Synthesis of multiantennary complex type N-Glycans by use of modular building blocks

Article abstract of DOI:10.1002/chem.200901908

A modular set of oligosaccharide building blocks was developed for the synthesis of multiantennary Nglycans of the complex type, which are commonly found on glycoproteins. The donor building blocks were laid out for the elongation of a core trisaccharide

Full text of DOI:10.1002/chem.200901908

DOI: 10.1002/chem.200901908
Synthesis of Multiantennary Complex Type N-Glycans by Use of Modular
Building Blocks
Carlo Unverzagt,* Gislinde Gundel, Steffen Eller, Ralf Schuberth, Joachim Seifert,
Harald Weiss, Mathꢀus Niemietz, Matthias Pischl, and Claudia Raps[a]
In memory of Professor Peter Welzel
Abstract: A modular set of oligosac-
charide building blocks was developed
for the synthesis of multiantennary N-
glycans of the complex type, which are
commonly found on glycoproteins. The
donor building blocks were laid out for
the elongation of a core trisaccharide
acceptor (b-mannosyl chitobiose) con-
veniently protected with a single ben-
zylidene moiety at the b-mannoside.
Through two consecutive regio- and
stereoselective couplings the donors
gave N-glycans with three to five an-
tennae in high yields. Due to the con-
sistent protection group pattern of the
donors the deprotection of the final
products can be performed by using a
general reaction sequence.
Keywords:
glycoproteins
carbohydrates
· glycosylation
·
·
oligosaccharides · regioselectivity
Introduction
identified N-glycans[2] and the demand for N-glycans on gly-
coarrays[12] we have developed a versatile set of building
blocks that allow the synthesis of the most frequent core
structures of complex type N-glycans.[13] Based on the pio-
neering work of Ogawa[14] and Paulsen[15] we have estab-
lished a robust and general method for the double regio-
and stereoselective glycosylation[16] of differently functional-
ized core trisaccharides,[17,18] each equipped with a benzyli-
dene protected b-mannose as a key component. After opti-
mizing this approach for biantennary N-glycans[19] the build-
ing blocks were modified for the convergent synthesis of
multiantennary N-glycans[20] with up to five branches.[13] Sev-
eral groups have developed a variety of additional strategies
for the chemical synthesis of N-glycans, which can be con-
ducted in solution[21–30] or on solid phase.[31–33]
The rapidly growing demand for recombinant therapeutic
glycoproteins[1] has improved the analytical tools[2–4] capable
of determining the vast number of structures of N-glycans
present in biological material. At the same time glycobiolo-
gists have revealed more details about the interplay of pro-
tein N-glycosylation[5] with cellular functions. Due to the mi-
croheterogeneity of N-glycoproteins only a few of these oli-
gosaccharides can be isolated from natural sources[6,7] in
more than analytical amounts and thus the biological roles
of N-glycans remain difficult to elucidate.[8] These circum-
stances have stimulated the chemical synthesis of N-glycans
as a method to provide sufficient quantities for biological
testing by using classical approaches[9] or miniaturized meth-
ods.[10] Seminal work utilizing chemically synthesized multi-
antennary fragments of N-glycans has revealed a multivalen-
cy effect for the asialoglycoprotein receptor, which also
shows preferences for certain types of branched N-gly-
cans.[11] In order to keep up with the growing number of
Results and Discussion
Based on the concept of modularizing the building blocks
for the antennae of branched N-glycans a robust and scal-
AHCTUNGTREGaNNUN ble synthesis for the donors C, D and E was developed
(Scheme 1). A seamless compatibility of these branched oli-
gosaccharide donors with the double regio- and stereoselec-
tive glycosylation approach for biantennary N-glycans was
attempted. The flexible use of key intermediates should thus
be provided and the chemical synthesis of the most frequent
patterns of branched N-glycans of the complex type should
[a] Prof. C. Unverzagt, Dr. G. Gundel, Dr. S. Eller, Dr. R. Schuberth,
Dr. J. Seifert, Dr. H. Weiss, M. Niemietz, M. Pischl, C. Raps
Bioorganische Chemie, Universitꢀt Bayreuth
Gebꢀude NW1, 95440 Bayreuth (Germany)
Fax : (+49)921-55-5365
E-mail: carlo.unverzagt@uni-bayreuth.de
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FULL PAPER
could be transferred to the
building blocks C, D and E by
adopting the protecting group
pattern of B and the anomeric
activation by using trichloro-
AHCTUNGTRENNUNG
acetimidates.[38] The general
use of acetyl or phthalimido
groups in these compounds
should ultimately facilitate
global deprotection of the final
N-glycans. Since the regioselec-
tive protection required benzyl
groups or other ethers in the
mannosyl part of the donors,
which might lead to b-linked
side products in the following
glycosylations,[14] these ethers
were generally replaced by
acetates in the donors C, D
and E (Scheme 1).
The synthesis of the 2,4-
branched donor C commenced
with the diallylated acceptor 5
(Scheme 2), which was initially
obtained by regioselective tri-
A
as
de-
scribed.[39] However, due to the
large number of side products
and the toxic properties of the
volatile reagent bistributyltin
oxide an alternative procedure
was sought. A solution was
found in a synthesis through
dibutylstannylene
and by employing the far less
toxic polymeric dibutyltin
acetals[40]
oxide in conjunction with
CsF,[41] which increases the
yield in the subsequent alkyla-
tion step. This approach
showed the desired 3,6-regiose-
lectivity and furnished the di-
Scheme 1. Flexible chemical synthesis of multiantennary complex type N-glycans can be accomplished by
using the modular set of oligosaccharide donors B–E. These building blocks react with the core trisaccharide
A in a double regio- and stereoselective coupling procedure. The cartoon represents the most frequent com-
plex type N-glycans found in vertebrates and a retrosynthetic disconnection of the penta-antennary structure
highlighted in a box.
AHCTUNGTRENNUNG
be facilitated. The antennae of these synthetic N-glycans
generally terminate with GlcNAc residues, which can be li-
berated by chemical deprotection and elongated by enzy-
matic glycosylation reactions by using glycosyltransferas-
es.[34] At the reducing end the synthetic N-glycans carry an
azido group, which allows rapid conjugation to peptides[35]
or linkers.[36]
sylate the acceptor 5 by using the trichloroacetimidate 2[38]
under activation with BF3·OEt2 gave the trisaccharide 7 in
35% yield whereas activation with TMS-triflate at ꢀ408C
raised the yield to 75%. A further increase was achieved
with the b-fluoride 1[42] to give the trisaccharide 7 in 81%
yield.
Subsequently, trisaccharide 7 was deallylated with PdCl2
in acetic acid at 708C. When attempting to acetylate the in-
termediate 3,6-diol with a 2:1 mixture of pyridine and acetic
anhydride, it was found that only the primary OH-6 reacted,
even when higher temperatures (808C) were applied. How-
ever, after adding DMAP[43] the very unreactive OH-3
group was slowly acetylated. The resulting trisaccharide 8
Synthesis of the branched donors C, D and E: When plan-
ning the synthesis of the three branched donors C, D and E
we aimed on maintaining the favorable reactivity and selec-
tivity found for the elongation of core trisaccharide A with
donor B.[19,37] It was assumed that the desired properties
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C. Unverzagt et al.
Scheme 2. a) 1) Bu2SnO, MeOH; 2) AllBr, CsF, DMF; 1)–2) 58%; b) 1) Bu2SnO, MeOH; 2) AllBr, CsF, DMF; 1)–2) 69%; c) 1+5, BF3·OEt2, CH2Cl2,
81%; d) 1) AcOH (95%), PdCl2, NaOAc, 708C; 2) Ac2O, pyridine, DMAP; 1)–2) 71%; e) 1) Pd/C, MeOH, AcOH; 2) trichloroacetonitrile, DBU,
CH2Cl2; 1)–2) 80%; f) 2+6, BF3·OEt2, CH2Cl2, ꢀ208C, 53%; g) 1) Pd/C, MeOH, AcOH; 2) Ac2O, pyridine; 1)–2) 70%; h) 1) hydrazine acetate, DMF;
2) trichloroacetonitrile, DBU, CH2Cl2; 1)–2) 68%; i) 1+4, BF3·OEt2, CH2Cl2, ꢀ108C, 89%; j) PdCl2, MeOH, 77%; k) Ac2O, pyridine, DMAP, 81%;
l) PdO/H2O, MeOH, AcOH, 82%; m) trichloroacetonitrile, DBU, CH2Cl2, 85%.
was debenzylated by catalytic hydrogenation, which gave
higher yields than the procedure with anhydrous FeCl3.[44]
Finally, the hemiacetal was converted to the imidate and tri-
saccharide donor C was isolated in 80% yield over two
steps.
ꢀ108C gave the tetrasaccharide 11 in 89% yield. Removal
of the allyl group was either carried out by using PdCl2 in
the presence of NaOAc in acetic acid (73% yield) or in an-
hydrous methanol at ambient temperature (77% yield) to
furnish compound 12. The latter conditions appear more
robust and less prone to side reactions. In analogy to the
synthesis of 8, the tetrasaccharide 12 also required the addi-
tion of DMAP to achieve complete acetylation; this indi-
cates a similarly low accessibility of OH-3. The fully protect-
ed compound 12a was debenzylated and the resulting hemi-
acetal was converted to the tetrasaccharide imidate E.
The synthesis of the 2,6-branched donor D was carried
out in analogy to the route described by Arnarp et al. by
using the benzylated acceptor 6.[45] Surprisingly, when at-
tempting the double glycosylation of 6 with the fluoride 1
about 10–20% of an a configured side product were ob-
tained, which could not be separated by flash chromatogra-
phy. However, coupling of the trichloroacetimidate 2 by
using borontrifluoride etherate at ꢀ208C gave the desired
trisaccharide 9 in 53% yield without this side reaction. On a
small scale the yield was raised to 60% by keeping the reac-
tion temperature between ꢀ40 and ꢀ508C. The trisacchar-
ide 9 was debenzylated by catalytic hydrogenation and ace-
tylated. Acetate 10 was selectively deprotected at the reduc-
ing end and converted to the trisaccharide imidate D.
For tetrasaccharide donor E the 3-O-allylated benzylman-
noside 4 was chosen as an acceptor. Compound 4 can be iso-
lated as a side product (23%) in the improved synthesis of 5
as shown above. By modifying the procedure this approach
might also be useful to obtain 4 as the major product. This
was achieved by selectively activating 3[46] by using only one
equivalent of dibutyltin oxide[47] followed by addition of al-
lylbromide and CsF in DMF, which resulted in a yield of
69% of the 3-O-monoallylated acceptor 4. A threefold gly-
cosylation of the triol 4 was attempted by treatment with
the fluoride donor 1. After optimizing the conditions it was
found that the use of six equivalents of the donor 1 at
Regioselective glycosylations leading to N-glycans: With the
donors C, D and E in hand the glycosylation of the core tri-
saccharide A and its coupling products was investigated.
Starting with the building blocks A and B the pentasacchar-
ide diol 13 was obtained after BF3·OEt2 mediated coupling
followed by acetylation and removal of the benzylidene
acetal.[19,48] Acceptor 13 was employed in a coupling reaction
with the branched donor D (Scheme 3). As desired a regio-
and stereoselective glycosylation occurred at the primary
OH-63 to furnish the triantennary octasaccharide N-glycan
14 in 91% yield. The initially reported yield[20] of 76% was
improved by increasing the concentration and raising the
starting temperature. Double glycosylation[19] was not found
under these conditions. The resulting downfield shift for the
13C NMR signal of C-63 (60.4 ppm for 13 vs. 66.3 ppm for
14) indicated glycosylation at OH-63. The newly formed a-
mannosidic linkage was confirmed after determination of
JC-1,H-1 for C-14ꢂ with a value of 176.2 Hz, which is typical for
a-mannosides.
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Synthesis of Multiantennary Complex Type N-Glycans
FULL PAPER
steric hindrance were found in these couplings. However,
when using the tribranched donor E the initial yields of 20
were quite low (ꢁ30%). Thus, the conditions were system-
atically optimized and it was found that higher yields could
be obtained by increasing the amounts of donor E and acti-
vator (BF3). The reaction showed temperature depenACHTUNGTRENNUNGdency
with an optimum at ꢀ108C, which provided the penta-an-
tennary decasaccharide 20 in 65% yield. Relative to the less
branched donors B and D the tetrasaccharide donor E gave
lower yields in the coupling with the hexasaccharide accept-
or 17 even after optimization. This indicates steric hindrance
for donor E due to the high degree of branching in conjunc-
tion with the three sterically demanding peripheral phthali-
mido groups.[13]
Scheme 3. The linear pentasaccharide acceptor 13 can be converted to
tri- or tetra-antennary N-glycans by regioselective coupling with donors
D or E.
We were pleased to see that even the bulky 2,4,6-substi-
tuted tetrasaccharide donor E reacted with the pentasac-
charide acceptor 13 selectively to give the tetra-antennary
nonasaccharide 15 in 63% yield under nonoptimized condi-
tions.
The incorporation of a branched a1,3-arm was achieved
by treating donor
C with the core trisaccharide A
(Scheme 4). This glycosylation also proceeded regio- and
stereoselectively (JC-1,H-1 =172.7 Hz for C-14) and gave hexa-
saccharide 16 in high yield (93%). After acetylation of the
OH-23 group the product was debenzylidenated to give hex-
asaccharide diol 17 in 81% over two steps. It is worth men-
tioning that the debenzylidenation reaction leading to the
branched hexasaccharide 17 should be conducted under less
acidic conditions compared to the analogous procedure for
the linear derivative 13, otherwise an unwanted deacetyla-
tion occurs. Glycosylation of 17 was investigated with the
three donors B, D and E. Selective coupling of the linear
donor B with the branched hexasaccharide diol 17 gave the
desired a1,6-linked octasaccharide 18 in 78% yield. When
using the branched donor D the tetra-antennary nonasac-
charide 19 was obtained in similar yield (88%). Regio- and
stereoselectivity were excellent in both cases and no signs of
Scheme 4. The branched hexasaccharide intermediate 17 can be convert-
ed to tri-, tetra- or penta-antennary N-glycans by regioselective coupling
with donors B, D or E.
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C. Unverzagt et al.
Avance 360, AMX 500 and DMX 500 instruments. Coupling constants
are reported in Hz. For mass spectra a Varian CH5 instrument was used
in the fast atom bombardment mode (FAB) with an m-nitrobenzylalco-
hol matrix (NBA). ESI-TOF mass spectra were recorded on a Micromass
LCT instrument coupled to an Agilent 1100 HPLC. Flash chromatogra-
phy was performed on silica gel 60 (230–400 mesh; Merck, Darmstadt,
Germany). The reactions were monitored by thin layer chromatography
on coated aluminum plates (silica gel 60 GF254; Merck, Darmstadt, Ger-
many). Spots were detected by UV-light or by charring with a 1:1 mixture
of H2SO4 (2n) and resorcine monomethylether (0.2%) in ethanol.
Deprotection of the synthetic multiantennary N-glycans is
achieved readily in a one-pot procedure.[34,48] Concomitant
deacetylation and dephthaloylation can be carried out by
heating the protected N-glycans with ethylenediamine in n-
butanol.[49] Under these conditions the anomeric azide re-
mains unaffected. Global acetylation and O-deacetylation
by using aqueous methylamine gives the deprotected N-gly-
cans. For the tetra-antennary nonasaccharide 19 this three-
step procedure was conducted in a one-pot manner followed
by a simple workup by using solid phase extraction on a
C18 SepPak (Waters) cartridge to furnish compound 21 in
86% yield (Scheme 5). The selective removal of benzyl
groups in the presence of an anomeric azide is not straight-
forward. Preferably after reduction of the anomeric azide to
an amine a spacer is attached and the benzyl groups can be
removed subsequently by catalytic hydrogenation. The N-
glycan spacer conjugates thus obtained, were used as accept-
ors for enzymatic elongation and for the synthesis of neogly-
coproteins.[34]
Benzyl 3-O-allyl-a-d-mannopyranoside (4) A mixture of benzylmanno-
side 3 (200 mg, 0.74 mmol) and dibutyltin oxide (184 mg, 0.74 mmol) was
stirred under reflux in absolute methanol (4 mL) for 4 h resulting in a
clear solution. The solvent was evaporated in vacuo followed by addition
of caesium fluoride (169 mg, 1.3 mmol) to the solid remainder, which was
dried under high vacuum for 16 h. The mixture was suspended in dry di-
methylformamide (1 mL) and allyl bromide (0.33 mL, 3.75 mmol) was
added at 08C and the solution was vigorously stirred at room tempera-
ture for 22 h. The reaction was quenched by addition of ethyl acetate
(2.4 mL) and water (40 mL). After filtration over Celite the solution was
concentrated in vacuo and purified by flash chromatography (hexane/ace-
tone, 1.5:1) to afford
4 (158 mg, 68.8%); Rf =0.10 (hexane/acetone
1
1.2:1); [a]2D3 =+108.0 (c=0.8, CH2Cl2); H NMR (360 MHz, [D6]DMSO):
ꢀ
d=7.47–7.20 (m, 5H, Ph), 5.90 (m, 1H, =CH ), 5.28 (dd, Jtrans =17.4 Hz,
J
gem =1.8 Hz, 1H, H2C=trans), 5.09 (dd, Jcis =10.4 Hz, Jgem =1.6 Hz, 1H,
H2C=cis), 4.91 (d, JOH,4 =6.1 Hz, 1H, OH-4), 4.79 (d, JOH,2 =4.9 Hz, 1H,
OH-2), 4.70 (d, J1,2 =1.3 Hz, 1H, H-1), 4.65 (d, Jgem =11.8 Hz, 1H, CH2O-
Bn), 4.56 (t,
JOH,6 =6.3 Hz, 1H, OH-6), 4.42 (d, Jgem =11.8 Hz, 1H,
CH2O-Bn), 4.09–4.03 (m, 2H, CH2O-All), 3.82 (m, J1,2 <1 Hz, J2,3 <1 Hz,
1H, H-2), 3.86 (m, 1H, H-6a), 3.52–3.33 (m, 4H, H-4, H-6b, H-5, H-3);
13
ꢀ
C NMR (90 MHz, [D6]DMSO): d=137.8 (C-i Ar), 136.1 (=CH ), 128.3,
127.9, 127.6 (C-Ar), 115.9 (=CH2), 99.0 (C-1), 79.0 (C-3), 74.4 (C-5), 70.3
(CH2O-All), 67.5 (CH2O), 67.1 (C-2), 66.2 (C-4), 61.2 (C-6); ESI-MS:
m/z calcd for C16H22O6: 310.14; found 333.5 [M+Na]+.
Benzyl 3,6-di-O-allyl-a-d-mannopyranoside (5): A mixture of benzyl-
mannoside
3
(12.4 g, 45.9 mmol) and dibutyltin oxide (34.3 g,
Scheme 5. One-pot deprotection of the tetra-antennary N-glycan 19.
137.7 mmol) was stirred under reflux in absolute methanol (300 mL) for
4 h resulting in a clear solution. The solvent was evaporated and dried
under high vacuum for 16 h. Cesium fluoride (20.9 g, 137.7 mmol) and
dry dimethylformamide (200 mL) were added to the remainder. The sus-
pension was stirred at 08C for 10 min and allyl bromide (40 mL,
459 mmol) was added and vigorously stirred at room temperature for
4 days. The reaction was quenched by addition of ethyl acetate (480 mL)
and water (8 mL). After filtration over Celite the solution was concen-
trated in vacuo and purified by flash chromatography (cyclohexane/ace-
tone 5:1) to afford 5 (9.3 g, 57.9%). Rf =0.65 (hexane/acetone 1:1).
Conclusions
In summary we have developed a modular set of building
blocks that can be used in a generally applicable double
regio- and stereoselective glycosylation approach for multi-
antennary N-glycans from two to five antennae. The yields
of the convergent glycosylation reactions were high and in
all cases only the formation of desired stereo- and re-
gioisomers was found. In the case of the tri- and tetra-an-
tennary products the glycosylation reactions were free of
steric hindrance. This demonstrates a high compatibility of
the donors with the corresponding acceptors. These donors
were successfully applied in the synthesis of other multian-
tennary N-glycans bearing additional core substituents.[13,50]
After one-pot deprotection the N-glycans can be further
modified and incorporated into neoglycoproteins.[39]
Benzyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!2)-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!4)]-3,6-di-O-allyl-a-d-mannopyranoside (7): A suspension of ac-
ceptor 5 (5.25 g, 15 mmol), fluoride 1 (20.1 g, 46 mmol) and ground mo-
lecular sieves 4 ꢃ (21 g) in absolute CH2Cl2 (215 mL) was stirred at am-
bient temperature for 30 min. Boron trifluoride ethyl etherate (1.0 mL,
8 mmol) was added over a period of 5 min. After 3 h (TLC: hexane/ace-
tone 1:2) the mixture was filtered over Celite, washed with CH2Cl2 and
extracted with dilute KHCO3. The organic phase was dried over MgSO4,
concentrated and purified by flash chromatography (hexane/acetone,
1.7:1) to afford 7 (14.3 g, 80.6%); Rf =0.32 (hexane/acetone 2:1); [a]D23
=
+33.8 (c=1, CH2Cl2); 1H NMR (250 MHz, [D6]DMSO): d=8.0–7.6 (m,
ꢀ
8H, Pht), 7.35–7.15 (m, 5H, Ar), 6.0–5.84 (m, 1H, =CH ), 5.60 (dd, J2,3
10.8 Hz, J3,4 =9.2 Hz, 1H, H-3’), 5.57 (dd, J2,3 =10.6 Hz, J3,4 =9.2 Hz, 1H,
H-3’’), 5.50 (d, J1,2 =8.5 Hz, 1H, H-1’’), 5.45 (d, J1,2 =8.5 Hz, 1H, H-1’),
=
ꢀ
5.22 (m, 1H, H2C=trans), 5.17–5.07 (m, 2H, =CH , H2C=cis), 5.01 (dd,
Experimental Section
J
4,5 =8.5 Hz, 1H, H-4’), 4.97 (dd, J4,5 =8.6 Hz, 1H, H-4’’), 4.81 (m, 1H,
H2C=cis), 4.67 (m, 1H, H2C=trans), 4.52 (d, J1,2 =1.7 Hz, 1H, H-1), 4.38
(d, Jgem =11.6 Hz, 1H, CH2O), 4.26–3.85 (m, 12H, CH2O, H-2, H-2’, H-
2’’, H-5’, H-5’’, H-6a,b’, H-6a,b’’), 3.61 (dd, J2,3 =3.2 Hz, J3,4 =7.9 Hz, 1H,
H-3), 3.34 (m, 1H, H-4), 3.26 (m, 1H, H-5), 2.91 (m, 2H, CH2O), 2.80
(dd, Jgem =10.7 Hz, Jvic <1.0 Hz, 1H, H-6a), 2.49 (m, 1H, H-6b), 2.09,
General methods: Solvents were dried according to standard methods.
Molecular sieves were activated prior to use by being heated under high
vacuum. Optical rotations were measured on a Perkin–Elmer 241 polar-
imeter at 589 nm. NMR spectra were recorded on Bruker AC 250,
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Synthesis of Multiantennary Complex Type N-Glycans
FULL PAPER
2.04, 2.00, 1.99, 1.79, 1.76 (6s, 18H, OAc); 13C NMR (62.5 MHz,
[D6]DMSO): d=169.9, 169.5, 169.2, 167.3 (C=O), 136.8 (C-i Ar), 135.5
5’’), 4.0–3.94 (m, 3H, H-2’’, H-5’, H-6b’), 3.84 (dd, Jgem =12.0 Hz, Jvic
1.0 Hz, 1H, H-6b’’), 3.79 (dd, J3,4 =J4,5 =9.6 Hz, 1H, H-4), 3.72 (dd, Jgem
<
=
ꢀ
ꢀ
(=CH ), 134.9 (C-4/5 Pht), 134.2 (=CH ), 130.6 (C-1/2 Pht), 128.2, 127.8,
127.6 (C-Ar), 123.5 (C-3/6 Pht), 116.2, 115.7 (CH2=), 97.2 (C-1’’), 96.0 (C-
1), 95.8 (C-1’), 75.6 (C-3), 74.1 (C-4), 72.4 (C-2), 70.8 (C-5’, C-5’’), 70.4
(CH2O-All), 69.9 (C-5, C-3’, C-3’’), 68.8 (CH2O-All), 68.6 (CH2O-Bn, C-
4’), 68.2 (C-4’’), 67.7 (C-6), 61.8 (C-6’), 61.5 (C-6’’), 54.7 (C-2’’), 53.8 (C-
2’), 20.3, 20.03, 19.95 (OAc); FAB-MS (NBA): m/z calcd for
C59H64N2O24: 1184.4; found 1191.4 [M+Li]+, 1207.6 [M+Na]+; elemental
analysis calcd (%) for C59H64N2O24 (1185.15): C 59.79, H 5.44, N 2.36;
found: C 59.79, H 5.44, N 2.36.
11.5 Hz, Jvic <1.0 Hz, 1H, H-6a), 3.62 (m, 1H, H-5), 3.16 (m, 1H, H-6b),
2.04, 2.03, 2.00, 1.98, 1.97, 1.77, 1.73, 1.44 (8s, 24H, OAc); 13C NMR
(125 MHz, [D6]DMSO): d=170.13, 170.09, 169.7, 169.6, 169.4, 169.2,
168.9, 167.5 (C=O), 156.8 (C=NH), 135.2, 134.9 (C-4/5 Pht), 130.4 (C-1/2
Pht), 123.8, 123.5 (C-3/6 Pht), 96.9 (C-1’’), 96.5 (C-1’), 93.5 (C-1), 90.0
(CCl3), 72.3 (C-2), 71.9 (C-4), 71.0 (C-5’), 70.8 (C-5’’), 69.9 (C-3’, C-5),
69.7 (C-3, C-3’’), 68.4 (C-4’), 68.3 (C-4’’), 61.8 (C-6’’), 61.7 (C-6’), 61.2 (C-
6), 54.5 (C-2’’), 53.8 (C-2’), 20.44, 20.40, 20.1, 20.0, 19.5 (OAc); ESI-MS:
m/z calcd for C52H54Cl3N3O26: 1241.21; found 1264.13 [M+Na]+.
Benzyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!2)-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!4)]-3,6-di-O-acetyl-a-d-mannopyranoside (8): Palladium chlo-
ride (2.8 g, 15.8 mmol) and sodium acetate (2.8 g, 34 mmol) were added
to a solution of trisaccharide 7 (8.0 g, 6.75 mmol) in acetic acid (530 mL,
95%). The reaction was stirred for 2.5 h at 708C. Subsequently, the pre-
cipitate was filtered off and the solvents were removed on a rotary evap-
orator. The remainder was dissolved in CH2Cl2 (300 mL) and extracted
with water (2ꢄ) and dilute KHCO3. The organic phase was dried over
MgSO4 and concentrated. Pyridine (50 mL), acetic anhydride (25 mL)
and p-dimethylaminopyridine (DMAP, 1 g) were added to the residue.
After 24 h the mixture was concentrated, dissolved in CH2Cl2 (300 mL)
and extracted with dilute HCl (3ꢄ) and dilute KHCO3. The organic
phase was dried over MgSO4, concentrated and purified by flash chroma-
tography (hexane/ethyl acetate 1:2) to afford 8 (5.73 g, 71.4%). Rf diol=
Benzyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!2)-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!6)]-3,4-di-O-benzyl-a-d-mannopyranoside (9): A suspension of
diol 6 (3.68 g, 8.2 mmol), imidate 2 (19.8 g, 34.2 mmol) and ground mo-
lecular sieves 4 ꢃ (10 g) in absolute CH2Cl2 (150 mL) was stirred for
60 min at ꢀ208C. Boron trifluoride ethyl etherate (1.0 mL, 8 mmol) was
added over 5 min. After 2 h (TLC: hexane/ethyl acetate 1:2) the reaction
was quenched with triethylamine (1.0 mL). The solids were filtered over
Celite and washed with CH2Cl2. The organic phase was washed with
dilute KHCO3, dried over MgSO4 and concentrated. The remainder was
purified by flash chromatography (hexane/ethyl acetate, 0.75:1) to afford
9 (5.58 g, 53.2%). Rf =0.45 (hexane/ethyl acetate 1:2); [a]2D3 =+5.7 (c=
0.5, CH2Cl2); 1H NMR (500 MHz, [D6]DMSO): d=7.9–7.6 (m, 8H, Pht),
7.3–6.95 (m, 15H, Ar), 5.68 (dd, J2,3 =10.3 Hz, 1H, H-3’), 5.61 (dd, J2,3
10.3 Hz, 1H, H-3’’), 5.40 (d, J1,2 =8.5 Hz, 1H, H-1’), 5.01 (dd, J3,4 =J4,5
9.7 Hz, 1H, H-4’), 4.93 (dd, J3,4 =J4,5 =9.7 Hz, 1H, H-4’’), 4.87 (d, J1,2
8.3 Hz, 1H, H-1’’), 4.55 (d, Jgem =11.4 Hz, 1H, CH2O), 4.54 (d, Jgem
=
=
=
=
0.21 (hexane/acetone 1:1); Rf acetate=0.30 (hexane/acetone 1:1); [a]D23
=
+15.6 (c=1, CH2Cl2); 1H NMR (500 MHz, [D6]DMSO): d=7.95–7.63
(m, 8H, Pht), 7.35–7.15 (m, 5H, Ar), 5.63 (dd, J2,3 =10.5 Hz, 1H, H-3’’),
5.60 (dd, J2,3 =10.8 Hz, 1H, H-3’), 5.40 (d, J1,2 =8.4 Hz, 1H, H-1’’), 5.35
(d, J1,2 =8.5 Hz, 1H, H-1’), 4.98 (dd, J3,4 =J4,5 =9.6 Hz, 1H, H-4’), 4.96
(dd, J3,4 =J4,5 =9.6 Hz, 1H, H-4’’), 4.81 (dd, J2,3 =3.5 Hz, J3,4 =9.3 Hz, 1H,
H-3), 4.37 (d, Jgem =11.4 Hz, 1H, CH2O), 4.33 (d, J1,2 <1.0 Hz, 1H, H-1),
4.24–4.18 (m, 3H, CH2O, H-6a’, H-6a’’), 4.08–4.03 (m, 3H, H-2, H-2’, H-
11.0 Hz, 1H, CH2O), 4.42 (d, J1,2 <1 Hz, 1H, H-1), 4.33 (d, Jgem =11.3 Hz,
1H, CH2O), 4.23–4.18 (m, 3H, CH2O, H-6a’, H-6a’’), 4.15–4.07 (m, 3H,
H-2, H-2’, H-6b’), 4.03–3.98 (m, 3H, CH2O, H-5’, H-6b’’), 3.94 (dd, 1H,
H-2’’), 3.85 (m, 1H, H-5’’), 3.72 (d, Jgem =11.3 Hz, 1H, CH2O), 3.55 (m,
2H, H-3, H-6a), 3.28 (m, 1H, H-5), 3.03 (dd, J3,4 =J4,5 =9.8 Hz, 1H, H-4),
2.45 (m, 1H, H-6b), 2.01, 2.00, 1.98, 1.95, 1.81, 1.77 (6s, 18H, OAc);
13C NMR (125 MHz, [D6]DMSO): d=170.0, 169.6, 169.3, 167.1 (C=O),
138.1, 137.9, 136.5 (C-i Ar), 134.9 (C-4/5 Pht), 130.4 (C-1/2 Pht), 128.2–
127.3 (C-Ar), 123.4 (C-3/6 Pht), 97.6 (C-1’’), 95.4 (C-1’), 95.2 (C-1), 77.4
(C-3), 73.8 (C-4, CH2O), 72.1 (C-2), 71.0 (C-5’), 70.9 (C-5’’), 70.5 (C-5),
69.8 (C-3’, C-3’’), 69.6 (CH2O), 69.3 (C-6), 68.6 (C-4’), 68.6 (C-4’’), 67.7
(CH2O), 61.8 (C-6’), 61.6 (C-6’’), 54.1 (C-2’’), 54.0 (C-2’), 20.44, 20.40,
20.16, 20.06 (OAc); ESI-MS: m/z calcd for C67H68N2O24: 1284.42; found
1307.65 [M+Na]+; elemental analysis calcd (%) for C67H68N2O24
(1285.27): C 62.61, H 5.33, N 2.18; found: C 62.69, H 5.40, N 2.01.
5’’), 4.0–3.94 (m, 3H, H-2’’, H-5’, H-6b’), 3.84 (dd, Jgem =10.8 Hz, Jvic
<
1.0 Hz, 1H, H-6b’’), 3.69–3.62 (m, 2H, H-4, H-6a), 3.41 (m, 1H, H-5),
3.06 (dd, Jgem =11.8 Hz, Jvic =3.7 Hz, 1H, H-6b), 2.04, 2.02, 2.00, 1.98,
1.97, 1.77, 1.73, 1.48 (8s, 24H, OAc); 13C NMR (125 MHz, [D6]DMSO):
d=170.0, 169.5, 169.2, 169.0, 167.2 (C=O), 136.6 (C-i Ar), 135.1, 134.8
(C-4/5 Pht), 130.4 (C-1/2 Pht), 123.7, 123.3 (C-3/6 Pht), 96.77 (C-1’’),
96.42 (C-1’), 96.35 (C-1), 73.6 (C-2), 72.3 (C-4), 70.8 (C-5’), 70.6 (C-5’’),
70.4 (C-3), 69.8 (C-3’), 69.7 (C-3’’), 69.0 (CH2O), 68.6 (C-4’), 68.2 (C-4’’),
67.5 (C-5), 61.9 (C-6’), 61.7 (C-6’’), 61.6 (C-6), 54.6 (C-2’’), 53.8 (C-2’),
20.5, 20.41, 20.36, 20.1, 20.0, 19.6 (OAc); ESI-MS: m/z calcd for
C57H60N2O28: 1188.34; found 1211.81 [M+Na]+; elemental analysis calcd
(%) for C57H60N2O28 (1189.1): C 57.58, H 5.09, N 2.36; found: C 57.37, H
5.13, N 2.13.
Acetyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!2)-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!6)]-3,4-di-O-acetyl-a-d-mannopyranose (10): Trisaccharide
9
(4.5 g, 3.5 mmol) was dissolved in methanol (100 mL). After addition of
acetic acid (1.0 mL) and Pd/C (10%, 5 g) the mixture was stirred under a
hydrogen atmosphere. After complete reaction (TLC: hexane/acetone
1:1) the catalyst was filtered off and the solvents were removed in
vacuum. The remainder was treated with pyridine (10 mL) and acetic an-
hydride (5 mL) and concentrated in vacuum after 2 h. The residue was
codistilled with toluene and purified by flash chromatography (hexane/
acetone 1:1) to afford 10 (2.78 g, 69.6%). Rf =0.13 (hexane/acetone 1:1);
[a]2D3 =ꢀ0.4 (c=0.5, CH2Cl2); 1H NMR (500 MHz, [D6]DMSO): d=7.95–
7.77 (m, 8H, Pht), 5.64 (dd, J2,3 =10.5 Hz, 1H, H-3’), 5.50–5.46 (m, 2H,
H-1, H-3’’), 5.42 (d, J1,2 =8.5 Hz, 1H, H-1’), 5.06 (d, J1,2 =8.5 Hz, 1H, H-
1’’), 4.99 (dd, J3,4 =J4,5 =9.5 Hz, 1H, H-4’), 4.91 (dd, J3,4 =J4,5 =9.7 Hz, 1H,
O-{O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!2)-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!4)]-3,6-di-O-acetyl-a-d-mannopyranosyl}-trichloroacetimidate
(C): Trisaccharide 8 (5.3 g, 4.8 mmol) was dissolved in methanol (70 mL).
After addition of acetic acid (7 mL) and Pd/C (10%, 5 g) the suspension
was stirred under
a hydrogen atmosphere. After complete reaction
(TLC: hexane/acetone 1:1) the catalyst was filtered off and the solvents
were evaporated. The remainder was taken up in CH2Cl2 (300 mL), ex-
tracted with dilute KHCO3, dried over MgSO4 and concentrated. The
hemiacetal was dissolved in absolute CH2Cl2 (50 mL) cooled to 08C and
treated with trichloroacetonitrile (4 mL, 39.9 mmol) and 1,8-diazabicyclo-
A
H-4’’), 4.77 (dd, J2,3 =2.9 Hz, J3,4 =9.8 Hz, 1H, H-3), 4.68 (dd, J4,5 =
hemiacetal (TLC: hexane/acetone 1:1) the solution was evaporated and
the residue was purified by flash chromatography (hexane/acetone 1:1)
to afford C (4.76 g, 79.8%). Rf hemiacetal=0.23 (hexane/acetone 2:1); Rf
imidate=0.31 (hexane/acetone 2:1); [a]2D3 =+22.6 (c=0.5, CH2Cl2);
1H NMR (500 MHz, [D6]DMSO): d=9.87 (s, 1H, NH), 7.92–7.68 (m,
8H, Pht), 5.70 (d, J1,2 <1.0 Hz, 1H, H-1), 5.63 (dd, J2,3 =10.7 Hz, 1H, H-
3’’), 5.61 (dd, J2,3 =10.9 Hz, 1H, H-3’), 5.48 (d, J1,2 =7.7 Hz, 1H, H-1’),
5.46 (d, J1,2 =8.3 Hz, 1H, H-1’’), 5.01 (dd, J3,4 =J4,5 =9.5 Hz, 1H, H-4’),
4.98 (dd, J3,4 =J4,5 =9.5 Hz, 1H, H-4’’), 4.94 (dd, J2,3 =3.5 Hz, J3,4 =9.3 Hz,
1H, H-3), 4.3–4.2 (m, 3H, H-2, H-6a’, H-6a’’), 4.13–4.08 (m, 2H, H-2’, H-
10.0 Hz, 1H, H-4), 4.22–4.17 (m, 2H, H-6a’, H-6a’’), 4.13–4.08 (m, 2H,
H-2, H-2’), 4.00–3.96 (m, 3H, H-5’, H-6b’, H-6b’’), 3.89 (dd, J2,3 =10.4 Hz,
1H, H-2’’), 3.83 (m, 1H, H-5’’), 3.58 (m, 1H, H-5), 3.40 (dd, Jgem
=
10.1 Hz, Jvic <1.0 Hz, 1H, H-6a), 2.91 (dd, Jvic =5.9 Hz, 1H, H-6b), 2.05,
2.00, 1.98, 1.89, 1.88, 1.82, 1.80, 1.77 (8s, 27H, OAc); 13C NMR
(125 MHz, [D6]DMSO): d=170.0, 169.6, 169.3, 169.2, 168.9, 167.8, 167.1
(C=O), 134.8 (C-4/5 Pht), 130.7 (C-1/2 Pht), 123.6 (C-3/6 Pht), 97.6 (C-
1’’), 95.9 (C-1’), 89.8 (JC-1,H-1 =177.6 Hz from a coupled HMQC spectrum,
C-1a), 72.4 (C-2), 71.02 (C-5’), 70.95 (C-5’’), 70.90 (C-5), 70.1 (C-3’’), 69.8
(C-3’), 69.0 (C-3), 68.6 (C-4’), 68.4 (C-4’’, C-6), 61.6 (C-6’, C-6’’), 53.8 (C-
Chem. Eur. J. 2009, 15, 12292 – 12302
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12297

C. Unverzagt et al.
2’, C-2’’), 20.5, 20.4, 20.3, 20.1, 20.0 (OAc); elemental analysis calcd (%)
for C52H56N2O27 (1141.01): C 54.74, H 4.95, N 2.46; found: C 54.47, H
5.04, N 2.49.
Benzyl
O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!2)]-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyra-
nosyl)-(1!4)]-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-gluco-
pyranosyl)-(1!6)]-a-d-mannopyranoside
(12):
a) Sodium
acetate
O-{O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!2)-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!6)]-3,4-di-O-acetyl-a-d-mannopyranosyl}-trichloroacetimidate
(D): Hydrazine acetate (400 mg) was added to a solution of trisaccharide
10 (2.61 g, 2.29 mmol) in DMF (10 mL). After complete reaction (TLC:
hexane/acetone 1:1) acetone (2 mL) was added and the volatiles were
distilled off. The remainder was taken up in CH2Cl2 (200 mL) and
washed with dilute HCl and KHCO3. The organic phase was dried over
MgSO4 and concentrated. Subsequently, the hemiacetal was dissolved in
absolute CH2Cl2 (20 mL) and trichloroacetonitrile (3 mL, 29.9 mmol) and
DBU (150 mL, 1 mmol) were added at 08C. After complete reaction
(TLC: hexane/acetone 1:1) the mixture was concentrated and purified by
flash chromatography (hexane/acetone 1:1) to afford D (1.94 g, 68.2%).
Rf hemiacetal=0.27 (hexane/acetone 1:1); Rf imidate=0.35 (hexane/ace-
tone 1:1); [a]2D3 =ꢀ8.9 (c=1, methanol); 1H NMR (500 MHz,
(531 mg, 6.4 mmol) and palladium chloride (345 mg, 1.92 mmol) were
added under an argon atmosphere to a solution of tetrasaccharide 11
(2.0 g, 1.28 mmol) in acetic acid (100 mL, 95%). The suspension was
stirred at 808C until the reaction was completed (TLC: hexane/acetone,
1.2:1). Subsequently, the precipitate was filtered off and the solvents
were removed on a rotary evaporator. The remainder was taken up in
water/dichloromethane and extracted with dilute KHCO3 and water. The
organic phase was dried over MgSO4 and concentrated. The remainder
was purified by flash chromatography (cyclohexane/ethyl acetate 1:1.2)
to afford 12 (1.41 g, 72.5%). Rf =0.09 (cyclohexane/ethyl acetate 1:1.5);
[a]2D3 = +12.9 (c=0.4, CH2Cl2).
b) Tetrasaccharide 11 (100 mg, 64 mmol) was dissolved in absolute metha-
nol (1.5 mL) under an argon atmosphere. Subsequently anhydrous palla-
dium chloride (55.3 mg, 0.31 mmol) was added and the suspension was
stirred for 60 min (TLC: cyclohexane/ethyl acetate 1:2). The solids were
removed by filtration over Celite and the filtrate was concentrated to
dryness. The remainder was dissolved in dichloromethane and extracted
with brine and KHCO3 (2m). After being dried over MgSO4 the organic
phase was concentrated. The remainder was purified by flash chromatog-
raphy (cyclohexane/ethyl acetate 1:1) to afford 12 (75 mg, 77.0%). Rf =
0.27 (cyclohexane/ethyl acetate 1:2); 1H NMR (360 MHz, [D6]DMSO):
d=8.00–6.80 (m, 17H, Pht, Ar), 5.63–5.52 (m, 2H, H-35’, H-37’’), 5.48 (dd,
[D6]DMSO): d=9.5 (s, 1H, NH), 7.97–7.77 (m, 8H, Pht), 5.71 (d, J1,2
<
1.0 Hz, 1H, H-1), 5.65 (dd, J2,3 =10.7 Hz, J3,4 =9.2 Hz, 1H, H-3’), 5.46 (m,
2H, H-1’, H-3’’), 5.42 (d, J1,2 =8.5 Hz, 1H, H-1’), 5.01 (m, 2H, H-1’’, H-
4’), 4.90 (dd, J3,4 =J4,5 =9.6 Hz, 1H, H-4’’), 4.84 (dd, J2,3 =2.9 Hz, J3,4
=
10.1 Hz, 1H, H-3), 4.75 (dd, J4,5 =9.9 Hz, 1H, H-4), 4.23 (m, 1H, H-2),
4.22–4.17 (m, 2H, H-6a’, H-6a’’), 4.13 (dd, 1H, H-2’), 4.04–3.95 (m, 3H,
H-5’, H-6b’, H-6b’’), 3.87 (dd, J1,2 =8.6 Hz, J2,3 =10.2 Hz, 1H, H-2’’), 3.81
(m, 1H, H-5’’), 3.63 (m, 1H, H-5), 3.43 (dd, Jgem =11.3 Hz, Jvic =2.1 Hz,
1H, H-6a), 2.88 (dd, Jvic =6.3 Hz, 1H, H-6b), 2.04, 2.00, 1.98, 1.89, 1.87,
1.80, 1.76 (7s, 24H, OAc); 13C NMR (125 MHz, [D6]DMSO): d=170.0,
169.6, 169.24, 169.17, 169.1, 166.9 (C=O), 156.6 (C=NH), 134.8 (C-4/5)
Pht, 130.6 (C-1/2) Pht, 123.5 (C-3/6) Pht, 97.8 (C-1’’), 95.9 (C-1’), 93.2 (C-
1), 89.8 (CCl3), 71.7 (C-2), 71.5 (C-5), 71.0 (C-5’), 70.9 (C-5’’), 70.0 (C-
3’’), 69.7 (C-3’), 68.8 (C-6), 68.7 (C-3), 68.5 (C-4’), 68.4 (C-4’’), 61.6 (C-6’,
C-6’’), 53.7 (C-2’), 53.6 (C-2’’), 20.4, 20.3, 20.23, 20.16, 20.08, 19.98 (OAc);
elemental analysis calcd (%) for C52H54Cl3N3O26 (1243.36): C 50.23, H
4.38, N 3.38; found: C 49.86, H 4.38, N 3.46.
J
2,3 =10.1 Hz, J3,4 =9.7 Hz, 1H, H-37’), 5.33–5.31 (m, 2H, H-15’, H-17’’),
4.95–4.88 (m, 2H, H-47’, H-45’), 4.82 (dd, J2,3 =J3,4 =9.7 Hz, 1H, H-47’’),
4.64 (d, J1,2 =9.2 Hz, 1H, H-17’), 4.42 (d, JOH,3 =8.2 Hz, 1H, OH-34’), 4.36
(d, J1,2 <1 Hz, 1H, H-14’), 4.27–4.17 (m, 2H, H-6a7’’, H-6a5’), 4.12–3.79 (m,
10H, H-25’, H-27’, H-27’’, H-55’, H-57’’, CH2O, H-6b5’, H-6a,b7’, H-6b7’’),
3.72–3.48 (m, 3H, H-24’, H-34’, CH2O), 3.44–3.21 (m, 3H, H-57’, H-54’, H-
6a4’), 2.97 (dd, J3,4 =J4,5 =8.8 Hz, 1H, H-44’), 2.13 (m, 1H, H-6b4’), 2.10–
1.70 (9s, 27H, OAc); 13C NMR (90 MHz, [D6]DMSO): d=170.0–166.9
(C=O), 136.6 (C-i Ph), 135.1 (C-4/5 Pht), 129.5 (C-1/2 Pht), 128.1–127.0
(C-Ar), 123.7, 123.5, 123.1 (C-3/6 Pht), 98.3 (C-17’), 95.9 (C-17’’), 95.1 (C-
15’), 94.7 (C-14’), 76.1 (C-44’), 75.7 (C-24’), 70.7 (C-55’), 70.6 (C-57’’), 70.4
(C-57’), 69.9 (C-54’), 69.6 (C-35’, C-37’’), 69.5 (C-64’, C-37’), 68.2 (C-47’, C-45’,
C-47’’), 67.4 (CH2O-Bn), 67.1 (C-34’), 61.4 (C-67’), 61.3 (C-67’’), 61.2 (C-65’),
53.9 (C-27’’), 53.7 (C-27’, C-25’), 20.4–20.0 (OAc); ESI-MS: m/z calcd for
C73H75N3O33: 1521.43; found 1544.51 [M+Na]+.
Benzyl
O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!2)]-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyra-
nosyl)-(1!4)]-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-gluco-
pyranosyl)-(1!6)]-3-O-allyl-a-d-mannopyranoside (11): A suspension of
triol 4 (118 mg, 0.38 mmol), fluoride 1 (1.0 g, 2.29 mmol) and ground mo-
lecular sieves 4 ꢃ (1.2 g) in absolute CH2Cl2 (2.4 mL) was stirred at am-
bient temperature and at ꢀ108C for 30 min at each temperature. Boron
trifluoride ethyl etherate (48 mL, 0.38 mmol, 1.0 equiv) was added over
5 min. After 4.5 h at ꢀ108C (TLC: hexane/ethyl acetate 1:2) the reaction
was diluted with CH2Cl2 and the solids were filtered over Celite. The or-
ganic phase was extracted with dilute KHCO3, dried over MgSO4 and
concentrated. The remainder was purified by flash chromatography
(hexane/acetone, 1.5:1) to afford 11 (529 mg, 88.8%). Rf =0.20 (hexane/
acetone, 1.2:1); [a]D23 =ꢀ83.7 (c=0.5, CH2Cl2); 1H NMR (360 MHz,
Benzyl
O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!2)]-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyra-
nosyl)-(1!4)]-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-gluco-
pyranosyl)-(1!6)]-3-O-acetyl-a-d-mannopyranoside (12a): Tetrasacchar-
ide 12 (1.06 g, 0.7 mmol) was dissolved in pyridine (10.0 mL) and acetic
anhydride (5.0 mL) at 08C. DMAP (150 mg, 1.2 mmol) was added and
the mixture was stirred for 2 days at ambient temperature. The solvents
were removed and the remainder was codistilled with toluene (3ꢄ). The
residue was taken up in CH2Cl2 and extracted with dilute HCl, dilute
KHCO3 and water. The organic phase was dried over MgSO4 and con-
centrated. The remainder was purified by flash chromatography (hexane/
acetone, 1.1:1) to afford 12a (881 mg, 81.0%); Rf =0.30 (hexane/acetone
1.1:1); [a]2D3 =+3.1 (c=0.4, CH2Cl2); 1H NMR (360 MHz, [D6]DMSO):
d=8.20–6.90 (m, 17H, Pht, Ar), 5.65–5.50 (m, 2H, H-35’, H-37’’), 5.43 (dd,
ꢀ
[D6]DMSO): d=8.10–6.90 (m, 17H, Pht, Ar), 5.81 (m, 1H, =CH ), 5.65–
5.45 (m, 3H, H-35’, H-37’’, H-37’), 5.40 (d, J1,2 =8.4 Hz, 1H, H-17’’), 5.30 (d,
J
1,2 =8.4 Hz, 1H, H-15’), 5.11 (m, 1H, =CH2 trans), 5.04–4.91 (m, 3H, H-
45’, H-47’’, =CH2 trans), 4.85 (dd, J3,4 =9.6 Hz, J4,5 =10.4 Hz, 1H, H-47’),
4.67 (d, J1,2 =5.7 Hz, 1H, H-17’), 4.30 (d, J1,2 =1.5 Hz, 1H, H-14’), 4.22–
3.81 (m, 16H, H-6a7’’, H-6a5’, H-6a7’, H-27’’, H-6b5’, H-25’, H-6b7’’, H-55’, H-
6b7’, H-57’’, H-27’, H-24’, H-6a4’, CH2O-All, CH2O-Bn), 3.76 (d, Jgem
11.2 Hz, 1H, CH2O-Bn), 3.56–3.43 (m, 2H, H-34’, H-57’), 3.37 (dd, J3,4
=
J
2,3 =9.9 Hz, J3,4 =11.1 Hz, 1H, H-37’), 5.30–5.25 (m, 2H, H-15’, H-17’’),
4.97–4.91 (m, 2H, H-45’, H-47’’), 4.79–4.73 (m, 2H, H-47’, H-34’), 4.64 (d,
J
1,2 =8.4 Hz, 1H, H-17’), 4.30–3.70 (m, 15H, H-14’, H-6a7’, H-6a7’’, CH2O-
=
9.2 Hz, J4,5 =9.3 Hz, 1H, H-44’’), 3.21 (dd, Jgem =10.5 Hz, 1H, H-6b4’), 3.04
(m, 1H, H-54’), 2.09–1.65 (9s, 27H, OAc); 13C NMR (90 MHz,
Bn, H-6a5’, H-55’, H-27’’, H-6b7’’, H-25’, H-57’’, H-24’, H-6b5’, CH2O-Bn, H-
27’, H-6b7’), 3.94 (dd, J4,5 =9.3 Hz, J5,6 =10.2 Hz, 1H, H-54’), 3.27–3.18 (m,
2H, H-44’, H-6a4’), 3.08 (dd, J4,5 =7.4 Hz, J5,6 =9.6 Hz, 1H, H-57’), 2.46 (m,
1H, H-6b4’), 2.30–1.60 (10s, 30H, OAc); 13C NMR (90 MHz,
[D6]DMSO): d=170.0, 169.5, 169.2 (C=O), 136.2 (C-i Ph), 135.2, 135.0,
134.8 (C-4/5 Pht), 130.4 (C-1/2 Pht), 128.2–127.7 (C-Ar), 123.9, 123.5,
123.3 (C-3/6 Pht), 98.4 (C-17’), 96.3 C-15’), 95.3 (C-17’’), 94.7 (C-14’), 73.0
(C-24’), 72.6 (C-44’), 70.6 (C-57’’), 70.5 (C-55’), 70.4 (C-57’), 69.6 (C-54’), 69.5
(C-34’), 69.5 (C-64’), 69.5 (C-37’’), 69.5 (C-35’), 69.4 (C-37’), 68.2 (C-47’’), 68.2
(C-47’), 68.1 (C-45’), 67.6 (CH2O-Bn), 61.5 (C-65’), 61.4 (C-67’), 61.3 (C-
ꢀ
[D6]DMSO): d=170.1, 169.9 (C=O), 136.5 ( CH=), 135.2, 135.1, 135.0
(C-4/5 Pht), 131.6, 130.9, 130.4 (C-i Ph), 128.7–127.6 (C-Ar), 123.8, 123.5,
123.0 (C-1/2 Pht), 123.7, 123.5, 123.1 (C-3/6 Pht), 116.5 (=CH2), 98.1 (C-
17’), 96.6 (C-17’’), 95.0 (C-15’), 94.9 (C-14’), 75.4 (C-34’), 74.3 (C-54’), 72.1
(C-24’), 70.7 (C-55’), 70.6 (C-57’), 70.6 (C-57’’), 69.6 (C-44’), 69.6 (C-37’), 69.5
(C-37’’), 69.4 (C-35’), 69.4 (C-64’), 69.3 (CH2O-All), 68.2 (C-47’), 68.2 (C-
47’’), 68.1 (C-45’), 67.6 (CH2O-Bn), 61.4 (C-65’), 61.4 (C-67’), 61.4 (C-67’’),
54.1 (C-27’), 53.8 (C-25’), 53.7 (C-27’’), 20.4–20.0 (OAc); ESI-MS: m/z
calcd for C76H79N3O33: 1561.46; found 1584.51 [M+Na]+.
12298
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ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 12292 – 12302

Synthesis of Multiantennary Complex Type N-Glycans
FULL PAPER
67’’), 54.2 (C-25’), 53.6 (C-27’), 53.7 (C-27’’), 20.4–19.9 (OAc); ESI-MS: m/z
calcd for C75H77N3O34: 1563.44; found 1586.54 [M+Na]+.
(m, 8H, H-21, H-27’, H-54, H-54’, H-6a2, H-6a,b4, H-6b5’), 3.58–3.52 (m,
2H, H-51, H-57’), 3.50–3.43 (m, 2H, H-6b2, H-6a4’), 3.39–3.34 (m, 2H, H-
33, H-6a1), 3.33–3.16 (m, 5H, H-43, H-52, H-55’, H-6b1, H-6a3), 2.99–2.88
(m, 3H, H-53, H-6b3, H-6b4’), 2.02, 2.01, 2.00, 1.99, 1.98, 1.96, 1.94, 1.91,
1.89, 1.80, 1.78, 1.76, 1.75, 1.69 (14s, 45H, OAc); 13C NMR (125 MHz,
[D6]DMSO): d=170.0, 169.9, 169.8, 169.5, 169.2, 169.1, 169.0, 167.6, 167.1
(C=O), 138.2, 138.1, 138.0, 137.95 (C-i Ar), 134.8, 134.7 (C-4/5 Pht),
130.8, 130.6, 130.5 (C-1/2 Pht), 128.1–127.0 (C-Ar), 123.6, 123.4 (C-3/6
Pht), 97.7 (JC-1,H-1 =176.2 Hz from a coupled HMQC spectrum, C-14a),
97.6 (C-17’), 96.9 (C-13), 96.64 (JC-1,H-1 =176.2 Hz from a coupled HMQC
spectrum, C-14’a), 96.62 (C-12), 96.0 (C-15), 95.9 (C-15’), 84.7 (C-11), 77.3
(C-42), 76.6 (C-31), 76.3 (C-33), 75.6 (C-32, C-51), 75.2 (C-41), 74.2 (C-52),
74.1 (C-53), 73.8 (CH2O), 73.30 (C-24), 73.29 (C-24’), 73.28, 72.3, 71.6
(CH2O), 71.0 (C-55), 70.8 (C-57’), 70.6 (C-55’), 70.3 (C-23), 70.0 (C-37’),
69.8 (C-35’), 69.6 (C-35), 69.5 (C-34’), 69.0 (C-34), 68.6 (C-45), 68.5 (C-54’),
68.3 (C-47’), 68.2 (C-45’), 68.1 (C-54), 68.0 (C-64’), 67.6 (C-62), 67.4 (C-61),
66.3 (C-63), 66.1 (C-43), 64.8 (C-44’), 64.7 (C-44), 61.9 (C-65), 61.8 (C-64),
61.5 (C-67’), 61.3 (C-65’), 55.8 (C-22), 54.6 (C-21), 53.9 (C-27’), 53.7 (C-25,
C-25’), 20.5, 20.3, 20.1, 20.0, 19.9 (OAc); FAB-MS (NBA): m/z calcd for
C146H150N8O60: 2974.9; found 2976 [M+H]+; elemental analysis calcd (%)
for C146H150N8O60 (2976.81): C 58.91, H 5.08, N 3.76; found: C 58.59, H
5.08, N 3.70.
O-{O-[(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-
(1!2)]-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!4)]-O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyra-
nosyl)-(1!6)]-3-O-acetyl-a-d-mannopyranosyl}-trichloroacetimidate (E):
Tetrasaccharide 12a (100 mg, 63.9 mmol) was dissolved in methanol
(10.8 mL). Acetic acid (1.2 mL) and palladium oxide hydrate (222 mg)
were added and the suspension was stirred under a hydrogen atmos-
phere. After complete reaction (TLC: CH2Cl2/methanol, 10:1) the cata-
lyst was filtered off and washed with methanol (3ꢄ). After removal of
the volatiles the remainder was purified by flash chromatography
(hexane/acetone 1.2:1) to afford the hemiacetal 12b (77 mg, 81.7%). Rf =
0.22 (CH2Cl2/methanol, 20:1); ESI-MS: m/z calcd for C68H71N3O34
:
1473.39; found 1496.1 [M+Na]+.
Hemiacetal 12b (4.60 g, 3.12 mmol) and trichloroacetonitrile (11.9 mL,
117 mmol) were dissolved in CH2Cl2 (250 mL) and stirred for 30 min at
08C. The reaction was started by addition of DBU (441 mL, 2.95 mmol).
After complete reaction (TLC: CH2Cl2/methanol, 20:1) the volatiles
were removed at ambient temperature. The remainder was dried in high
vacuum and purified by flash chromatography (hexane/acetone, 1.2:1) to
afford E (4.3 g, 85.3%). Rf =0.35 (CH2Cl2/methanol 20:1); [a]2D3 =+15.4
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-
O-(3,4,6-tri-O-acetyl-a-d-mannopyranosyl)-(1!3)-O-{3,4,6-tri-O-acetyl-
2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-O-[3,4,6-tri-O-acetyl-
2-deoxy-2-phthalimido-b-d-glucopyranosyl-(1!4)]-O-[3,4,6-tri-O-acetyl-
2-deoxy-2-phthalimido-b-d-glucopyranosyl-(1!6)]-O-(3-O-acetyl-a-d-
mannopyranosyl)-(1!6)}-O-(2-O-acetyl-b-d-mannopyranosyl)-(1!4)-O-
(3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!4)-3,6-
di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosylazide (15): Penta-
1
(c=0.4, CH2Cl2); H NMR (500 MHz, [D6]DMSO): d=9.50 (s, 1H, NH),
8.20–7.55 (m, 12H, Pht), 5.67 (d, J1,2 <1 Hz, 1H, H-14’), 5.62 (dd, J2,3
=
9.9 Hz, J3,4 =8.8 Hz, 1H, H-35’), 5.49–5.38 (m, 3H, H-37’’, H-37’, H-15’),
5.30 (d, J1,2 =8.4 Hz, 1H, H-17’’), 4.99–4.94 (m, 3H, H-45’, H-47’’, H-34’),
4,77 (dd, J3,4 =10.0 Hz, J4,5 =9.4 Hz, 1H, H-47’), 4.72 (d, J1,2 =8.0 Hz, 1H,
H-17’), 4.28–4.23 (m, 3H, H-6a7’’, H-6a7’, H-6a5’), 4.08–3.90 (m, 7H, H-24’,
H-25’, H-6b5’, H-27’’, H-55’, H-57’’, H-6b7’), 3.88 (dd, Jgem =12.0 Hz, 1H, H-
6b7’’), 3.78 (dd, J1,2 =8.0 Hz, J2,3 =9.5 Hz, 1H, H-27’), 3.67 (dd, J4,5 =9.4 Hz,
saccharide 13 (200 mg, 105 mmol), donor
E (540 mg, 334 mmol) and
J
5,6 =8.5 Hz, 1H, H-54’), 3.40 (dd, J3,4 =8.8 Hz, J4,5 =9.4 Hz, 1H, H-44’),
ground molecular sieves 4 ꢃ (800 mg) in absolute CH2Cl2 (30 mL) were
stirred for 30 min at ꢀ458C. Boron trifluoride ethyl etherate (4 mL,
32.5 mmol) was added over 5 min. The reaction was carried out for 3 h
(TLC: hexane/acetone 1:1) and gradually warmed to ꢀ58C. Subsequent-
ly, the solids were removed by filtration over Celite and washed with
CH2Cl2. After removal of the solvent the remainder was purified by flash
chromatography (cyclohexane/acetone 1.2:1) to afford 15 (224 mg,
63.3%). Rf =0.29 (cyclohexane/acetone 1:1); [a]2D3 =ꢀ25.1 (c=0.5,
CH2Cl2); 1H NMR (500 MHz, [D6]DMSO): d=8.04–7.40 (m, 24H, Pht),
7.30–6.70 (m, 20H, Ar), 5.67 (dd, J2,3 =10.4 Hz, J3,4 =12.2 Hz, 1H, H-37’’),
5.60 (d, J4,OH =3.7 Hz, 1H, OH-43), 5.51 (dd, J2,3 =10.4 Hz, J3,4 =11.0 Hz,
3.18 (dd, Jgem =11.2 Hz, 1H, H-6a4’), 3.15 (dd, J4,5 =9.4 Hz, J5,6 =10.0 Hz,
1H, H-57’), 2.61 (dd, Jgem =11.2 Hz, J5,6 =9.4 Hz, 1H, H-6b4’), 2.07–1.70
(10s, 30H, OAc); 13C NMR (125 MHz, [D6]DMSO): d=170.0, 169.6,
169.2, 167.1, 166.9 (C=O), 135.1, 134.8 (C-4/5 Pht), 130.4 (C-1/2 Pht),
123.7, 123.3 (C-3/6 Pht), 98.7 (C-17’), 96.6 (C-17’’), 95.8 (C-15’), 93.2 (C-14’),
72.3 (C-44’, C-54’), 71.5 (C-54’), 70.9 (C-55’), 70.5 (C-57’’), 70.4 (C-57’), 69.6
(C-37’’), 69.4 (C-64’, C-35’), 69.3 (C-37’), 68.9 (C-34’), 68.1 (C-45’), 68.0 (C-
47’), 67.7 (C-47’’), 61.4 (C-67’), 61.2 (C-65’), 60.9 (C-67’’), 54.1 (C-27’’), 53.4
(C-25’), 53.2 (C-27’), 20.00, 19.7 (OAc); ESI-MS: m/z calcd for
C70H71Cl3N4O34: 1616.30; found 1639.41 [M+Na]+.
1H, H-35), 5.44–2.91 (m, 69H, H-17’’, H-35’ H-15, H-11, H-37’, H-12, H-47’’,
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-
O-(3,4,6-tri-O-acetyl-a-d-mannopyranosyl)-(1!3)-O-{3,4,6-tri-O-acetyl-
2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-O-[(3,4,6-tri-O-
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!6)]-O-(3,4-di-O-
acetyl-a-d-mannopyranosyl)-(1!6)}-O-(2-O-acetyl-b-d-mannopyrano-
syl)-(1!4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl-
,
H-45, H-43, H-45’, CH2O, H-34, H-14, H-47’, H-33, H-13, H-17’, CH2O,
CH2O, CH2O, CH2O, H-15’, CH2O, H-23, CH2O, CH2O, H-6a7’’, H-6a7’, H-
6a5, H-27’’, H-24’, H-31, H-14’, H-41, H-55, H-22, H-6b7’’, H-25, H-6b7’, H-57’’,
H-32, H-53, H-27’, H-6a5’, H-6b5, H-21, H-25’, H-6a,b3, H-6a2, H-44’, H-24,
H-51, H-6a,b4, H-6b2, H-6b5’, H-6a1, H-54, H-34’, H-6a4’, H-42, H-6b1, H-55’,
H-52, H-44, H-54’, H-6b4’, H-57’), 2.08–1.67 (17s, 51H, OAc); 13C NMR
(125 MHz, [D6]DMSO): d=170.1–167.2 (C=O), 138.1, 138.0, 137.9 (C-i
Ar), 135.0, 134.9, 134.7 (C-4/5 Pht), 130.8, 130.7, 130.6, 130.5 (C-1/2 Pht),
128.3–127.1 (C-Ar), 123.6, 123.5, 123.4 (C-3/6 Pht), 97.84 (C-13), 97.80 (C-
14), 97.7 (C-15’), 96.9 (C-15), 96.5 (C-14’), 96.0 (C-12), 95.6 (C-17’), 95.5 (C-
17’’), 84.6 (C-11), 78.3 (C-32), 76.2 (C-34’), 75.8 (C-31), 75.3 (C-51), 74.7 (C-
41), 73.8 (C-42), 73.4 (CH2O), 73.3 (C-54’), 72.9 (C-44), 72.9 (CH2O), 72.8
(C-24), 72.6 (C-23), 72.0, 71.4 (CH2O), 70.7 (C-57’’), 70.5 (C-24’), 70.41 (C-
55), 70.40 (C-55’), 70.1 (C-34), 69.6 (C-35), 69.5 (C-64), 69.5 (C-37’’), 69.4
(C-35’), 69.1 (C-37’), 68.9 (C-33), 68.7 (C-54), 68.3 (C-47’’), 68.1 (C-57’), 68.0
(C-52), 67.9 (C-45), 67.89 (C-45’), 67.8 (C-53), 67.7 (C-47’), 67.4 (C-62), 67.2
(C-61), 65.7 (C-64’), 64.7 (C-44’), 64.4 (C-43), 61.6 (C-67’’), 61.5 (C-63, C-65),
61.1 (C-67’), 60.8 (C-65’), 55.8 (C-22), 54.3 (C-25), 54.2 (C-21), 53.7 (C-25’),
53.4 (C-27’), 53.39 (C-27’’), 20.4, 20.2, 20.1, 20.0, 19.8 (OAc); ESI-MS: m/z
calcd for C164H167N9O68: 3349.99; found 3350.73 [M+H]+.
ACHTUNGTRENNUNGazide (14): A suspension of pentasaccharide 13 (661 mg, 349 mmol),
donor D (650 mg, 523 mmol) and ground molecular sieves 4 ꢃ (1 g) in ab-
solute CH2Cl2 (65 mL) was stirred for 30 min at ꢀ358C. Boron trifluoride
ethyl etherate (17 mL, 138 mmol) was added over 5 min. The reaction was
carried out over 4 h (TLC: cyclohexane/acetone, 1.5:1) and gradually
warmed to +18C. Subsequently, the solids were removed by filtration
over Celite and washed with CH2Cl2. After removal of the solvent the re-
mainder was purified by flash chromatography (cyclohexane/acetone
1.6:1) to afford 14 (940 mg, 90.6%). Rf =0.26 (cyclohexane/acetone,
1.6:1); [a]2D3 =ꢀ13.3 (c=0.5, CH2Cl2); 1H NMR (500 MHz, [D6]DMSO):
d=8.02–7.60 (m, 20H, Pht) 7.30–6.72 (m, 20H, Ar), 5.71 (dd, J2,3 =J3,4
=
10.0 Hz, 1H, H-35), 5.43 (m, 2H, H-35’, H-37), 5.40–5.35 (m, 2H, H-15,
OH-43), 5.25 (d, J1,2 =9.5 Hz, 1H, H-11), 5.14 (d, J1,2 =8.0 Hz, 1H, H-12),
5.11 (d, J1,2 =8.2 Hz, 1H, H-15’), 5.08–5.02 (m, 2H, H-23, H-45), 4.98 (dd,
J
3,4 =J4,5 =10.1 Hz, 1H, H-44), 4.93 (d, J1,2 =8.3 Hz, 1H, H-17’), 4.90–4.78
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-
O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!
4)]-O-(3,6-di-O-acetyl-a-d-mannopyranosyl)-(1!3)-O-(4,6-O-benzyli-
dene-b-d-mannopyranosyl)-(1!4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthali-
mido-b-d-glucopyranosyl)-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-
b-d-glucopyranosylazide (16): A suspension of trisaccharide A (558 mg,
(m, 5H, H-14, H-34’, H-45’, H-47’, CH2O), 4.76 (dd, J2,3 =2.4 Hz, 1H, H-34),
4.69 (dd, J3,4 =J4,5 =10.2 Hz, 1H, H-44’), 4.54 (d, J1,2 <1.0 Hz, 1H, H-13),
4.52–4.42 (m, 3H, CH2O), 4.38 (d, Jgem =12.9 Hz, 1H, CH2O), 4.33–4.17
(m, 8H, CH2O, H-14’, H-24, H-25, H-6a5, H-6a7’), 4.07–3.84 (m, 11H, H-22,
H-24’, H-25’, H-31, H-32, H-41, H-42, H-55, H-6b5, H-6a5’, H-6b7’), 3.83–3.60
Chem. Eur. J. 2009, 15, 12292 – 12302
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12299

C. Unverzagt et al.
451 mmol), imidate C (840 mg, 676 mmol) and ground molecular sieves
4 ꢃ (1.3 g) in absolute CH2Cl2 (10 mL) was stirred for 30 min at ꢀ258C.
Boron trifluoride ethyl etherate (10 mL, 81 mmol) was added over 5 min
and the solution was stirred continuously for 1 h (TLC: hexane/acetone
1:1). The solids were removed by filtration over Celite and washed with
CH2Cl2. After concentration the remainder was purified by flash chroma-
tography (hexane/acetone, 1.2:1) to afford 16 (975 mg, 93.2%). Rf =0.24
(C-51), 75.0 (C-41), 74.3 (C-52), 73.8, 73.7 (CH2O), 72.4 (C-24), 72.1, 71.7
(CH2O), 71.6 (C-44), 70.9 (C-55), 70.5 (C-23), 70.4 (C-57), 69.9 (C-37), 69.8
(C-35), 68.7 (C-34), 68.5 (C-45), 68.2 (C-47), 67.7 (C-54), 67.63 (C-61), 67.60
(C-62), 66.8 (C-43), 61.9 (C-65, C-64), 61.5 (C-67), 60.4 (C-63), 55.9 (C-22),
54.6 (C-21), 54.5 (C-27), 53.7 (C-25), 20.4, 20.1, 20.04, 20.00, 19.7 (OAc);
ESI-MS: m/z calcd for C114H115N7O43: 2269.70; found 2288.94 [M+H3O]+.
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-
O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!
4)]-O-(3,6-di-O-acetyl-a-d-mannopyranosyl)-(1!3)-O-[(3,4,6-tri-O-
acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-O-(3,4,6-tri-O-
acetyl-a-d-mannopyranosyl)-(1!6)]-O-(2-O-acetyl-b-d-mannopyrano-
syl)-(1!4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyrano-
syl)-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl-
1
(hexane/acetone 2:1); [a]D23 =ꢀ16.0 (c=0.5, CH2Cl2); H NMR (500 MHz,
[D6]DMSO): d=8.0–6.8 (m, 41H, Pht, Ar), 5.62 (dd, J2,3 =10.5 Hz, J3,4
=
9.5 Hz, 1H, H-37), 5.61 (s, 1H, =CH-Ph), 5.43 (dd, J2,3 =J3,4 =9.8 Hz, 1H,
H-35), 5.42 (d, J1,2 =8.9 Hz, 1H, H-17b), 5.29 (d, J1,2 =9.5 Hz, 1H, H-11b),
5.23 (d, J1,2 =8.8 Hz, 1H, H-12b), 5.22 (d, J2,OH =3.7 Hz, 1H, OH-23), 5.0–
4.95 (m, 2H, H-47, H-34), 4.89 (dd, J3,4 =J4,5 =9.4 Hz, 1H, H-45), 4.85 (d,
J
J
1,2 =8.7 Hz, 1H, H-15b), 4.83 (d, Jgem =11.8 Hz, 1H, CH2O), 4.78 (d,
AHCTUNGERTGaNNUN zide (18): A suspension of hexasaccharide 17 (600 mg, 264 mmol), imi-
gem =11.8 Hz, 1H, CH2O), 4.63 (d, J1,2 <1.0 Hz, 1H, H-14), 4.54 (d, Jgem
=
date B (342 mg, 394 mmol) and ground molecular sieves 4 ꢃ (900 mg) in
absolute CH2Cl2 (60 mL) was stirred for 30 min at ꢀ358C. Boron trifluor-
ide ethyl etherate (12 mL, 98 mmol) was added over 5 min and the reac-
tion was allowed to gradually warm up to +58C over a period of 3 h
(TLC: cyclohexane/acetone 1:1). Subsequently, the solids were removed
by filtration over Celite and washed with CH2Cl2. After concentration
the remainder was purified by flash chromatography (cyclohexane/ace-
tone, 1.4:1) to afford 18 (614 mg, 78.1%). Rf =0.3 (cyclohexane/acetone
11.8 Hz, 1H, CH2O), 4.49 (d, Jgem =11.8 Hz, 1H, CH2O), 4.45–4.36 (m,
5H, H-13, CH2O), 4.24 (dd, Jgem =12.1 Hz, Jvic =4.2 Hz, 1H, H-6a7), 4.17–
4.06 (m, 9H, H-32, H-41, H-31, H-57, H-25, H-27, H-22, H-6a5, H-6a3), 4.03–
3.72 (m, 9H, H-42, H-43, H-24, H-6b7, H-21, H-54, H-6a4, H-6b5, H-6a2),
3.68–3.61 (m, 3H, H-23, H-44, H-51), 3.57–3.44 (m, 4H, H-6b2, H-6b3, H-
6a1, H-33), 3.38–3.30 (m, 2H, H-6b1, H-52), 3.1–3.0 (m, 2H, H-53, H-6b4),
2.27 (m, 1H, H-55), 2.04, 2.02, 2.0, 1.98, 1.97, 1.77, 1.73, 1.51 (8s, 24H,
OAc); 13C NMR (125 MHz, [D6]DMSO): d=170.1, 169.9, 169.62, 169.57,
169.3, 169.2, 168.8, 167.4, 167.1 (C=O), 138.3, 138.1, 138.0, 137.8 (C-i Ar),
135.1, 134.8 (C-4/5 Pht), 130.7, 130.6, 130.4 (C-1/2 Pht), 128.3–126.6 (C-
Ar), 123.4 (C-3/6 Pht), 101.2 (=CH-Ph), 99.6 (C-13b), 97.8 (JC-1,H-1 =172.7
from a coupled HMQC spectrum, C-14a), 97.0 (C-17b), 96.5 (C-12b), 95.8
(C-15b), 84.9 (C-11b), 78.0 (C-33), 77.2 (C-43), 76.9 (C-42), 76.2 (C-31), 75.8
(C-32), 75.7 (C-51), 75.0 (C-41), 74.5 (C-52), 73.8, 73.6 (CH2O), 72.9 (C-24),
72.4 (C-44), 72.3, 71.6 (CH2O), 70.8 (C-55), 70.6 (C-57), 70.2 (C-34), 69.8
(C-23, C-35, C-37), 68.2 (C-45, C-47), 67.8 (C-63), 67.7 (C-62), 67.6 (C-61),
67.4 (C-54), 66.1 (C-53), 61.8 (C-67), 61.7 (C-64), 61.3 (C-65), 56.0 (C-22),
54.6 (C-21, C-27), 53.7 (C-25), 20.6, 20.4, 20.0, 19.9, 19.0 (OAc); ESI-MS:
m/z calcd for C119H117N7O42: 2315.72; found 2334.66 [M+H3O]+.
1
1:1); [a]2D3 =+4.0 (c=0.5, CH2Cl2); H NMR (500 MHz, [D6]DMSO): d=
7.95–7.66 (m, 20H, Pht), 7.30–6.72 (m, 20H, Ar), 5.63–5.55 (m, 3H, H-35,
H-35’, H-37), 5.53 (d, JOH,4 =4.6 Hz, 1H, OH-43), 5.43 (d, J1,2 =8.1 Hz, 1H,
H-17), 5.30–5.24 (m, 3H, H-11, H-15, H-15’), 5.14 (d, J1,2 =8.0 Hz, 1H, H-
12), 5.07 (dd, J1,2, J2,3 <1.0 Hz, 1H, H-23), 5.02–4.90 (m, 5H, H-34’, H-44’,
H-45, H-45’, H-47), 4.85 (d, Jgem =12.4 Hz, 1H, CH2O), 4.74 (d, J1,2
<
1.0 Hz, 1H, H-14), 4.71 (m, 1H, H-34), 4.62 (d,
Jgem =12.4 Hz, 1H,
CH2O), 4.57 (d, J1,2 <1.0 Hz, 1H, H-13), 4.47, 4.41 (2d, Jgem =12.2 Hz, 2H,
CH2O), 4.39–4.29 (m, 5H, CH2O, H-14’), 4.22–3.85 (m, 18H, H-22, H-24,
H-24’, H-25, H-25’, H-27, H-31, H-32, H-41, H-42, H-55, H-57, H-6a4, H-6a,b5,
H-6a5’, H-6a,b7), 3.8–3.7 (m, 4H, H-21, H-44, H-54, H-6b5’), 3.65–3.36 (m,
13H, H-33, H-43, H-51, H-54’, H-55’, H-6a1, H-6a,b2, H-6a,b3, H-6b4, H-
6a,b4’), 3.32–3.19 (m, 3H, H-52, H-53, H-6b1), 2.025, 2.021, 1.99, 1.98, 1.97,
1.93, 1.81, 1.78, 1.77, 1.74, 1.70, 1.64 (12s, 45H, OAc); 13C NMR
(125 MHz, [D6]DMSO): d=170.1, 170.05, 169.9, 169.8, 169.6, 169.4, 169.2,
169.1, 167.9, 167.1 (C=O), 138.1, 138.0 (C-i Ar), 135.1, 134.9 (C-4/5 Pht),
130.7, 130.6, 130.5 (C-1/2 Pht), 128.2–127.0 (C-Ar), 123.8, 123.4 (C-3/6
Pht), 97.7 (JC-1,H-1 =177.6 Hz from a coupled HMQC spectrum, C-14a),
97.1 (JC-1,H-1 =177.6 Hz from a coupled HMQC spectrum, C-14’a), 96.8
(C-12), 96.7 (C-13), 96.4 (C-15’), 96.2 (C-15), 95.3 (C-17), 84.8 (C-11), 76.7
(C-31, C-42), 76.0 (C-33), 75.8 (C-32), 75.7 (C-51), 75.3 (C-41), 74.3 (C-52),
74.2 (C-53), 73.9 (CH2O), 73.8 (C-24’), 73.6 (C-24), 73.5, 72.3 (CH2O), 71.7
(C-44), 71.6 (CH2O), 71.0 (C-55), 70.7 (C-55’), 70.5 (C-57), 70.4 (C-23), 69.9
(C-37, C-35’), 69.7 (C-35, C-34’), 68.7 (C-34), 68.5 (C-45), 68.4 (C-45’), 68.2
(C-47), 67.8 (C-54), 67.53 (C-62, C-54’), 67.48 (C-61), 67.3 (C-63), 67.1 (C-
43), 64.3 (C-44’), 61.9 (C-65, C-64), 61.6 (C-64’), 61.4 (C-65’, C-67), 55.7 (C-
22), 54.6 (C-21), 54.5 (C-27), 53.8 (C-25’), 53.7 (C-25), 20.5, 20.4, 20.3, 20.25,
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-
O-[(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!
4)]-O-(3,6-di-O-acetyl-a-d-mannopyranosyl)-(1!3)-O-(2-O-acetyl-b-d-
mannopyranosyl)-(1!4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-
glucopyranosyl)-(1!4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glu-
copyranosylazide (17): Hexasaccharide 16 (1288 mg, 556 mmol) was dis-
solved in pyridine (10 mL) and acetic anhydride (5 mL). The mixture was
concentrated after 16 h and codistilled (3ꢄ) with toluene. The acetylated
hexasaccharide was taken up in acetonitrile (200 mL) and a solution con-
taining p-toluenesulfonic acid (2 g) in acetonitrile (20 mL) was added.
After 1 h (TLC: hexane/acetone 1:1) the reaction was neutralized with
pyridine and evaporated to dryness. The residue was dissolved in CH2Cl2
(500 mL) and extracted with water, dilute KHCO3 and water. The organ-
ic phase was dried over MgSO4, concentrated and purified by flash chro-
matography (hexane/acetone, 1.1:1) to afford 17 (1020 mg, 80.8%). Rf =
0.23 (hexane/acetone 1:1); [a]2D3 =+2.0 (c=0.5, CH2Cl2); 1H NMR
(500 MHz, [D6]DMSO): d=7.95–7.63 (m, 16H, Pht), 7.35–6.72 (m, 20H,
Ar), 5.63 (dd, J2,3 =10.4 Hz, 1H, H-37), 5.59 (dd, J2,3 =10.2 Hz, 1H, H-35),
5.43 (d, J1,2 =8.3 Hz, 1H, H-17), 5.42 (d, JOH,4 =5.6 Hz, 1H, OH-43), 5.28
20.18, 20.1, 20.0, 19.8 (OAc); ESI-MS: m/z calcd for C146H150N8O60
:
2974.9; found 2997.9 [M+Na]+.
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-
O-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl-(1!4)]-
O-(3,6-di-O-acetyl-a-d-mannopyranosyl)-(1!3)-O-{3,4,6-tri-O-acetyl-2-
deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-O-[(3,4,6-tri-O-acetyl-
2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!6)]-O-(3,4-di-O-acetyl-
a-d-mannopyranosyl)-(1!6)}-O-(2-O-acetyl-b-d-mannopyranosyl)-(1!
4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!
4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosylazide (19):
A suspension of hexasaccharide acceptor 17 (721 mg, 320 mmol), imidate
D (590 mg, 261 mmol) and ground molecular sieves 4 ꢃ (530 mg) in abso-
lute CH2Cl2 (55 mL) was stirred for 30 min at ꢀ458C. Boron trifluoride
ethyl etherate (16 mL, 130 mmol) was added over 5 min. The reaction was
carried out for 3 h (cyclohexane/acetone, 1.5:1) and gradually warmed to
ꢀ58C. Subsequently, the solids were removed by filtration over Celite
and washed with CH2Cl2. After removal of the solvent the remainder was
purified by flash chromatography (cyclohexane/acetone, 1.7:1) to afford
19 (932 mg, 87.6%). Rf =0.19 (cyclohexane/acetone, 1.5:1); [a]2D3 =ꢀ2.1
(c=0.5, CH2Cl2); 1H NMR (500 MHz, [D6]DMSO): d=8.04–7.60 (m,
(d, J1,2 =9.8 Hz, 1H, H-11), 5.25 (d, J1,2 =9.9 Hz, 1H, H-15), 5.20 (d, J1,2
8.8 Hz, 1H, H-12), 5.08 (dd, J1,2, J2,3 <1.0 Hz, 1H, H-23), 4.99 (dd, J3,4
=
=
J
J
4,5 =9.2 Hz, 1H, H-47), 4.97 (dd, J3,4 =J4,5 =9.3 Hz, 1H, H-45), 4.89 (d,
gem =11.9 Hz, 1H, CH2O), 4.80 (d, J1,2 <1.0 Hz, 1H, H-14), 4.79 (d, Jgem
=
11.3 Hz, 1H, CH2O), 4.71 (m, 1H, H-34), 4.61 (d, J1,2 <1.0 Hz, 1H, H-13),
4.48 (s, 2H, CH2O), 4.62 (t, J6,OH =5.2 Hz, 1H, OH-63), 4.40 (s, 2H,
CH2O), 4.37 (d, Jgem =12.7 Hz, 1H, CH2O), 4.29 (d, Jgem =11.9 Hz, 1H,
CH2O), 4.22–3.90 (m, 14H, H-22, H-24, H-25, H-27, H-31, H-32, H-41, H-42,
H-57, H-6a4, H-6a,b5, H-6a,b7), 3.85 (m, 1H, H-55), 3.8–3.3 (m, 13H, H-21,
H-33, H-43, H-44, H-51, H-54, H-6a,b1, H-6a,b2, H-6a,b3, H-6b4), 3.24 (m,
1H, H-52), 3.03 (m, 1H, H-53), 2.02, 1.97, 1.75, 1.74, 1.66, 1.60 (6s, 27H,
OAc); 13C NMR (125 MHz, [D6]DMSO): d=170.1, 170.0, 169.9, 169.6,
169.4, 169.2, 167.4, 167.1 (C=O), 138.4, 138.1, 137.9 (C-i Ar), 135.1, 134.8
(C-4/5 Pht), 130.6, 130.5 (C-1/2 Pht), 128.2–127.1 (C-Ar), 123.8, 123.4 (C-
3/6 Pht), 97.4 (C-14), 97.0 (C-13), 96.5 (C-12), 96.2 (C-15), 95.1 (C-17), 84.8
(C-11), 76.6 (C-32), 76.5 (C-53), 76.3 (C-42), 76.20 (C-31), 76.16 (C-33), 75.7
12300
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 12292 – 12302

Synthesis of Multiantennary Complex Type N-Glycans
FULL PAPER
24H, Pht), 7.30–6.70 (m, 20H, Ar), 5.64 (m, 1H, H-35), 5.62 (m, 1H, H-
37), 5.48–5.39 (m, 3H, H-17, H-35’, H-37’), 5.31 (d, J1,2 =8.5 Hz, 1H, H-15),
5.29 (d, JOH,4 =5.2 Hz, 1H, OH-43), 5.25 (d, J1,2 =9.5 Hz, 1H, H-11), 5.18–
5.12 (m, 2H, H-12, H-15’), 5.04–4.97 (m, 3H, H-23, H-45, H-47), 4.94 (d,
69.9 (C-34’), 69.58 (C-37’’), 69.56 (C-37), 69.55 (C-35), 69.5 (C-35’), 69.1 (C-
37’), 68.7 (C-54’), 68.5 (C-34), 68.2 (C-52), 68.2 (C-57’), 68.1 (C-45), 67.9 (C-
45’), 67.9 (C-47), 67.8 (C-47’’), 67.6 (C-47’), 67.56 (C-54), 67.3 (C-62), 67.2
(C-61), 65.6 (C-63), 64.6 (C-43), 61.8 (C-64), 61.5 (C-65), 61.45 (C-67’’),
61.11 (C-67), 61.10 (C-67’), 60.7 (C-65’), 55.8 (C-22), 54.3 (C-27’’, C-21), 54.2
(C-27), 53.6 (C-25’), 53.4 (C-25, C-27’), 21.5–19.7 (OAc); ESI-MS: m/z
calcd for C182H184N10O76: 3725.08; found 3748.10 [M+Na]+.
J
1,2 =8.2 Hz, 1H, H-17), 4.89 (dd, J3,4 =J4,5 =9.5 Hz, 1H, H-45’), 4.85–4.80
(m, 3H, H-47’, H-34’, CH2O), 4.76–4.70 (m, 3H, H-14, H-34, H-44’), 4.50–
4.42 (m, 3H, H-13, CH2O), 4.39 (d, Jgem =12.2 Hz, 1H, CH2O), 4.37–3.82
(m, 25H, CH2O, H-14’, H-22, H-24, H-24’, H-25, H-25’, H-27, H-31, H-32, H-
41, H-42, H-55, H-57, H-6a4, H-6a,b5, H-6a5’, H-6a,b7, H-6a,b7’), 3.80–3.62
(m, 6H, H-21, H-27’, H-44, H-54, H-54’, H-6b5’), 3.59 (m, 1H, H-6a2), 3.57–
3.50 (m, 2H, H-51, H-57), 3.48–3.36 (m, 4H, H-6a1, H-6b2, H-6b4, H-6a4’),
3.30–3.17 (m, 6H, H-33, H-43, H-52, H-55’, H-6b1, H-6a3), 3.04–2.94 (m,
2H, H-6b3, H-6b4’), 2.90 (m, 1H, H-53), 2.02, 2.015, 2.01, 2.0, 1.98, 1.96,
1.92, 1.78, 1.77, 1.76, 1.75, 1.74, 1.73, 1.68, 1.63 (15s, 51H, OAc);
13C NMR (125 MHz, [D6]DMSO): d=170.1–167.2 (C=O), 138.3, 138.1,
138.0 (C-i Ar), 135.1, 134.9, 134.6 (C-4/5 Pht), 130.9, 130.7, 130.6, 130.5
(C-1/2 Pht), 128.2–127.0 (C-Ar), 123.8, 123.7, 123.4 (C-3/6 Pht), 97.9 (JC-
1,H-1 =180.0 Hz from a coupled HMQC spectrum, C-14a), 97.7 (C-17’),
96.70 (C-12, JC-1,H-1 =162.7 Hz from a coupled HMQC spectrum, C-13b),
96.65 (JC-1,H-1 =172.6 Hz from a coupled HMQC spectrum, C-14’a), 96.2
(C-15), 96.0 (C-15’), 95.3 (C-17), 84.7 (C-11), 77.0 (C-42), 76.8 (C-31), 76.6
(C-33), 75.7 (C-51), 75.5 (C-32), 75.3 (C-41), 74.3 (C-53), 74.2 (C-52), 73.9
(CH2O), 73.4 (C-24), 73.3 (C-24’), 73.2, 72.3 (CH2O), 71.6 (CH2O, C-44),
71.0 (C-55), 70.8 (C-57’), 70.7 (C-55’), 70.5 (C-57), 70.3 (C-23), 70.1 (C-37’),
69.9 (C-35’, C-37), 69.8 (C-35), 69.6 (C-34’), 68.8 (C-34), 68.6 (C-45), 68.5
(C-54’), 68.4 (C-47’), 68.3 (C-45’), 68.2 (C-47), 67.9 (C-54, C-64’), 67.4 (C-61,
C-62), 66.5 (C-63), 66.2 (C-43), 64.8 (C-44’), 61.9 (C-64, C-65), 61.5 (C-67’),
61.4 (C-65’, C-67), 55.8 (C-22), 54.6 (C-21), 54.5 (C-27), 53.9 (C-27’), 53.8
(C-25, C-25’), 20.4, 20.2, 20.1, 20.0, 19.8 (OAc); ESI-MS: m/z calcd for
C164H167N9O68: 3349.99; found 3372.5 [M+Na]+.
O-(2-Acetamido-2-deoxy-b-d-glucopyranosyl)-(1!2)-O-[2-acetamido-2-
deoxy-b-d-glucopyranosyl-(1!4)]-O-a-d-mannopyranosyl-(1!3)-O-{2-
acetamido-2-deoxy-b-d-glucopyranosyl)-(1!2)-O-[(2-acetamido-2-deoxy-
b-d-glucopyranosyl)-(1!6)]-O-a-d-mannopyranosyl-(1!6)}-O-b-d-man-
nopyranosyl-(1!4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-b-d-gluco-
pyranosyl)-(1!4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-d-glucopyrano-
sylazide (21): Nonasaccharide 19 (900 mg, 0.27 mmol) was dissolved in n-
butanol (90 mL) and ethylenediamine (27.5 mL) and stirred at 908C for
16 h (TLC: isopropanol/1m ammonium acetate 2:1). The volatiles were
distilled off and the remainder was dried in vacuo until the weight re-
mained constant. Subsequently, pyridine (90 mL) and acetic anhydride
(45 mL) were added. After 16 h (TLC: CH2Cl2/methanol, 10:1) the mix-
ture was concentrated and codistilled with toluene (3ꢄ). The remainder
was dried in high vacuum and methylamine (90 mL, 40% in H2O) was
added. After stirring for 2 h at ambient temperature (TLC: isopropanol/
1m ammonium acetate 2:1) the solution was evaporated and dried in
high vacuum. The remainder was dissolved in water (50 mL) and passed
in portions of 10 mL over three connected SepPak cartridges (Waters).
Elution of each batch was performed with water (30 mL) followed by
acetonitrile/water (10 mL each, ratios 1:19, 1:9 and 1:6.6). The product
was eluted with acetonitrile/water (30 mL, 4:1) and lyophilized yielding
21 (485.2 mg, 85.7%). Rf amine=0.55 (isopropanol/1m ammoniumace-
tate 2:1); Rf peracetate=0.60 (CH2Cl2/methanol, 10:1); Rf 21=0.67 (iso-
propanol/1m ammoniumacetate 2:1); [a]2D3 =ꢀ23.8 (0.4, H2O); 1H NMR
(500 MHz, D2O [D6]DMSO as internal standard): d=7.41–6.89 (m, 20H,
Ar), 4.98 (d, J1,2 <1.0 Hz, 1H, H-14), 4.78–2.95 (m, 69H, CH2O, CH2O,
H-14’, CH2O, CH2O, H-11, CH2O, H-13, H-15, H-17, CH2O, H-17’, CH2O,
CH2O, H-12, H-15’, H-24, H-6a4’, H-34, H-23, H-6a7, H-6a3, H-6a7’, H-42, H-
6a5, H-41, H-43, H-24’, H-54, H-6a4, H-6a1, H-6b7, H-21, H-27, H-22, H-6b7’,
H-34’, H-6b5, H-25, H-44, H-27’, H-6a2, H-54’, H-6a,b5’, H-6b1, H-31, H-6b3,
H-6b4, H-51, H-35, H-32, H-37, H-37’, H-6b4’, H-33, H-6b2, H-25’, H-57, H-35’,
O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-
O-[3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl-(1!4)]-
O-(3,6-di-O-acetyl-a-d-mannopyranosyl)-(1!3)-O-{3,4,6-tri-O-acetyl-2-
deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!2)-O-[3,4,6-tri-O-acetyl-2-
deoxy-2-phthalimido-b-d-glucopyranosyl-(1!4)]-O-[3,4,6-tri-O-acetyl-2-
deoxy-2-phthalimido-b-d-glucopyranosyl-(1!6)]-O-(3-O-acetyl-a-d-
mannopyranosyl)-(1!6)}-O-(2-O-acetyl-b-d-mannopyranosyl)-(1!4)-O-
(3,6-di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosyl)-(1!4)-3,6-
di-O-benzyl-2-deoxy-2-phthalimido-b-d-glucopyranosylazide (20): Hexa-
saccharide 17 (200 mg, 88 mmol), donor E (428 mg, 264 mmol) and ground
molecular sieves 4 ꢃ (600 mg) in absolute CH2Cl2 (32 mL) were stirred
for 30 min at 08C. The suspension was cooled to ꢀ108C and boron tri-
fluoride ethyl etherate (5.8 mL, 47 mmol) was added over 5 min. The reac-
tion was stirred at ꢀ108C (TLC: hexane/acetone 1:1). After a further 3 h
boron trifluoride ethyl etherate (2.5 mL, 20.3 mmol) was added and 1 h
later boron trifluoride ethyl etherate (2.5 mL, 20.3 mmol) and donor E
(100 mg, 62 mmol) were added. The reaction was continued for 1 h and
the solids were removed by filtration over Celite and washed with
CH2Cl2. After removal of the solvent the remainder was purified by flash
chromatography (cyclohexane/ethyl acetate 1:2) to afford 20 (213 mg,
64.9%). Rf =0.07 (cyclohexane/ethyl acetate 1:2); [a]2D3 =+0.2 (c=0.5,
CH2Cl2); 1H NMR (500 MHz, [D6]DMSO): d=8.04–7.40 (m, 28H, Pht),
7.30–6.65 (m, 20H, Ar), 5.67–3.00 (m, 75H, H-37, H-35, H-37’’, H-17, H-35’,
H-15, H-17’’, H-11, H-37’, H-12, OH-43, H-45, H-47’’, H-23, H-47, CH2O, H-
45’, H-34’, H-47’, H-34, H-14, H-17’, H-13, CH2O, CH2O, H-15ꢂ, CH2O, CH2O,
CH2O, H-24, CH2O, CH2O, H-6a5, H-6a7’, H-6a7’’, H-6a7, H-6a4’, H-57’’, H-
25, H-31, H-6b7, H-41, H-27, H-22, H-14’, H-57, H-27’’, H-6b5, H-6a4, H-55,
H-25’, H-27’, H-32, H-6a5’, H-6b7’’, H-6b7’, H-21, H-44, H-54, H-24’, H-6a2, H-
43, H-51, H-6b5’, H-6b2, H-6b4, H-6a1, H-54’, H-6a3, H-6b1, H-55’, H-42, H-
33, H-52, H-44’), 2.72 (m, 1H, H-53), 2.55 (m, 1H, H-6b3), 2.28–2.20 (m,
2H, H-6b4’, H-57’), 2.18–1.68 (19s, 57H, OAc); 13C NMR (125 MHz,
[D6]DMSO): d=170.1–167.2 (C=O), 138.80, 138.75, 138.72 (C-i Ar),
135.8, 135.6, 135.5 (C-4/5 Pht), 131.5, 131.3, 131.26, 131.1 (C-1/2 Pht),
129.1–127.8 (C-Ar), 124.5, 124.4, 124.2 (C-3/6 Pht), 97.8 (C-15’), 97.7 (C-
14), 97.6 (C-13), 96.8 (C-17’’), 96.5 (C-14’), 96.0 (C-12), 95.6 (C-15), 95.5 (C-
17’), 94.9 (C-17), 84.4 (C-11), 78.3 (C-32), 76.7 (C-33), 75.8 (C-31), 75.3 (C-
51), 74.6 (C-41), 73.6 (C-42), 73.4 (CH2O), 73.3 (C-53), 72.9 (C-44’), 72.7
(CH2O), 72.6 (C-24’), 72.04 (C-24), 72.03 (CH2O), 71.4 (C-44, CH2O), 70.6
(C-55), 70.5 (C-57), 70.4 (C-55’), 70.3 (C-23), 70.13 (C-57’’), 70.10 (C-64’),
H-47, H-45, H-55, H-47’, H-45’, H-57’, H-44’, H-53, H-52), 2.88 (dd, J4,5
=
7.9 Hz, 1H, H-55’), 2.09–1.38 (6s, 18H, NAc); 13C NMR (125 MHz, D2O
[D6]DMSO as internal standard): d=174.9, 174.7, 174.6, 174.4, 174.1 (C=
O), 138.3, 138.1, 137.4, 137.2 (C-i Ar), 129.2–128.1 (C-Ar), 101.9 (C-17),
101.8 (C-17’), 100.7 (C-12), 100.5 (C-13), 100.2 (C-15), 99.9 (C-15’), 99. 5 (C-
14), 97.0 (C-14’), 88.8 (C-11), 81.0 (C-33), 80.4 (C-31), 80.3 (C-32), 78.3 (C-
44), 77.6 (C-42), 76.9 (C-24’), 76.5 (C-24), 76.3 (C-51), 76.21 (C-57), 76.16
(C-55), 76.1 (C-57’), 75.8 (C-55’), 75.4 (C-41), 74.6 (C-53), 74.12, 74.06,
(CH2O), 74.03 (C-37’), 73.98 (C-52), 73.91 (C-35), 73.7 (C-37), 73.32
(CH2O), 73.29 (C-35’), 73.23 (CH2O), 72.1 (C-54), 71.8 (C-54’), 70.7 (C-23),
70.3 (C-47’), 70.2 (C-45), 70.10 (C-47), 70.06 (C-64’), 69.89 (C-34’), 69.88 (C-
45’), 68.4 (C-34), 68.3 (C-62), 68.0 (C-61), 67.7 (C-44’), 65.8 (C-43), 65.6 (C-
63), 61.4 (C-64), 61.1 (C-65), 61.0 (C-67’), 60.9 (C-67), 60.5 (C-65’), 55.9 (C-
27, C-27’, C-25’), 55.7 (C-25), 55.2 (C-22), 53.9 (C-21), 20.4, 20.2, 20.1, 20.0,
19.8 (NAc); ESI-MS: m/z calcd for C94H133N9O45: 2107.84; found 2130.8
[M+Na]+.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft, the
Fonds der Deutschen Chemischen Industrie and the Leonhard-Lorenz-
Stiftung.
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Received: July 9, 2009
Published online: October 5, 2009
12302
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ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 12292 – 12302