DMF (10×). The Fmoc deprotec-
tions were performed by treatment with piperidine (20%) in
DMF (20 min). Each Fmoc amino acid Pfp ester and Dhbt-OH
(3 mol equiv., 1.5 mol equiv. for building block 24) and the
building blocks 6, 10, 16 and 30, TBTU and N-ethyldiiso-
propylamine (1.5 mol equiv.) were dissolved in DMF (0.75 cm3)
and the solutions were transferred to the respective wells. After
10–18 h, the reaction mixtures were removed and the wells were
washed with DMF. The synthesis cycle was repeated to com-
plete the assembly of each glycopeptide 31–90 by using the
appropriate amino acids. After removal of the last Fmoc
2a
2b
Fmoc CH), 4.52 (1 H, dd, JCH α,CH β 2.0, Thr CHα), 4.45 (1 H,
d, JCH a,CH b 12.2, CHa Teoc), 4.43 (1 H, dd, JCH β,CH γ 6.6, Thr
CHβ), 4.39 (2 H, m, Fmoc CH2), 4.35 (1 H, d, CHb Teoc),
4.30–4.22 (3 H, m, 5-H and 6Ј-H2), 4.07 (1 H, m, 5Ј-H), 3.89
(1 H, dd, 6-Ha), 3.78 (1 H, dd, 2Ј-H), 3.66 (1 H, dd, 6-Hb),
3.60 (1 H, dd, 2-H), 2.07 and 2.06 (6 H, 2 s, 2 × COCH3),
1.50 (9 H, s, But), 1.33 (3 H, d, Thr CHγ) (Found: C, 57.0;
H, 5.0; N, 5.4. C63H64Cl3N5O20 requires C, 57.4; H, 4.9;
N, 5.3%).
J. Chem. Soc., Perkin Trans. 1, 1997
2367

View Article Online
Table 3 Synthesized glycopeptides
Substance
Formula
Yield [mg (%)]
Substance
Formula
Yield [mg (%)]
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
C49H83N11O22
C49H83N11O22
C49H83N11O22
C49H83N11O22
C49H83N11O22
C57H96N12O27
C55H93N11O27
C55H93N11O27
C55H93N11O27
C55H93N11O27
C55H93N11O27
C69H116N12O37
C63H106N12O32
C63H106N12O32
C63H106N12O32
C63H106N12O32
C63H106N12O32
C85H142N14O47
C77H129N13O42
C77H129N13O42
C48H77N10O24
C48H77N10O24
C48H77N10O24
C54H87N13O29
C54H87N13O29
C54H87N13O29
C62H100N14O34
C62H100N14O34
C62H100N14O34
C57H96N12O27
4.8 (41)
3.8 (19)
2.6 (19)
3.8 (21)
4.3 (24)
5.6 (31)
6.6 (37)
8.4 (41)
9.3 (43)
6.6 (38)
7.5 (41)
10.0 (34)
8.2 (52)
7.8 (50)
6.5 (48)
6.8 (41)
9.8 (59)
14.2 (53)
12.1 (43)
8.6 (34)
4.6 (19)
7.2 (31)
4.2 (17)
5.6 (38)
6.1 (43)
7.8 (44)
6.8 (35)
4.3 (22)
4.8 (31)
9.4 (33)
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
C57H96N12O27
C57H96N12O27
C57H96N12O27
C57H96N12O27
C73H122N14O37
C65H109N13O32
C65H109N13O32
C65H109N13O32
C65H109N13O32
C65H109N13O32
C65H109N13O32
C65H109N13O32
C89H148N16O47
C81H135N15O42
C81H135N15O42
C57H96N12O27
C57H96N12O27
C57H96N12O27
C57H96N12O27
C57H96N12O27
C73H112N14O37
C56H90N14O29
C56H90N14O29
C56H90N14O29
C64H103N15O34
C64H103N5O34
C64H103N5O34
C56H90N14O29
C56H90N14O29
C56H90N14O29
4.1 (16)
11.0 (36)
7.0 (28)
7.8 (33)
12.1 (44)
9.0 (25)
7.9 (24)
7.6 (30)
9.4 (37)
10.1 (39)
8.3 (30)
11.2 (43)
10.7 (26)
8.8 (31)
9.7 (37)
7.5 (41)
8.4 (45)
6.6 (37)
7.8 (43)
5.7 (31)
8.1 (41)
2.3 (9)
3.4 (14)
7.8 (29)
8.1 (52)
6.4 (40)
7.7 (48)
5.6 (43)
4.8 (40)
6.0 (43)
4 R. S. Brealier, R. W. Rockwell, R. Dahiya and Y. S. Kim, Cancer
Res., 1990, 50, 1775.
5 I. Brockhausen, in Glycoproteins: Biosynthesis of O-glycans of
N-acetylgalactosamin-α-Ser/Thr linkage type, ed. J. Montreuil,
H. Schachter and J. F. G. Vliegenthart, Elsevier, Amsterdam, 1995,
p. 201.
6 S. Rio-Anneheim, H. Paulsen, M. Meldal and K. Bock, J. Chem.
Soc., Perkin Trans. 1, 1995, 1071.
7 T. Bielfeldt, S. Peters, M. Meldal, K. Bock and H. Paulsen, J. Chem.
Soc., Perkin Trans. 1, 1992, 1163.
8 H. Paulsen, T. Bielfeldt, S. Peters, M. Meldal and K. Bock, Liebigs
Ann. Chem., 1994, 369; 381.
9 H. Paulsen, S. Peters, T. Bielfeldt, M. Meldal and K. Bock,
Carbohydr. Res., 1995, 268, 17.
10 E. Meinjohanns, M. Meldal, A. Schleyer, H. Paulsen and K. Bock,
J. Chem. Soc., Perkin Trans. 1, 1996, 985.
11 R. R. Schmidt and W. Kinzy, Adv. Carbohydr. Chem. Biochem.,
1994, 50, 21.
groups, the resins were washed successively with DMF (8×) and
dichloromethane (5×), dried, and transferred to Eppendorf
tubes.
The resins were treated with 95% aq. TFA (2 cm3) for 2 h at
room temp., were then filtered off and washed with TFA (1 cm3,
3×). The solutions were concentrated, and co-distilled first with
toluene and then with toluene–methanol (3:1). The residues
were dissolved in methanol (1 cm3) and a solution of 1%
methanolic sodium methoxide was added until pH 7–8. The
reaction mixtures were stirred at room temp. for 6–9 h. The
solutions were neutralized with acetic acid, filtered, evaporated
and purified by preparative RP-HPLC [buffer A–buffer B
95:5→85:5 (20 min)→50:50 (30 min)]. The yields of
the substances are given in Table 3. All glycopeptides 31–90
1
were characterized by 1D and 2D H NMR spectroscopy, and
1
the H NMR data are available as supplementary material on
12 K. Frische, M. Meldal, O. Werdelin, S. Mouritsen, T. Jensen,
L. Galli-Stampino and K. Bock, J. Pept. Sci., 1996, 2, 212.
13 H. Paulsen, A. Schleyer, N. Mathieux, M. Meldal and K. Bock,
J. Chem. Soc., Perkin Trans. 1, 1997, 281.
14 H. Paulsen and M. Paal, Carbohydr. Res., 1984, 135, 53.
15 M. Meldal, C. B. Holm, G. Bojesen, M. H. Jacobsen and A. Holm,
Int. J. Pept. Protein Res., 1993, 41, 250.
request.
Acknowledgements
This research work was supported by EU SCIENCE program
(grant SCI*-CT92-0765). We thank Dr V. Sinnwell for record-
ing the NMR spectra.
16 R. B. Wang, J. Am. Chem. Soc., 1972, 94, 1328.
17 H. Paulsen and K. Adermann, Liebigs Ann. Chem., 1989, 751.
References
1 M. Verma and E. A. Davidson, Glycoconjugate J., 1994, 11, 172.
2 J. Taylor-Papadimitriou and A. A. Epenetos, TIB TECH, 1994, 12,
227.
3 Y. S. Kim, J. Gum, Jr. and I. Brockhausen, Glycoconjugate J., 1996,
13, 693.
Paper 7/01742A
Received 13th March 1997
Accepted 16th April 1997
2368
J. Chem. Soc., Perkin Trans. 1, 1997