Synthesis of an Fmoc-Asn-heptasaccharide building block and its application to chemoenzymatic glycopeptide synthesis

Article abstract of DOI:10.1016/j.carres.2010.02.022

The Fmoc-protected heptasaccharide asparagine building block β-GlcNAc-(1→2)-α-Man-(1→3)-[β-GlcNAc-(1→2) -α-Man-(1→6)]β-Man-(1→4)-β-GlcNAc-(1→4) -β-GlcNAc-(Fmoc)Asn was obtained by chemical synthesis. Two flexible strategies were developed with optimized c

Full text of DOI:10.1016/j.carres.2010.02.022

Carbohydrate Research 345 (2010) 1306–1315
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of an Fmoc-Asn-heptasaccharide building block and its application
to chemoenzymatic glycopeptide synthesis
*
Stefano Mezzato, Carlo Unverzagt
Bioorganische Chemie, Gebäude NWI, Universität Bayreuth, 95440 Bayreuth, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The Fmoc-protected heptasaccharide asparagine building block b-GlcNAc-(1?2)-a-Man-(1?3)-[b-Glc-
NAc-(1?2)-a-Man-(1?6)]b-Man-(1?4)-b-GlcNAc-(1?4)-b-GlcNAc-(Fmoc)Asn was obtained by chemi-
cal synthesis. Two flexible strategies were developed with optimized conditions for the simultaneous
debenzylation of the sugar and the amino acid part. The heptasaccharide asparagine building block is a
partial structure of many glycoproteins and can be used for glycopeptide synthesis in solution and on
the solid phase. In this work the heptasaccharide asparagine was elongated in solution to an Fmoc-gly-
copentapeptide methylester. After chemical cleavage of the Fmoc group the methylester was removed
enzymatically by chymotrypsin. The use of b-(1?4)-galactosyltransferase and a-(2?6)-sialyltransferase
in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor
resulting in an undecasaccharide glycopentapeptide.
Received 29 January 2010
Received in revised form 22 February 2010
Accepted 24 February 2010
Available online 17 March 2010
Keywords:
Glycopeptide
Glycoprotein
Galactosyltransferase
Sialyltransferase
N-Glycan
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
transferase and a-(2?6)-sialyltransferase gave the corresponding
glycopeptide A bearing a biantennary N-glycan undecasaccharide.
With the growing demand for human recombinant glycoprotein
therapeutics the synthesis of defined glycoforms of these valuable
compounds is receiving an increasing interest. Due to the inherent
heterogeneity of expressed glycoproteins and the lack of appropri-
ate high-resolution separation methods, chemical synthesis1–3 has
advanced in recent years to become a viable option for the prepa-
ration of pure glycoforms. However, the synthesis of a glycoprotein
is not a straightforward task and relies on many factors, notably
the given protein sequence and the number and kind of glycosyla-
tion sites.4 In particular, N-glycoproteins have challenged scientists
and a few examples have been successfully synthesized recently.5–10
Currently, the most flexible approach to synthetic N-glycoproteins
appears to be based on native chemical ligation,11 which typically
requires the synthesis of glycopeptides and glycopeptide thioesters
on the solid phase. In principle these glycopeptides can be assem-
bled either by a late introduction of the glycosyl amine following
the Lansbury protocol12 or by an incorporation of an Fmoc-pro-
tected glycosyl amino acid.13 Here we wish to report the synthesis
of the Fmoc-Asn building block 1, which is suitable for the
synthesis of complex-type N-glycopeptides in solution14 and on
the solid phase15 (Scheme 1). This glycosyl amino acid contains a
deprotected biantennary N-glycan heptasaccharide, which was
elongated in solution to a heptasaccharide glycopentapeptide.
Enzymatic oligosaccharide extension16 by b-(1?4)-galactosyl-
2. Results and discussion
The synthesis of N-glycopeptides or N-glycopeptide thioesters
with the complexity of compound A or B was demonstrated by uti-
lizing building block 1.14,15 The Fmoc-protected glycosyl amino acid
1 was obtained from the chemically synthesized heptasaccharide
azide 217–20 following two routes, which were optimized and differ
from our initial report.14 An alternative approach21 to compound 1
was established using a glycopeptide isolated from egg yolk.22
The heptasaccharide 2 can be synthesized according to the dou-
ble regio- and stereoselective glycosylation approach23 and subse-
quent removal of the base-labile protecting groups.18 Thus
heptasaccharide 2 retains an anomeric azide and four benzyl
ethers. This protection pattern requires the selective reduction of
the azide moiety and coupling to an amino acid prior to the deben-
zylation of the sugar part.17 A selective debenzylation in the pres-
ence of an azide would be desirable; however, for oligosaccharides
no promising method has been established. Due to the susceptibil-
ity of the Fmoc group to catalytic hydrogenation24 the introduction
of an Fmoc-Asp-OBzl residue was a priori discarded and an N-ben-
zyloxycarbonyl group was employed for the aspartic acid deriva-
tives
3 and 4. A subsequent global debenzylation of the
heptasaccharide-Asn conjugates was envisioned followed by the
installation of the Fmoc moiety.
Prior to the attachment of Z-Asp-OBzl 3, heptasaccharide azide
2 (Scheme 2) was reduced in methanol using 1,3-propanedithiol
* Corresponding author. Tel.: +49 921 55 2670; fax: +49 921 55 5365.
E-mail address: carlo.unverzagt@uni-bayreuth.de (C. Unverzagt).
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.02.022

S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
1307
OH
OH
O
OH
O
OH OH
OH
OH
OH
O
O
HOOC
O
HO
O
H
N
H
N
O
O
O
O
OH
HO
HO
HO
O
O
O
O
NH2
N
H
N
H
HO
HO
AcHN
HO
NHAc
OH
HO
OH OH
O
HO
OH
O
HOOC
O
O
O
O
HO
HO
O
O
OH
HO
HO
HO
OH
NHAc
O
AcHN
O
O
HO
NHAc
OH
HO
HO
O
O
NH
HO
A
NHAc
OH
synthesis in solution
O
HO
HO
HO
HO
O
O
O
H
HO
N
O
HO
OH
NHAc
O
O
O
HO
HO
HO
HO
HO
HO
O
O
O
OH
OH
NHAc
O
O
O
NHAc
HO
HO
NH
O
O
O
HO
1
NHAc
OH
H
2N
NH
H2N
NH
NH2
NH2
solid phase synthesis
HN
O
HN
O
O
O
O
H
N
H
H
N
H
N
H2N
N
S
O
N
H
N
H
N
N
H
N
H
H
HO
HO
O
O
O
O
O
O
O
O
O
O
HO
HO
HO
HO
HO
HO
S
OH
NHAc
O
O
O
HO
HO
HO
HO
HO
HO
O
O
OH
NHAc
O
OH
O
O
NHAc
HO
HO
NH
O
O
O
HO
B
NHAc
OH
Scheme 1.
and DIPEA,25 which ensures basic conditions, thus preventing ano-
merization of the glycosyl amine. Special attention is needed when
coupling the aspartic acid side chain with the glycosyl amine. The
unprotected hydroxyl groups of the sugar moiety are susceptible to
acylation, therefore only a minimal excess of amino acid should be
used. Additionally, an appropriate solvent is required to properly
dissolve both the polar heptasaccharide and the hydrophobic
aspartic acid derivative 3. In our initial report, the coupling of
Z-Asp-OBzl (3) to the glycosyl amine was accomplished via an
intermediate pentafluorophenyl ester.26 A more convenient proto-
col employs the in situ activation of the aspartic acid side chain
using PyBOP (benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phospho-
nium hexafluorophosphate). After a short preactivation, 4 equiv
of 3 gave full acylation of the glycosyl amine in NMP (N-methyl-
pyrrolidone) within 10 min. The majority of the reagents were sep-
arated from the resulting glycosyl amino acid 5 by solid-phase
extraction (SPE), and subsequently 5 was purified by RP-HPLC. A
preliminary isolation of 5 by SPE permitted faster HPLC purifica-
tion, furnishing pure 5 in 74% yield. Long coupling times and excess
amino acid are undesirable, as random esterification occurred at
one of the hydroxyl functions. The monoacylated by-products,
approximately 6% overall yield, were separated by SPE in a fraction
eluting after 5.
amino acid 7. Electrospray-ionization mass spectrometry indicated
that the methyl ester 7b of 7 had formed (m/z +CH3) along with an
aspartimide 7a (m/z –H2O). Succinimide formation of aspartic acid
derivatives is a known problem in peptide synthesis,27,28 and b-ben-
zyl esters of aspartic acid undergo both acid- and base-catalyzed
ring closure.29 Nevertheless, aspartimide formation from a glycosyl
asparagine a-benzyl ester has not been described so far. It is likely
that the acetic acid renders the benzyl ester susceptible to transeste-
rification and to intramolecular nucleophilic attack by the nitrogen
on the b-amide. The formation of a glycosylated aspartimide after
activation of the a
-carboxylate was reported.30,31
To overcome the side reactions during hydrogenolysis different
solvent systems were tested. Among these, a mixture of THF and
water proved to be the most effective. No acid was required and
both components were non-nucleophilic. Indeed, in this solvent
the side products found in methanol–acetic acid were not gener-
ated. Hence, further purification was not necessary and the palla-
dium catalyst was simply filtered off after hydrogenation to
afford 7 nearly quantitatively. The N-terminus of 7 was protected
using Fmoc-OSu and triethylamine, without affecting either the
hydroxyl or the carboxyl functions.32 The target building block 1
was isolated by solid-phase extraction removing unreacted
Fmoc-OSu and further purified by RP-HPLC.
The hydrogenolysis of compound 5 required special conditions
and the choice of the solvent was fundamental for the outcome of
the reaction. Initially, PdO-hydrate was used as a catalyst in metha-
nol containing 10% of acetic acid. After hydrogenolysis of 5, two
by-products were found along with the fully unprotected glycosyl
A second approach to the glycosyl amino acid 1 was conducted
using Z-Asp-OtBu 4. The tert-butyl ester is stable to hydrogenation
and should reduce the risk for both transesterification and
succinimide formation. Furthermore a tert-butyl ester should allow
for a selective acetylation of the complete carbohydrate part at a

1308
S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
O
O
H
H
N
O
N
O
O
O
O
O
O
O
HO
HO
O
O
O
O
O
HO
HO
HO
OH
OH
HO
3
4
NHAc
O
O
O
HO
HO
HO
HO
HO
HO
OBn
OH
NHAc
O
O
O
NHAc
BnO
HO
O
N3
O
BnO
NHAc
2
OBn
2
5
2
6
1. propanedithiol, DIPEA, MeOH
2. 3, PyBOP, DIPEA, NMP
(1.-2.:74%, RP-HPLC)
1. propanedithiol, DIPEA, MeOH
2. 4, PyBOP, DIPEA, NMP
(1.-2.: 84%, RP-HPLC)
O
HO
HO
HO
O
O
H
O
O
O
R
O
N
HO
5'
5
4'
4
HO
O
O
HO
NHAc
O
O
O
HO
HO
HO
HO
HO
OBn
HO
OH
NHAc
O
O
O
NHAc
5 R = Bn
BnO
HO
O
NH
2
3
1
O
O
BnO
6 R
= tBu
NHAc
OBn
5
6
H2, Pd, HOAc, THF, H2O
(quantitative)
H2, Pd, THF, H2O
(quantitative)
8
9
Fmoc-OSu, Et3N, dioxane, H2O
(60%)
7
O
HO
HO
HO
O
O
H
O
R1
O
N
HO
HO
O
HO
R
NHAc
O
O
O
O
HO
HO
HO
HO
HO
O
OH
HO
R1
OH
NHAc
7 R
O
= H,
= H
O
NHAc
HO
HO
O
NH
O
8 R = H, R1 = tBu
HO
O
9 R = Fmoc, R1 = tBu
NHAc
OH
7
1
9
Fmoc-OSu, Et3N, dioxane, H2O
(63%, RP-HPLC)
TFA
(72%, RP-HPLC)
1
Scheme 2.
later stage (e.g., compound 9) by preventing unwanted activation of
the -carboxyl group via a mixed anhydride. For this approach,
Z-Asp-OtBu 4 was liberated from the corresponding dicyclo-
hexylammonium salt. Azide 2 was reduced as described above
and subsequently coupled with 4 preactivated with PyBOP in
NMP. Again, the coupling was complete within a few minutes. It
a

S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
1309
is worth mentioning that if water was added before solvent re-
moval, only 2% of over acylated product was isolated after solid-
phase extraction. In the coupling experiments where the excess of
activated amino acid was not hydrolyzed in situ, up to 10% of the
glycosyl amino acid had undergone unwanted esterification by
the aspartic acid at one hydroxyl group, mainly during concentra-
tion of the coupling solution.
Compound 6 can be hydrogenated more safely than the corre-
sponding benzyl ester 5. No aspartimide formation could be de-
tected when 6 was dissolved in methanol–acetic acid, whereas
the benzyl ester 5 gave rise to ring closure under the same condi-
tions. Unexpectedly, hydrogenolysis of 6 in THF–water proceeded
much slower than 5. While the Z group was quickly cleaved, the
benzyl ethers remained intact even after prolonged hydrogenation,
as confirmed by mass spectrometry. It can be assumed that the lib-
erated amino group interferes with the palladium catalyst,
whereas in the case of 7 the amino group is protonated in the zwit-
terionic amino acid. Fully debenzylated 8 was finally obtained after
addition of 5% acetic acid to the THF–water hydrogenation solvent.
The tert-butyl ester of 8 was completely stable under these condi-
tions and the amino group was subsequently masked with an Fmoc
function to furnish 9.
was finally cleaved with TFA and Fmoc heptasaccharide asparagine
1 was obtained in 72% yield after RP-HPLC. The structure was iden-
tical to the batch synthesized via 5, as confirmed by NMR, MS and
retention times on HPLC.
With the Fmoc-heptasaccharide-Asn building block 1 in hand
the reactivity in peptide couplings was probed on the partial se-
quence 21–25 of bovine ribonuclease. After activation of 1 using
TBTU–HOBt (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroni-
um tetrafluoroborate–1-hydroxybenzotriazole) in NMP the cou-
pling to tyrosine methyl ester 10 was complete within 10 min.
The resulting glycopeptide 11 was isolated in 74% after RP-HPLC.
Removal of the Fmoc group was achieved in NMP containing 10%
of piperidine. Notably the Fmoc-deprotection required a reaction
time of 30 min indicating a steric hindrance of the substrate. Under
these conditions the methyl ester was not affected and the inter-
mediate glycopeptide-amine 12 was isolated by RP-HPLC. The seg-
ment coupling of 12 with the Fmoc-protected serine tripeptide 13
required some optimization. A test reaction indicated that the acti-
vation of 13 with TBTU–HOBt gave only slow coupling with 12.
When the stronger coupling reagent HATU was employed in the
presence of collidine34 the formation of the glycopentapeptide pro-
ceeded to completion within 10 min and the protected glycopep-
tide 14 was obtained in 70% yield after HPLC purification. The
HPLC peak for 14 showed a satellite peak (10–15%), which was effi-
ciently removed during purification indicating that some racemi-
zation35 might have occurred during the segment coupling.
Despite the unprotected hydroxyl groups of the heptasaccharide
part only the amino group was found to react in a chemoselective
manner (Scheme 3).
Compound 9 is a versatile intermediate, which is amenable to
O-acetylation without side reactions, because both the C- and the
N-terminus are blocked.33 Additionally, the tert-butyl and the
Fmoc protective groups are orthogonal, providing greater flexibil-
ity than the strategy mentioned above. However, hydroxyl protec-
tion of the glycosyl amino acid33 was not required because 1 could
be successfully coupled on a solid support.15 The tert-butyl ester
OH
OH
OMe
H2N
O
10
OH
OH
OH
O
O
H
O
O
O
N
OMe
HO
HO
HO
HO
R
N
H
TBTU, HOBt, NMP, DIPEA, 10
NHAc
O
OH
O
O
O
O
1
O
OH
HO
HO
HO
HO
OH
NHAc
O
O
O
HO
NHAc
HO
O
O
NH
HO
11 R = Fmoc
12 R = H
NHAc
OH
OH
O
OH
O
O
H
N
OH
N
N
H
Fmoc
OH
H
HO
OH
O
OH
O
13
OH
OH
OH
O
O
O
H
N
H
N
O
R
O
O
O
HO
HO
HO
HO
13, HATU, collidine, NMP
N
N
N
H
R1
HO
HO
H
H
NHAc
O
HO
12
OH
O
O
O
O
OH
HO
HO
OH
NHAc
O
14 R = Fmoc, R1 = Me
O
O
HO
NHAc
HO
O
R1
= Me
O
O
NH
15 R
= H,
HO
16 R = H, R1 = H
NHAc
OH
1. UDP-Gal, galactosyltransferase, alkaline phosphatase
2. CMP-Neu5Ac, α2, 6-sialyltransferase, alkaline phosphatase
A
Scheme 3.

1310
S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
Prior to the envisioned enzymatic elongation of the sugar part
of the final glycopeptide A can be viewed in the 500 MHz 1H NMR
spectrum (Scheme 4). The assignments of the 1H and 13C reso-
nances of the synthetic building blocks and glycopeptides were
carried out by 2D-NMR methods43 including TOCSY, NOESY,
HMQC, HMQC–COSY, HMQC–DEPT,44 and HMQC–TOCSY.
In summary, we have optimized the attachment of aspartic acid
onto partially protected synthetic N-glycans. These glycoconju-
gates were deprotected and converted to their Fmoc derivatives.
The glycosyl amino acid 1 can be applied either to solid-phase syn-
thesis or to the solution synthesis leading to glycopeptides in good
yields and high purity. Efficient methods for the generation of com-
plex glycopeptides and derivatives thereof are a prerequisite for
the further development of chemical synthesis of homogeneous
glycoproteins.
the Fmoc group and the methyl ester were removed because these
functions tend to be unstable in the presence of free amino groups
found in proteins. At first, the Fmoc group was cleaved in NMP con-
taining 5% of piperidine followed by enzymatic hydrolysis of the
methyl ester with the protease chymotrypsin without intermedi-
ate purification. In contrast to the alkaline hydrolysis of methyl es-
ters36 their enzymatic hydrolysis proceeds without racemization.
Chymotrypsin is selective for the cleavage of peptides and peptide
esters after aromatic amino acids or leucine residues.37 In the pep-
tide sequence of intermediate 15, only one cleavage site is present
at the C-terminal methyl ester, which allows for an aqueous reac-
tion medium and extended reaction times. Generally, proteases
convert the more reactive esters faster than the corresponding
amides. In the case of additional cleavage sites within the peptide
sequence, the amidase activity of a protease can be greatly reduced
by conducting the reaction in the presence of an organic cosolvent
(e.g., 50% DMF) thus ensuring selective hydrolysis of the ester over
the amide.38 After complete chemical and enzymatic deprotection,
the free glycopeptide 16 was isolated by gel filtration in 96% yield.
The stepwise elongation of the terminal GlcNAc moieties of the
heptasaccharide part of 16 was accomplished by enzymatic sugar
transfer39–42 in a one-pot manner.16 Dual galactosylation was
achieved by using bovine b-(1?4)-galactosyltransferase, UDP-gal-
actose, and the reactivity-enhancing alkaline phosphatase.16
According to TLC analysis the reaction had already proceeded to
3. Experimental
3.1. General methods
TLC was performed on silica gel plates (60-F254; E. Merck,
Darmstadt) and visualized by spraying with 0.5 M H2SO4 in EtOH
containing 0.1% orcinol. UDP-galactose, cytidine-50-monophos-
pho-N-acetylneuraminic acid, bovine b-(1?4)-galactosyltransfer-
ase (E.C. 2.4.1.22.),
a-(2?6)-sialyltransferase (E.C. 2.4.99.1.), and
bovine serum albumin were obtained from Sigma Chemical Co.
Calf intestinal alkaline phosphatase (molecular biology grade)
(E.C. 3.1.3.1.) was obtained from Boehringer Mannheim. Amino
acids, peptides, and other reagents were purchased from Bachem
(Läufelfingen, Switzerland). PyBOP was purchased from Iris Biotech
(Marktredwitz, Germany).
For size exclusion chromatography a Pharmacia Hi Load Super-
dex 30 column (600 ꢀ 16 mm) was used on a Pharmacia LKB gra-
dient system 2249 equipped with a Pharmacia LKB Detector
VWM 2141 (Freiburg, Germany). Analytical and preparative HPLC
was performed on either a Pharmacia Äkta Purifier 10 or a Pharma-
cia Äkta Basic device.
completion within 2 h at 37 °C. The subsequent dual
a-(2?6)-
sialylation was carried out by adding -(2?6)-sialyltransferase
a
and CMP-sialic acid to the reaction mixture. However, this conver-
sion required longer incubation times and a second addition of en-
zyme and sugar nucleotide. In the subsequent separation of the
reaction mixture by gel filtration, only 3% of monosialylated nona-
saccharide was found indicating that the enzymatic galactosylation
was nearly quantitative. The desired undecasaccharide-glycopen-
tapeptide A was obtained in 91% yield, which corresponds to an
average yield per enzymatic sugar transfer of over 97%. The purity
Scheme 4. 500 MHz-1H NMR of A in D2O.

S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
1311
0
Solid-Phase Extraction (SPE) was performed with Waters Sep-
Pak Vac C18 cartridges. NMR spectra were recorded on Bruker
AMX 500 and DRX 500 instruments. For spectra recorded in D2O
the HOD signal (4.81 ppm) was used as a reference. ESI-TOF mass
spectra were recorded on a Micromass LCT instrument coupled
to an Agilent 1100 HPLC. MALDI-TOF mass spectra were recorded
on a Voyager Biospectrometry workstation (Vestec/Perseptive)
MALDI-TOF mass spectrometer, using 2,5-dihydroxybenzoic acid
(DHB) as a matrix. Specific rotation was recorded on a Perkin–El-
mer Polarimeter 241.
(C-41), 74.4 (C-52), 74.2 (C-54), 73.9 (C-35, C-35 ), 73.8 (CH2O),
0
0
73.6 (C-54 ), 73.2, 72.2, 71.8 (CH2O), 70.5 (C-45 ), 70.3 (C-45), 69.9
0
(C-34, C-34 ), 69.1 (C-23), 68.7 (C-62), 68.3 (C-61), 67.6 (C-44), 67.1
0
(C-44 ), 66.0 (CH2O, C-63), 65.6 (CH2O), 64.9 (C-43), 61.5 (C-64),
0
0
0
61.1 (C-64 ), 61.0 (C-65), 60.8 (C-65 ), 55.6 (C-25, C-25 ), 55.3 (C-
22), 53.2 (C-21), 50.4 (
(NAc); ESI-MS (m/z) calcd for C97H126N6O40 [M]+: 2014.80; found:
2037.81 [M+Na]+, 1030.41 [M+2Na]2+
aC Asn), 36.8 (bC Asn), 23.2, 22.9, 22.7
.
3.3. N4-{O-(2-Acetamido-2-deoxy-b-
-mannopyranosyl-(1?3)-[O-(2-acetamido-2-deoxy-b-
copyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b- -man-
nopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b- -glucopyran-
-glucopyranosyl)}- -aspar-
D
-glucopyranosyl)-(1?2)-O-
a-
D
D
-glu-
3.2. N4-{O-(2-Acetamido-2-deoxy-b-
-mannopyranosyl-(1?3)-[O-(2-acetamido-2-deoxy-b-
copyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b- -man-
nopyranosyl-(1?4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-
b- -glucopyranosyl)-(1?4)-(2-acetamido-3,6-di-O-benzyl-2-de-
oxy-b- -asparagine
-glucopyranosyl)}-N2-benzyloxycarbonyl-
D
-glucopyranosyl)-(1?2)-O-
-glu-
a-D
D
a-
D
D
D
a-D
D
osyl)-(1?4)-(2-acetamido-2-deoxy-b-
D
L
agine 7
D
D
L
Protected glycosyl amino acid 5 (129 mg, 64.0 lmol) and PdO-
benzyl ester 5
hydrate (190 mg) were suspended in THF–water (10 mL, 1:1).
The reaction flask was purged with argon and the mixture was stir-
red vigorously under a hydrogen atmosphere for 41 h (TLC: isopro-
panol–1 M NH4OAc 2:1). The solvent was removed in vacuo and
after dilution with water, the catalyst was filtered off through a
syringe filter unit (Millipore, IC Millex-LG, 25 mm external diame-
Heptasaccharide 2 (294 mg, 173 lmol, 1 equiv) was dissolved
in freshly distilled MeOH (9 mL) and ethyldiisopropylamine (DI-
PEA) (305 L, 1.75 mmol) was added. The reaction flask was
purged with argon and 1,3-propanedithiol (915 l, 9.11 mmol)
l
l
was added. The solution was stirred for 2 h at room temperature
(TLC: isopropanol–1 M NH4OAc 4:1), concentrated in vacuo, and
the resulting glycosyl amine was dried under high vacuum. Amino
ter, 0.2 lm pore size). The clear solution was lyophilized affording
crude 7 (89.8 mg, 98%), which was used in the next step without
purification. ½a D23
ꢂ0.3 (c 1, H2O); Rf 5 = 0.91 (isopropanol–1 M
ꢁ
acid 3 (256 mg, 716
were dissolved in NMP (4 mL) and treated with DIPEA (245
l
mol, 4 equiv) and PyBOP (373 mg, 716
l
mol)
L,
NH4OAc 2:1); Rf 7 = 0.27 (isopropanol–1 M NH4OAc 2:1); 1H NMR
l
(500 MHz, D2O): d = 5.17 (d, J1,2 <1.0 Hz, 1H, H-14), 5.12 (d,
0
1.41 mmol). After 10 min the preactivation mixture was added to
the glycosyl amine and the solution was stirred for 30 min at room
temperature. The mixture was concentrated under high vacuum
and the residue was isolated by solid-phase extraction (one 10 g
cartridge), in order to remove the majority of the reagents. The so-
lid was dissolved in acetonitrile–water (100 mL, 1:4) and loaded
onto the cartridge. Impurities were washed off with acetonitrile–
water (200 mL, 30% acetonitrile), and 5 was eluted with acetoni-
trile–water (200 mL, 2:3). The fractions containing the product
were pooled, lyophilized, and further purified by RP-HPLC (col-
umn: Macherey-Nagel Nucleogel RP 100-10 (250 ꢀ 21 mm), elu-
ent: 32% acetonitrile–water, flow rate: 8 mL/min) to afford 5
J1,2 = 9.7 Hz, 1H, H-11), 4.97 (d, J1,2 <1.0 Hz, 1H, H-14 ), 4.82 (d, J1,2
<1.0 Hz, 1H, H-13), 4.66 (d, J1,2 = 7.6 Hz, 1H, H-12), 4.60 (m, 2H,
0
H-15, H-15 ), 4.30 (dd, J2,3 = 1.9 Hz, 1H, H-23), 4.24 (dd,
0
J2,3 = 1.9 Hz, 1H, H-24), 4.16 (dd, J2,3 = 1.9 Hz, 1H, H-24 ), 4.03–
0
3.45 (m, 40H,
a
CH Asn, H-21, H-22, H-25, H-25 , H-31, H-32, H-33,
0
0
0
0
H-34, H-34 , H-35, H-35 , H-41, H-42, H-43, H-44, H-44 , H-45, H-45 ,
0
0
H-51, H-52, H-53, H-54, H-54 , H-55, H-55 , H-6ab1, H-6ab2, H-6ab3,
0
0
H-6ab4, H-6ab4 , H-6ab5, H-6ab5 ), 2.92 (dd, Jgem = 16.6 Hz,
Jvic = 4.1 Hz, 1H, bCHa Asn), 2.79 (dd, Jvic = 7.3 Hz, 1H, bCHb Asn),
2.13, 2.10, 2.06 (3s, 12H, NAc); 13CNMR (125 MHz, D2O, DMSO-d6
as internal standard): d = 177.0, 176.4, 176.3, 176.2, 174.8 (C@O),
0
102.8 (C-12), 102.0 (C-13), 101.2 (C-15, C-15 ), 101.1 (C-14), 98.6
0
(268 mg, 77%). ½a D23
ꢁ
ꢂ0.3 (c 0.5, MeOH); Rf glycosyl amine = 0.51
(C-14 ), 82.0 (C-33), 81.1 (C-42), 80.4 (C-41), 79.7 (C-11), 78.1 (C-
0
0
(isopropanol–1 M NH4OAc 4:1); Rf 5 = 0.77 (isopropanol–1 M
NH4OAc 4:1); 1H NMR (500 MHz, [D6] DMSO): d = 8.44 (d,
24), 78.0 (C-24 ), 77.8 (C-51), 77.4 (C-55, C-55 ), 76.0 (C-52), 75.9
0
0
(C-53), 75.1 (C-54), 75.0, 74.9 (C-35, C-35 ), 74.44 (C-54 ), 74.39 (C-
0
0
JNH,1 = 8.8 Hz, 1H,
c
NH Asn), 7.99 (d, JNH,2 = 7.1 Hz, 1H, NH-22),
31), 73.6 (C-32), 71.8 (C-23), 71.5 (C-45, C-45 ), 71.1 (C-34 ), 71.0
0
0
7.83 (d, JNH,2 = 8.9 Hz, 1H, NH-21), 7.58 (m, 3H, NH-25, NH-25 ,
NH-urethane), 7.37–7.18 (m, 20H, Ar), 5.11, 5,06 (2d,
Jgem = 12.8 Hz, 2H, CH2O), 5.05–4.94 (m, 11H, H-11, H-14, OH-35,
(C-34), 68.93 (C-44 ), 68.90 (C-44), 67.4 (C-63), 67.3 (C-43), 63.3,
0
0
63.2 (C-64, C-64 ), 62.2 (C-65, C-65 ), 61.54 (C-62), 61.48 (C-61),
0
56.9 (C-25, C-25 ), 56.5 (C-22), 55.2 (C-21), 53.2 (
aC Asn), 38.5 (bC
0
0
OH-35 , OH-43, OH-45, OH-45 , CH2O), 4.75 (d, JOH,4 = 4.8 Hz, 1H,
Asn), 23.9, 23.8, 23.7 (NAc); ESI-MS (m/z) calcd for C54H90N6O38
[M]+: 1430.53; found: 1431.45 [M+H]+, 1453.44 [M+Na]+. Asparti-
mide by-product 7a: ESI-MS (m/z) calcd for C54H88N6O37 [M]+:
1412.52; found: 1435.44 [M+Na]+. Methyl ester by-product 7b:
0
OH-44), 4.71 (d, JOH,4 = 4.8 Hz, 1H, OH-44 ), 4.66 (d, J1,2 <1.0 Hz,
0
1H, H-14 ), 4.62–4.40 (m, 12H,
a
CH Asn, H-12, H-13, OH-23, OH-
0
64, OH-65, OH-65 , CH2O), 4.36 (d, J1,2 = 8.1 Hz, 1H, H-15), 4.31 (d,
0
Jgem = 12.2 Hz, 1H, CH2O), 4.23 (d, J1,2 = 8.2 Hz, 1H, H-15 ), 4.19
ESI-MS (m/z) calcd for C
55H92N6O38 [M]+: 1430.53; found:
0
(m, 1H, OH-64 ), 3.99–3.94 (m, 2H, H-23, OH-34), 3.90 (d,
1445.60 [M+H]+, 1467.57 [M+Na]+.
0
JOH,3 = 8.0 Hz, 1H, OH-34 ), 3.88–3.83 (m, 2H, H-24, H-41), 3.78–
0
0
3.21 (m, 34H, H-21, H-22, H-24 , H-25, H-25 , H-31, H-32, H-33,
3.4. N4-{O-(2-Acetamido-2-deoxy-b-
D
-glucopyranosyl)-(1?2)-O-
-glu-
-man-
-glucopyran-
-glucopyranosyl)}-N2-(9-
-asparagine 1
0
0
0
H-34, H-34 , H-35, H-35 , H-42, H-43, H-44, H-44 , H-51, H-52, H-54,
a
-
D
-mannopyranosyl-(1?3)-[O-(2-acetamido-2-deoxy-b-
copyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b-
nopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b-
osyl)-(1?4)-(2-acetamido-2-deoxy-b-
fluorenylmethoxycarbonyl)-
D
0
0
0
H-54 , H-6ab1, H-6ab2, H-6ab3, H-6ab4, H-6ab4 , H-6ab5, H-6ab5 ),
a-D
D
0
0
3.14–3.02 (m, 5H, H-45, H-45 , H-53, H-55, H-55 ), 2.67 (dd,
Jgem = 16.1 Hz, Jvic = 5.0 Hz, 1H, bCHa Asn), 2.50 (dd, Jvic = 7.2 Hz,
1H, bCHb Asn), 1.80, 1.77, 1.70 (3s, 12H, NAc); 13C NMR
(125 MHz, DMSO-d6): d = 171.2 (C@O ester), 169.7, 169.5, 169.4,
D
D
L
169.3 (C@O NAc), 155.8 (C@O urethane), 139.2, 138.5, 138.4,
The glycosyl amino acid 7 (90.2 mg, 63.0
water (9 mL) and treated with TEA (18 L, 129
Fmoc-OSu (70.0 mg, 207 mol) in dioxane (6 mL) was added and
the reaction mixture was allowed to stir for 2 h at room tempera-
ture (TLC: isopropanol–1 M NH4OAc 2:1). Subsequently, a second
l
mol) was dissolved in
0
136.7, 135.9 (C-i Ar), 128.3–126.9 (C-Ar), 101.5 (C-15 ), 101.1 (C-
l
lmol). A solution of
0
15), 100.2 (C-13), 99.8 (C-14), 99.6 (C-12), 97.6 (C-14 ), 81.5 (C-33),
l
0
81.4 (C-31), 80.8 (C-32), 79.2 (C-24 ), 78.8 (C-24), 78.5 (C-11), 77.0
0
(C-55), 76.9 (C-55 ), 76.1 (C-51), 75.9 (C-42), 75.5 (C-53), 74.6

1312
S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
portion of Fmoc-OSu (19.5 mg, 57.8
l
mol) and TEA (10
l
L,
preactivation mixture was added to the glycosyl amine and the
solution was stirred for 2 h at room temperature. Water (10 mL)
was added to hydrolyze excess amino acid active ester and the reac-
tion mixture was concentrated under high vacuum. The crude prod-
uct was purified by solid-phase extraction (one 10-g cartridge): the
residue was dissolved in acetonitrile–water (100 mL, 1:9) and
loaded onto the cartridge. Impurities were washed off with acetoni-
trile–water (200 mL, 30% acetonitrile) and 6 was eluted with aceto-
nitrile–water (200 mL, 2:3). The fractions containing the product
71.7
lmol) were added. After 4 h the reaction was quenched with
acetic acid (24
lL, pH 5) and lyophilized. The residue was diluted
with water (100 mL), centrifuged, and the supernatant was loaded
onto a 5-g Waters RP cartridge in order to remove the majority of
the reagents by solid-phase extraction. Impurities were washed off
with water (100 mL) and 1 was eluted with acetonitrile–water
(100 mL, 1:9). The fractions containing the product were pooled,
lyophilized, and further purified by RP-HPLC (column: YMC-Pack
ODS S-5
lm (250x20 mm), gradient: 27–55% acetonitrile (0.1% tri-
were pooled and lyophilized affording 6 (289 mg, 85.9%). ½a D23
ꢁ
fluoroacetic acid), flow rate: 9.5 mL/min) to afford 1 (65.6 mg,
ꢂ1.7 (c 1.3, MeOH); Rf glycosyl amine = 0.51 (isopropanol–1 M
63%). ½a 2D3
ꢂ3.7 (c 0.3, H2O); Rf = 0.75 (isopropanol–1 M NH4OAc
ꢁ
NH4OAc 4:1); Rf 6 = 0.72 (isopropanol–1 M NH4OAc 4:1); 1H NMR
2:1); 1H NMR (500 MHz, D2O, DMSO-d6 as internal standard):
(500 MHz, DMSO-d6): d = 8.38 (d, JNH,1 = 8.6 Hz, 1H, cNH Asn),
d = 7.7–7.3 (m, 8H, Fmoc), 4.93 (d, J1,2 <1.0 Hz, 1H, H-14), 4.81 (d,
8.00 (d, JNH,2 = 7.1 Hz, 1H, NH-22), 7.93 (d, JNH,2 = 8.8 Hz, 1H, NH-
0
J1,2 = 9.5 Hz, 1H, H-11), 4.73 (d, J1,2 <1.0 Hz, 1H, H-14 ), 4.56 (d, J1,2
0
0
21), 7.59 (m, 3H, NH-25, NH-25 , NH-urethane), 7.37–7.18 (m, 25H,
0
Ar), 5.02–4.87 (m, 11H, H-11, H-14, OH-35, OH-35 , OH-43, OH-45,
0
<1.0 Hz, 1H, H-13), 4.40–4.35 (m, 3H, H-12, H-15, H-15 ), 4.28 (m,
2H, CH2O), 4.10 (m, 1H, H-9 Fmoc), 4.05 (m, 1H, H-23), 3.99 (m,
OH-45 , CH2O), 4.77 (d, JOH,4 = 4.8 Hz, 1H, OH-44), 4.72 (d,
0
0
0
1H, H-24), 3.91 (m, 1H, H-24 ), 3.77–3.20 (m, 40H,
a
CH Asn, H-21,
JOH,4 = 4.8 Hz, 1H, OH-44 ), 4.65 (d, J1,2 <1.0 Hz, 1H, H-14 ), 4.62–
0
0
0
0
H-22, H-25, H-25 , H-31, H-32, H-33, H-34, H-34 , H-35, H-35 , H-41,
4.44 (m, 12H,
a
CH Asn, H-12, H-13, OH-23, OH-64, OH-65, OH-65 ,
0
0
0
H-42, H-43, H-44, H-44 , H-45, H-45 , H-51, H-52, H-53, H-54, H-54 ,
CH2O), 4.36 (d, J1,2 = 8.3 Hz, 1H, H-15), 4.34 (d, Jgem = 10.1 Hz, 1H,
0 0
0
0
H-55, H-55 , H-6ab1, H-6ab2, H-6ab3, H-6ab4, H-6ab4 , H-6ab5, H-
CH2O), 4.23 (d, J1,2 = 8.2 Hz, 1H, H-15 ), 4.19 (m, 1H, OH-64 ), 3.99–
0
0
6ab5 ), 2.54 (dd, Jgem = 15.2 Hz, Jvic = 2.6 Hz, 1H, bCHa Asn), 2.37
3.94 (m, 2H, H-23, OH-34), 3.93 (d, JOH,3 = 7.5 Hz, 1H, OH-34 ),
(dd, Jvic = 8.7 Hz, 1H, bCHb Asn), 1.88, 1.86 (2s, 12H, NAc); 13C
NMR (125 MHz, D2O, DMSO-d6 as internal standard): d = 176.2,
176.0, 174.8 (C@O), 159.1 (C@O urethane), 145.4, 142.5, 129.7,
129.1, 126.8, 121.8 (C-Ar), 102.9 (C-12), 102.0 (C-13), 101.2 (C-14,
3.88–3.83 (m, 2H, H-24, H-41), 3.78–3.21 (m, 34H, H-21, H-22, H-
0
0
0
0
24 , H-25, H-25 , H-31, H-32, H-33, H-34, H-34 , H-35, H-35 , H-42, H-
0
0
43, H-44, H-44 , H-51, H-52, H-54, H-54 , H-6ab1, H-6ab2, H-6ab3,
0
0
0
H-6ab4, H-6ab4 , H-6ab5, H-6ab5 ), 3.14–3.02 (m, 5H, H-45, H-45 ,
0
0
0
C-15, C-15 ), 98.6 (C-14 ), 82.0 (C-33), 81.1 (C-42), 80.3 (C-41), 79.8
H-53, H-55, H-55 ), 2.67 (dd, Jgem = 16.1 Hz, Jvic = 5.0 Hz, 1H, bCHa
0
0
(C-11), 78.02 (C-24), 77.95 (C-24 ), 77.7 (C-51), 77.4 (C-55, C-55 ),
Asn), 2.50 (dd, Jvic = 7.2 Hz, 1H, bCHb Asn), 1.80, 1.77, 1.72 (3s,
12H, NAc), 1.34 (1s, 9H, tBu); 13C NMR (125 MHz, DMSO-d6):
d = 170.5 (C@O ester), 169.7, 169.5, 169.4, 169.3 (C@O NAc), 155.8
0
75.9 (C-52, C-53), 75.1 (C-54), 75.95, 74.85 (C-35, C-35 ), 74.4 (C-
0
0
54 ), 74.3 (C-31), 73.5 (C-32), 71.7 (C-23), 71.5 (C-45, C-45 ), 71.0
0
0
(C-34, C-34 ), 68.9 (C-44, C-44 ), 68.1 (CH2O), 67.4 (C-63), 67.2 (C-
(C@O urethane), 139.2, 138.5, 138.4, 136.9, (C-i Ar), 128.3–126.9
0
0
0
43), 63.3, 63.2 (C-64, C-64 ), 62.2 (C-65, C-65 ), 61.5 (C-62), 61.3 (C-
(C-Ar), 101.5 (C-15 ), 101.2 (C-15), 100.2 (C-13), 99.9 (C-14, C-12),
0
0
0
61), 56.9 (C-25, C-25 ), 56.5 (C-22), 55.4 (C-21), 54.0 (
aC Asn), 48.4
97.6 (C-14 ), 81.5 (C-33), 81.4 (C-31), 80.7 (C-32), 79.3 (C-24 ), 78.8
0
(C-9 Fmoc), 39.9 (bC Asn), 23.9, 23.8, 23.6 (NAc); ESI-MS (m/z)
(C-24), 78.4 (C-11), 77.0 (C-55), 76.9 (C-55 ), 76.1 (C-51), 75.9 (C-
calcd for C69H100N6O40 [M]+: 1652.60; found: 1675.49 [M+Na]+,
42), 75.5 (C-53), 74.6 (C-41), 74.4 (C-52), 74.2 (C-54), 73.9 (C-35, C-
0
0
0
1691.42 [M+K]+, 849.25 [M+2Na]2+
.
35 ), 73.8 (CH2O), 73.6 (C-54 ), 73.2, 72.2, 71.8 (CH2O), 70.5 (C-45 ),
0
70.3 (C-45), 69.9 (C-34, C-34 ), 69.1 (C-23), 68.7 (C-62), 68.3 (C-61),
0
67.6 (C-44), 67.0 (C-44 ), 65.9 (C-q tBu), 65.4 (CH2O, C-63), 64.9
3.5. N2-Benzyloxycarbonyl-
L
-aspartic acid
a
-tert-butyl ester 4
a-tert-butyl ester dicy-
0
0
(C-43), 61.5 (C-64), 61.1 (C-64 ), 61.0 (C-65), 60.8 (C-65 ), 55.6 (C-22,
0
C-25, C-25 ), 53.2 (C-21,) 50.9 (
a
C Asn), 37.0 (bC Asn), 27.5 (CH3
N2-Benzyloxycarbonyl-
L-aspartic acid
tBu), 23.2, 22.9, 22.7 (NAc); ESI-MS (m/z) calcd for C94H128N6O40
clohexylamine salt (Bachem) (5 g, 9.91 mmol) was dissolved in
CH2Cl2 (300 mL), washed with 5% KHSO4 (3ꢀ) and water, dried
over MgSO4, filtered, and concentrated in vacuo affording 4
(3.17 g, 99%). Rf = 0.62 (CH2Cl2–MeOH–HOAc 90:8:2); ESI-MS (m/
[M]+: 1980.82; found: 1981.90 [M+H]+, 991.39 [M+2H]2+
.
3.7. N4-{O-(2-Acetamido-2-deoxy-b-
-mannopyranosyl-(1?3)-[O-(2-acetamido-2-deoxy-b-
copyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b- -man-
nopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b- -glucopyran-
-glucopyranosyl)}- -aspar-
D-glucopyranosyl)-(1?2)-O-
z) calcd for
C
16H21NO6 [M]+: 323.14; found: positive mode
a-D
D
-glu-
345.90 [M+Na]+, negative mode 322.12 [MꢂH]ꢂ, 645.20 [2MꢂH]ꢂ.
a-D
D
D
3.6. N4-{O-(2-Acetamido-2-deoxy-b-
-mannopyranosyl-(1?3)-[O-(2-acetamido-2-deoxy-b-
copyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b- -man-
>nopyranosyl-(1?4)-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-b-
-glucopyranosyl)-(1?4)-(2-acetamido-3,6-di-O-benzyl-2-deoxy-
b- -asparagine tert-
-glucopyranosyl)}-N2-benzyloxycarbonyl-
D-glucopyranosyl)-(1?2)-O-
osyl)-(1?4)-(2-acetamido-2-deoxy-b-
D
L
a-
D
D
-glu-
agine tert-butyl ester 8
a-D
D
Protected glycosyl amino acid 6 (288 mg, 145 lmol) and PdO-
D
hydrate (453 mg) were suspended in THF–water–HOAc (11 mL,
10:10:1). The reaction flask was purged with argon and the mix-
ture was stirred vigorously under a hydrogen atmosphere for
3 days (TLC: isopropanol–1 M NH4OAc 2:1). THF was removed in
vacuo and after dilution with water (100 mL) the catalyst was fil-
tered off through a syringe filter unit (Millipore, IC Millex-LG,
D
L
butyl ester 6
Heptasaccharide azide 2 (289 mg, 170 lmol, 1 equiv) was dis-
solved in freshly distilled MeOH (10 mL) and ethyldiisopropylamine
(300 L, 1.72 mmol) was added. The reaction flask was purged with
argon and 1,3-propanedithiol (868 mol, 8.65 mmol) was subse-
l
25 mm external diameter, 0.2 lm pore size). The clear solution
l
was lyophilized affording crude 8 (211.7 mg, 97.9%), which was
quently added. The reaction mixture was stirred for 2 h at room
temperature (TLC: isopropanol–1 M NH4OAc 4:1). The solvent was
removed in vacuo and the resulting glycosyl amine was dried under
high vacuum. Amino acid 4 (330 mg, 1.02 mmol, 6 equiv) and Py-
used in the next step without purification. ½a D23
ꢁ
+1.8 (c 2.5, H2O);
Rf 6 = 0.83 (isopropanol–1 M NH4OAc 2:1); Rf 8 = 0.51 (isopropa-
nol–1 M NH4OAc 2:1); 1H NMR (500 MHz, D2O, DMSO-d6 as inter-
nal standard): d = 5.17 (d, J1,2 <1.0 Hz, 1H, H-14), 5.12 (d,
0
BOP (531 mg, 1.02
with ethyldiisopropylamine (350
l
mol) were dissolved in NMP (5 mL) and treated
L 2.01 mmol). After 10 min, the
J1,2 = 9.8 Hz, 1H, H-11), 4.97 (d, J1,2 <1.0 Hz, 1H, H-14 ), 4.82 (d, J1,2
l
<1.0 Hz, 1H, H-13), 4.66 (d, J1,2 = 7.7 Hz, 1H, H-12), 4.60 (m, 2H,

S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
1313
0
H-15, H-15 ), 4.30 (dd, J2,3 = 1.9 Hz, 1H, H-23), 4.24 (dd, J2,3 = 1.7 Hz,
tBu), 23.9, 23.8, 23.6 (NAc). ESI-MS (m/z) calcd for C73H108N6O40
[M]+: 1708.66; found: 1709.58 [M+H]+, 855.34 [M+2H]2+
0
1H, H-24), 4.15 (dd, J2,3 = 1.7 Hz, 1H, H-24 ), 4.03–3.45 (m, 40H,
aCH
.
0
0
Asn, H-21, H-22, H-25, H-25 , H-31, H-32, H-33, H-34, H-34 , H-35, H-
0
0
0
35 , H-41, H-42, H-43, H-44, H-44 , H-45, H-45 , H-51, H-52, H-53, H-54,
3.9. N4-{O-(2-Acetamido-2-deoxy-b-
-mannopyranosyl-(1?3)-[O-(2-acetamido-2-deoxy-b-
copyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b- -man-
nopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b- -glucopyran-
osyl)-(1?4)-(2-acetamido-2-deoxy-b-
-glucopyranosyl)}-N2-(9-
fluorenylmethoxycarbonyl)- -asparagine 1
D-glucopyranosyl)-(1?2)-O-
0
0
0
H-54 , H-55, H-55 , H-6ab1, H-6ab2, H-6ab3, H-6ab4, H-6ab4 , H-6ab5,
a-
D
D
-glu-
0
H-6ab5 ), 3.11 (dd, Jgem = 17.5 Hz, Jvic = 4.9 Hz, 1H, bCHa Asn), 2.97
a-D
D
(dd, Jvic = 4.9 Hz, 1H, bCHb Asn), 2.17, 2.10, 2.06 (3s, 12H, NAc),
1.53 (1s, 9H, tBu); 13C NMR (125 MHz, D2O, DMSO-d6 as internal
standard): d = 176.3, 176.1, 173.0, 169.4 (C@O), 102.8 (C-12),
D
D
L
0
0
101.9 (C-13), 101.1 (C-15, C-15 , C-14), 98.5 (C-14 ), 81.9 (C-33),
0
81.1 (C-42), 80.2 (C-41), 79.7 (C-11), 78.0 (C-24), 77.8 (C-24 ), 77.6
0
Glycosyl amino acid 9 (108 mg, 63.2 lmol) was treated with
(C-51), 77.3 (C-55, C-55 ), 75.9 (C-52), 75.8 (C-53), 75.1 (C-54),
TFA (8 mL). The reaction mixture was allowed to stir for 5 min at
room temperature (TLC: isopropanol–1 M NH4OAc 2:1), subse-
quently the TFA was removed in vacuo and the solid was dried un-
der high vacuum. The residue was diluted with water, lyophilized,
0
0
74.9, 74.8 (C-35, C-35 ), 74.4 (C-54 ), 74.2 (C-31), 73.5 (C-32), 71.7
0
0
0
(C-23), 71.4 (C-45, C-45 ), 71.0 (C-34 ), 70.9 (C-34), 68.9 (C-44 ),
0
68.8 (C-44), 67.4 (C-63), 67.2 (C-43), 63.2, 63.1 (C-64, C-64 ), 62.1
0
0
(C-65, C-65 , C-q tBu), 61.5 (C-62), 61.4 (C-61), 56.8 (C-25, C-25 ),
and purified by RP-HPLC (column: YMC-Pack ODS S-5 lm
56.4 (C-22), 55.2 (C-21), 52.3 (
aC Asn), 36.5 (bC Asn), 28.5 (CH3
(250 ꢀ 20 mm), gradient: 27–55% acetonitrile (0.1% trifluoroacetic
tBu), 23.9, 23.7, 23.6 (NAc); ESI-MS (m/z) calcd for C58H98N6O38
[M]+: 1486.59; found: positive mode 1487.49 [M+H]+, 1509.52
[M+Na]+, negative mode 1485.46 [M – H]-.
acid), flow rate: 9.5 mL/min) affording 1 (75.3 mg, 72.1%).
3.10. N4-{O-(2-Acetamido-2-deoxy-b-
O- -mannopyranosyl-(1?3)-O-[(2-acetamido-2-deoxy-b-
glucopyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b-
mannopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b- -gluco-
-glucopyranosyl)}-
-asparaginyl- -tyrosine-
D-glucopyranosyl)-(1?2)-
a
-
D
D-
a-
D
D-
3.8. N4-{O-(2-Acetamido-2-deoxy-b-
-mannopyranosyl-(1?3)-[O-(2-acetamido-2-deoxy-b-
copyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b- -man-
nopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b- -glucopyran-
osyl)-(1?4)-(2-acetamido-2-deoxy-b-
-glucopyranosyl)}-N2-(9-
fluorenylmethoxycarbonyl)- -asparagine tert-butyl ester 9
D
-glucopyranosyl)-(1?2)-O-
-glu-
D
a-
D
D
pyranosyl)-(1?4)-(2-acetamido-2-deoxy-b-
D
N2-(9-fluorenylmethoxycarbonyl)-
L
L
a-D
D
D
methylester 11
D
L
Glycosyl amino acid 1 (16 mg, 9.68
ter hydrochloride (4 mg, 17.3 mol), 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) (5.9 mg,
lmol), L-tyrosine methyles-
l
Glycosyl amino acid 8 (189 mg, 127
water (18 mL) and treated with triethylamine (56
A solution of Fmoc-OSu (181 mg, 537 mol) in dioxane (16 mL)
was added. The reaction mixture was allowed to stir for 90 min
at room temperature (TLC: isopropanol–1 M NH4OAc 2:1), the
lmol) was dissolved in
l
L, 402 mol).
l
18.4
(450
l
l
mol), and HOBt (2.8 mg, 18
L). The coupling was started by addition of a mixture of eth-
L, 61 mol) and NMP (100 L). After
lmol) were dissolved in NMP
l
yldiisopropylamine (10.7
l
l
l
complete reaction (5–10 min) (TLC: isopropanol–1 M NH4OAc
2:1) the mixture was dried in vacuum and the residue was purified
by RP-HPLC (column: Macherey-Nagel Nucleogel RP 100-8
(300 ꢀ 7.7 mm), gradient: 25–42 % acetonitrile, flow rate: 2 mL/
reaction was subsequently quenched with acetic acid (50 ll, pH
5), and the reaction mixture was lyophilized. The residue was di-
luted with water (50 mL), centrifuged, and the supernatant was
loaded onto a 5-g RP cartridge in order to remove the majority of
the reagents by solid-phase extraction. Impurities were washed
off with acetonitrile–water (100 mL, 2:8) and 9 was eluted with
acetonitrile–water (100 mL, 3:7). The fractions containing the
product were pooled and lyophilized affording 9 (130 mg, 59.8%).
min) and lyophilized to afford 11 (13.1 mg, 73.9%). ½a D23
ꢂ10.0 (c
ꢁ
0.2, H2O); Rf = 0.73 (isopropanol–1 M NH4OAc 2:1); 1H NMR
(500 MHz, D2O, acetone-d6 (20% v/v) as internal standard):
d = 7.62–7.10 (m, 8H, Fmoc), 6.78, 6.56 (2 m, 4H, Tyr), 5.0 (d, J1,2
<1.0 Hz, 1H, H-14), 4.87 (d, J1,2 = 9.4 Hz, 1H, H-11), 4.80 (d, J1,2
a 2D3
ꢁ
ꢂ5.5 (c 1.1, H2O); Rf = 0.78 (isopropanol–1 M NH4OAc 2:1);
0
<1.0 Hz, 1H, H-14 ), 4.63 (d, J1,2 <1.0 Hz, 1H, H-13), 4.46 (d,
½
1H NMR (500 MHz, D2O, DMSO-d6 as internal standard): d = 7.4–
6.6 (m, 8H, Fmoc), 4.93 (d, J1,2 <1.0 Hz, 1H, H-14), 4.81 (d,
0
J1,2 = 6.8 Hz, 1H, H-12), 4.44–4.40 (m, 3H,
a
CH Tyr, H-15, H-15 ),
4.36 (m, 1H,
aCH Asn), 4.22 (m, 1H, CH2O), 4.13 (m, 2H, CH2O,
40
0
J1,2 = 9.5 Hz, 1H, H-11), 4.73 (d, J1,2 <1.0 Hz, 1H, H-1 ), 4.56 (d, J1,2
H-23), 4.05 (m, 1H, H-24), 3.97 (m, 2H, H-24 , H-9 Fmoc), 3.83–
50
0
<1.0 Hz, 1H, H-13), 4.40–4.35 (m, 3H, H-12, H-15, H-1 ), 4.28 (m,
3.24 (m, 42H, CH3O, H-21, H-22, H-25, H-25 , H-31, H-32, H-33,
2H, CH2O), 4.16 (m, 1H, H-9 Fmoc), 4.05 (m, 1H, H-23), 3.99 (m,
0
0
0
0
H-34, H-34 , H-35, H-35 , H-41, H-42, H-43, H-44, H-44 , H-45, H-45 ,
40
0
0
1H, H-24), 3.91 (m, 1H, H-2 ), 3.77–3.20 (m, 40H,
a
CH Asn, H-21,
H-51, H-52, H-53, H-54, H-54 , H-55, H-55 , H-6ab1, H-6ab2, H-6ab3,
50
40
50
0
0
H-22, H-25, H-2 , H-31, H-32, H-33, H-34, H-3 , H-35, H-3 , H-41,
H-6ab4, H-6ab4 , H-6ab5, H-6ab5 ), 2.85–2.68 (m, 2H, bCHab Tyr),
2.60–2.38 (m, 2H, bCHab Asn), 1.93, 1.91 (2s, 12H, NAc). 13C
NMR (125 MHz, D2O, acetone-d6 as internal standard): d = 174.8,
174.7, 173.2, 172.4 (C@O), 155.1 (C-p Tyr), 143.8, 141.2 (C-Ar),
0
0
0
H-42, H-43, H-44, H-44 , H-45, H-45 , H-51, H-52, H-53, H-54, H-54 ,
0
0
H-55, H-55 , H-6ab1, H-6ab2, H-6ab3, H-6ab4, H-6ab4 , H-6ab5, H-
0
6ab5 ), 2.50 (dd, Jgem = 15.2 Hz, Jvic = 2.6 Hz, 1H, bCHa Asn), 2.30
(dd, Jvic = 8.7 Hz, 1H, bCHb Asn), 1.86, 1.73 (2s, 12H, NAc), 1.01
(1s, 9H, tBu); 13C NMR (125 MHz, D2O, DMSO-d6 as internal stan-
dard): d = 176.2, 176.0, 174.8 (C@O), 159.1 (C@O urethane),
145.4, 142.2, 129.7, 129.1, 126.8, 121.8 (C-Ar), 102.9 (C-12), 102.0
130.7 (C-o Tyr), 128.3, 127.8, 127.6, 125.4, 120.4 (C-Ar), 115.7 (C-
0
m Tyr), 101.7 (C-12), 100.8 (C-13), 100.0 (C-15, C-15 ), 99.9 (C-14),
0
97.3 (C-14 ), 80.8 (C-33), 79.9 (C-42), 79.1 (C-41), 78.6 (C-11), 76.8
0
0
(C-24), 76.7 (C-24 ), 76.5 (C-51), 76.2 (C-55, C-55 ), 74.7 (C-52, C-
50
40
0
0
(C-13), 101.2 (C-14, C-15, C-1 ), 98.6 (C-1 ), 82.0 (C-33), 81.1 (C-
53), 73.9 (C-54), 73.7, 73.6 (C-35, C-35 ), 73.2 (C-54 ), 73.0 (C-31),
40
0
0
42), 80.3 (C-41), 79.8 (C-11), 78.07 (C-24), 77.93 (C-2 ), 77.7 (C-
72.3 (C-32), 70.5 (C-23), 70.3 (C-45, C-45 ), 69.8 (C-34, C-34 ), 67.7
50
0
51), 77.4 (C-55, C-5 ), 76.0 (C-52, C-53), 75.1 (C-54), 74.99, 74.87
(C-44, C-44 ), 67.1 (CH2O), 66.0 (C-43, C-63), 62.0 (C-64), 61.9 (C-
50
40
0
0
0
(C-35, C-3 ), 74.4 (C-31, C-5 ), 73.6 (C-32), 71.8 (C-23), 71.5 (C-45,
64 ), 61.0 (C-65, C-65 ), 60.3 (C-62), 60.1 (C-61), 55.7 (C-25, C-25 ),
0
0
0
C-45 ), 71.1 (C-34, C-34 ), 68.9 (C-44, C-44 ), 68.1 (CH2O), 67.4 (C-
55.2 (C-22), 54.4 (
a
C Tyr), 54.1 (C-21), 53.0 (CH3O), 51.5 (
aC Asn),
0
63), 67.2 (C-43), 63.4 (C-q tBu), 63.3, 63.2 (C-64, C-64 ), 62.2 (C-65,
0
47.0 (C-9 Fmoc), 37.3 (bC Asn), 36.0 (bC Tyr), 22.7, 22.6, 22.4
C-65), 61.6 (C-62), 61.3 (C-61), 56.9 (C-25, C-25 ), 56.5 (C-22), 55.4
(NAc); MALDI-TOF-MS (m/z) calcd for
C
79H111N7O42 [M]+:
(C-21), 54.0 (
aC Asn), 48.4 (C-9 Fmoc), 39.9 (bC Asn), 28.7 (CH3
1829.67; found: 1853.77 [M+Na]+.

1314
S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
0
3.11. N-(9-Fluorenylmethoxycarbonyl)-
L
-seryl-
L
-seryl-
L
-serine
2H, H-15, H-15 ), 4.40 (m, 1H,
aCH Tyr), 4.36 (m, 1H,
a
CH SerC),
ethyldiisopropylammonium salt 13
4.30 (m, 1H,
4.17 (m, 1H,
aCH SerB), 4.25 (d, Jvic = 6.0 Hz, 2H, CH2O Fmoc),
a
CH SerA), 4.14 (m, 1H, H-23), 4.09 (t, 1H, H-9 Fmoc),
0
Commercially available
(40 mg, 143 mol) was dissolved in water (1.2 mL) in the presence
of ethyldiisopropylamine (20 L). A solution of Fmoc-OSu (80 mg,
237 mol) in acetonitrile (1 mL) was added followed by pH adjust-
L
-seryl-
L
-seryl-
L
-serine (Bachem)
4.06 (m, 1H, H-24), 3.97 (m, 1H, H-24 ), 3.83–3.27 (m, 48H, CH3O,
0
l
bCH2 SerA,B,C, H-21, H-22, H-25, H-25 , H-31, H-32, H-33, H-34,
0
0
0
0
l
H-34 , H-35, H-35 , H-41, H-42, H-43, H-44, H-44 , H-45, H-45 , H-51,
0
0
l
H-52, H-53, H-54, H-54 , H-55, H-55 , H-6ab1, H-6ab2, H-6ab3,
0 0
ment to a value of 8.5 with ethyldiisopropylamine. After 2.5d the
solution was evaporated, taken up in water (20 mL), and loaded
onto two Sep Pak cartridges. The salts were eluted with water
(20 mL). The desired peptide 13 was eluted with 10% and 30% ace-
tonitrile (20 mL each), lyophilized, and used for the coupling with
12. Rf 13 = 0.62 (isopropanol–1 M NH4OAc 4:1). 1H NMR (500 MHz,
D2O, acetone-d6 as internal standard): d = 7.95–7.46 (m, 8H, Fmoc),
H-6ab4, H-6ab4 , H-6ab5, H-6ab5 ), 2.85 (dd, Jgem = 14.0 Hz,
Jvic = 6.5 Hz, 1H, bCHa Tyr), 2.79 (dd, Jvic = 7.5 Hz, 1H, bCHb Tyr),
2.63 (dd, Jgem = 15.7 Hz, Jvic = 4.4 Hz, 1H, bCHa Asn), 2.53 (dd,
Jvic = 6.5 Hz, 1H, bCHb Asn), 1.93, 1.92 , 1.84 (3s, 12H, NAc); 13C
NMR (125 MHz, D2O, acetone-d6 as internal standard): d = 174.7,
174.5, 173.2, 172.7, 172.4, 172.0, 171.7, 171.3 (C@O), 155.2 (C-p
Tyr), 143.9, 141.3 (C-Ar), 130.8 (C-o Tyr), 128.4, 128.0, 127.8,
125.5, 120.4 (C-Ar), 115.7 (C-m Tyr), 101.7 (C-12), 100.8 (C-13),
4.64–4.60 (m, 1H,
aCH SerA), 4.53–4.50 (m, 2H, CH2O Fmoc), 4.44
0
0
(m, 3H, CH SerB,C, H-9 Fmoc) 4.03–3.90 (m, 6H, bCH2 SerA,B,C),
a
100.0 (C-15, C-15 ), 99.9 (C-14), 97.4 (C-14 ), 80.8 (C-33), 79.9 (C-
0
42), 79.3 (C-41), 78.7 (C-11), 76.9 (C-24), 76.8 (C-24 ), 76.5 (C-51),
0
3.84–3.77 (m, 2H, CH iPr), 3.31–3.26 (m, 2H, CH2 Et), 1.45–1.41
(m, 15H, CH3); 13C NMR (125 MHz, D2O, acetone-d6 as internal
standard): d = 143.9, 141.2, 128.4, 127.8, 125.5, 120.4 (C-Ar), 67.2
(CH2O Fmoc), 62.1 (bC SerB), 61.7 (bC SerC), 61.5 (bC SerA), 56.8
76.3 (C-55, C-55 ), 74.8 (C-52, C-53), 73.9 (C-54), 73.8, 73.7 (C-35,
0
0
C-35 ), 73.2 (C-54 ), 73.1 (C-31), 72.3 (C-32), 70.5 (C-23), 70.4 (C-
0
0
0
45, C-45 ), 69.9 (C-34, C-34 ), 67.7 (C-44, C-44 ), 67.3 (CH2O), 66.2
0
(aC SerB,C), 54.6
a
(C SerA), 54.6 (CH iPr), 47.0 (C-9 Fmoc), 42.7
(C-63), 66.1 (C-43), 62.1, 62.0 (C-64, C-64 ), 61.8 (bC SerA), 61.3
0
(CH2 Et), 18.0, 16.6 (CH3 iPr), 12.4 (CH3 Et), Ser A, B, C were not as-
signed to the peptide sequence.
(bC SerB,C), 61.0 (C-65, C-65 ), 60.3 (C-62), 60.2 (C-61), 57.1 (
a
C
0
SerA), 56.1
a
(C SerB), 56.0 (
a
C SerC), 55.7 (C-25, C-25 ), 55.2 (C-
22), 54.8 (
a
C Tyr), 54.1 (C-21), 52.9 (CH3O), 50.2 (
a
C Asn), 47.0
3.12. N4-{O-(2-Acetamido-2-deoxy-b-
O-
glucopyranosyl)-(1?2)-O-
D
-glucopyranosyl)-(1?2)-
(C-9 Fmoc), 36.9 (bC Asn), 36.2 (bC Tyr), 22.7, 22.6, 22.4 (NAc),
Ser A, B, C were not assigned to the peptide sequence; MALDI-
TOF-MS (m/z) calcd for C88H126N10O48 [M]+: 2090.77; found:
2114.6 [M+Na]+.
a-D-mannopyranosyl-(1?3)-O-[(2-acetamido-2-deoxy-b-D-
a
-
D
-mannopyranosyl-(1?6)]-O-b-D-
mannopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b-
pyranosyl)-(1?4)-(2-acetamido-2-deoxy-b- -glucopyranosyl)}-
-asparaginyl- -tyrosine-methylester 12
D-gluco-
D
L
L
3.14. N4-{O-(2-Acetamido-2-deoxy-b-
O- -mannopyranosyl-(1?3)-O-[(2-acetamido-2-deoxy-b-
glucopyranosyl)-(1?2)-O- -mannopyranosyl-(1?6)]-O-b-
mannopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b- -glucopyr-
anosyl)-(1?4)-(2-acetamido-2-deoxy-b-
-glucopyranosyl)}-N2-
-seryl- -seryl- -seryl)- -asparaginyl- -tyrosine 16
D-glucopyranosyl)-(1?2)-
a
-
D
D-
Fmoc-glycopeptide 11 (13 mg, 7.1
lmol) was dissolved in NMP
a
-
D
D-
(400 L) and deprotected by addition of piperidine (40
l
lL). After
D
1 h (TLC: isopropanol–1 M NH4OAc 2:1) the volatiles were evapo-
rated in vacuum and the remainder was purified by RP-HPLC (col-
umn: Macherey-Nagel Nucleogel RP 100-8 (300 ꢀ 7.7 mm),
gradient: 2–50 % acetonitrile, flow rate: 2 mL/min). The fractions
containing the desired product were lyophilized and afforded 12
(9.9 mg, 86.7%), which was used for the synthesis of 14. Rf = 0.48
D
(L
L
L
L
L
Fmoc glycopeptide methylester 12 (5.4 mg, 2.58
dissolved in NMP (200 L) and piperidine (10 L) was added. After
1 h (TLC: isopropanol–1 M NH4OAc 2:1) the volatiles were removed
in vacuum. The remainder was taken up in water (600 L) and chy-
motrypsin(0.8 mg dissolved in 300 L of water) was added. After 1 h
lmol) 14 was
l
l
(isopropanol–1 M NH4OAc 2:1); C64H101N7O40
.
l
l
3.13. N4-{O-(2-Acetamido-2-deoxy-b-
O-
glucopyranosyl)-(1?2)-O-
D
-glucopyranosyl)-(1?2)-
(TLC: isopropanol–1 M NH4OAc 2:1) the mixture was centrifuged.
The supernatant was concentrated in vacuum to a volume of
400 lL and purified by gel filtration (column: Pharmacia Hi Load
a-D-mannopyranosyl-(1?3)-O-[(2-acetamido-2-deoxy-b-D-
a
-
D
-mannopyranosyl-(1?6)]-O-b-
D
-
mannopyranosyl-(1?4)-O-(2-acetamido-2-deoxy-b-
anosyl)-(1?4)-(2-acetamido-2-deoxy-b-
-glucopyranosyl)}-N2-
(9-fluorenylmethoxycarbonyl- -seryl- -seryl- -seryl)- -asparag-
inyl- -tyrosine-methylester 14
D
-glucopyr-
Superdex 30 (600 ꢀ 16 mm) eluent: 100 mM NH4HCO3, flow rate
D
750
l
L/min) and lyophilized to afford 16 (4.6 mg, 96.0%). ½a D23
ꢂ5.6
ꢁ
L
L
L
L
(c 0.5, H2O); Rf 15 = 0.44 (isopropanol–1 M NH4OAc 2:1); Rf
16 = 0.32 (isopropanol–1 M NH4OAc 2:1); 1H NMR (500 MHz, D2O,
DMSO-d6 as internal standard): d = 6.91 (d, Jvic = 8.5 Hz, 2H, H-o
L
Deprotected glycopeptide 12 (9.9 mg, 6.2
Ser-Ser-OH ꢀ EtNiPr2 13 (10 mg, 15.9 mol), and O-(7-aza-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium-hexafluorophos-
phate (HATU) (6 mg, 15.8 mol) were dissolved in NMP (500 L).
2,4,6-Collidine (2.1 L, 15.9 mol) dissolved in NMP (10 l) was
lmol), Fmoc-Ser-
Tyr), 6.63 (d, 2H, H-m Tyr), 4.92 (d, J1,2 <1.0 Hz, 1H, H-14), 4.81 (d,
0
l
J1,2 = 9.6 Hz, 1H, H-11), 4.72 (d, J1,2 <1.0 Hz, 1H, H-14 ), 4.57 (d, J1,2
<1.0 Hz, 1H, H-13), 4.51 (t, Jvic = 6.5 Hz, 1H,
a
a
CH Asn), 4.41 (d,
CH SerA), 4.35 (2d,
CH SerB),
l
l
J1,2 = 7.8 Hz, 1H, H-12), 4.36 (t, Jvic = 5.3 Hz, 1H,
0
l
l
l
J1,2 = 8.6 Hz, 2H, H-15, H-15 ), 4.27 (t, Jvic = 5.4 Hz, 1H,
a
added. The reaction was complete after 5–10 min (TLC: isopropa-
nol–1 M NH4OAc 2:1) and the reaction mixture was evaporated
in vacuum and purified by RP-HPLC (column: Macherey-Nagel
Nucleogel RP 100-8 (300 ꢀ 7.7 mm), gradient: 20–40% acetonitrile,
flow: 2 mL/min). The fractions containing the product were
4.15 (dd, Jvic = 7.1, Jvic = 5.4 Hz, 1H, a
CH Tyr), 4.05 (m, 1H, H-23),
0
3.98 (m, 1H, H-24), 3.90 (m, 1H, H-24 ), 3.78–3.22 (m, 46H,
a
CH SerC,
0
0
bCH2 SerA,B,C, H-21, H-22, H-25, H-25 , H-31, H-32, H-33, H-34, H-34 ,
0
0
0
H-35, H-35 , H-41, H-42, H-43, H-44, H-44 , H-45, H-45 , H-51, H-52,
0
0
H-53, H-54, H-54 , H-55, H-55 , H-6ab1, H-6ab2, H-6ab3, H-6ab4, H-
0
0
lyophilized affording 14 (9.0 mg, 69.9 %). ½a D23
ꢁ
ꢂ0.3 (c 0.15,
6ab4 , H-6ab5, H-6ab5 ), 2.86 (dd, Jgem = 14.0 Hz, Jvic = 4.9 Hz, 1H,
bCHa Tyr), 2.69 (dd, Jvic = 7.5 Hz, 1H, bCHb Tyr), 2.56 (dd,
Jgem = 16.1 Hz, Jvic = 5.4 Hz, 1H, bCHa Asn), 2.45 (dd, Jvic = 7.4 Hz,
1H, bCHb Asn), 1.88, 1.85 , 1.80 (3s, 12H, NAc); 13C NMR (125 MHz,
D2O, DMSO-d6 as internal standard): d = 176.1, 176.0, 174.0, 172.9,
MeOH–H2O 5:1); Rf = 0.68 (isopropanol–1 M NH4OAc 2:1); 1H
NMR (500 MHz, D2O, acetone-d6 (25% v/v) as internal standard):
d = 7.70–7.20 (m, 8H, Fmoc), 6.88, 6.43 (2 m, 4H, Tyr), 5.0 (d, J1,2
<1.0 Hz, 1H, H-14), 4.86 (d, J1,2 = 9.6 Hz, 1H, H-11), 4.81 (d, J1,2
0
<1.0 Hz, 1H, H-14 ), 4.63 (d, J1,2 <1.0 Hz, 1H, H-13), 4.60 (m, 1H,
172.5, 172.0 (C@O), 155.7 (C-p Tyr), 132.3 (C-o Tyr), 130.8 (C-i
0
a
CH Asn), 4.47 (d, J1,2 = 7.7 Hz, 1H, H-12), 4.43 (2d, J1,2 = 8.2 Hz,
Tyr), 116.8 (C-m Tyr), 102.8 (C-12), 102.0 (C-13), 101.2 (C-15, C-15 ),

S. Mezzato, C. Unverzagt / Carbohydrate Research 345 (2010) 1306–1315
3. Hojo, H.; Nakahara, Y. Biopolymers 2007, 88, 308–324.
1315
0
101.1 (C-14), 98.5 (C-14 ), 82.0 (C-33), 81.1 (C-42), 80.3 (C-41), 79.7 (C-
40
50
4. Tan, Z.; Shang, S.; Halkina, T.; Yuan, Y.; Danishefsky, S. J. J. Am. Chem. Soc. 2009,
131, 5424–5431.
5. Yamamoto, N.; Tanabe, Y.; Okamoto, R.; Dawson, P. E.; Kajihara, Y. J. Am. Chem.
Soc. 2008, 130, 501–510.
6. Piontek, C.; Ring, P.; Harjes, O.; Heinlein, C.; Mezzato, S.; Lombana, N.; Pöhner,
C.; Püttner, M.; Varon Silva, D.; Martin, A.; Schmid, F. X.; Unverzagt, C. Angew.
Chem., Int. Ed. 2009, 48, 1936–1940.
7. Piontek, C.; Varon Silva, D.; Heinlein, C.; Pöhner, C.; Mezzato, S.; Ring, P.;
Martin, A.; Schmid, F. X.; Unverzagt, C. Angew. Chem., Int. Ed. 2009, 48, 1941–
1945.
11), 78.0 (C-24), 77.9 (C-2 ), 77.7 (C-51), 77.4 (C-55, C-5 ), 75.9 (C-52,
0
0
C-53), 75.1 (C-54), 74. 9, 74.8 (C-35, C-35 ), 74.5 (C-54 ), 74.3 (C-31),
0
0
73.5 (C-32), 71.7 (C-23), 71.5 (C-45, C-45 ), 71.0 (C-34, C-34 ), 68.9
0
0
(C-44, C-44 ), 67.4 (C-63), 67.2 (C-43), 63.2 (C-64 , C-64 , bC SerC),
0
62.6 (bC SerA, B), 62.2 (C-65, C-65 ), 61.52 (C-62), 61.45 (C-61), 57.9
0
(aC Tyr), 57.0 (
a
C SerA), 56.9 (
a
C SerB, C-25, C-25 ), 56.7 (
a
C SerC),
56.6 (C-22), 55.2 (C-21), 51.7 (
a
C Asn), 38.22 (bC Tyr), 38.16 (bC
Asn), 23.9, 23.8, 23.7 (NAc), Ser A, B, C were not assigned to the pep-
tide sequence; MALDI-TOF-MS (m/z) calcd for C72H114N10O46 [M]+:
1854.7; found: 1879.5 [M+Na]+.
8. Nagorny, P.; Fasching, B.; Li, X.; Chen, G.; Aussedat, B.; Danishefsky, S. J. J. Am.
Chem. Soc. 2009, 131, 5792–5799.
9. Huang, W.; Li, C.; Li, B.; Umekawa, M.; Yamamoto, K.; Zhang, X.; Wang, L. X. J.
Am. Chem. Soc. 2009, 131, 2214–2223.
10. Heidecke, C. D.; Ling, Z.; Bruce, N. C.; Moir, J. W.; Parsons, T. B.; Fairbanks, A. J.
Chembiochem 2008, 9, 2045–2051.
11. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science 1994, 266, 776–
779.
12. Cohen-Ansfield, S. T.; Lansbury, P. T. J. Am. Chem. Soc. 1993, 115, 10531.
13. Herzner, H.; Reipen, T.; Schultz, M.; Kunz, H. Chem. Rev. 2000, 100, 4495–
44538.
14. Unverzagt, C. Tetrahedron Lett. 1997, 38, 5627–5630.
15. Mezzato, S.; Schaffrath, M.; Unverzagt, C. Angew. Chem., Int. Ed. 2005, 44, 1650–
1654.
16. Unverzagt, C.; Kunz, H.; Paulson, J. C. J. Am. Chem. Soc. 1990, 112, 9308–9309.
17. Unverzagt, C. Angew. Chem., Int. Ed. 1996, 35, 2350–2353.
18. Unverzagt, C. Carbohydr. Res. 1997, 305, 423–431.
19. Unverzagt, C. Chem. Eur. J. 2003, 9, 1369–1376.
20. Unverzagt, C.; Eller, S.; Mezzato, S.; Schuberth, R. Chem. Eur. J. 2008, 14, 1304–
1311.
3.15. N4-{O-(5-Acetamido-3,5-dideoxy-
non-2-ulopyranosylonic acid)-(2?6)-b- -galactopyranosyl-(1?4)-
O-2-acetamido-2-deoxy-b- -glucopyranosyl)-(1?2)-O- -man-
nopyranosyl-(1?3)-O-[(5-acetamido-3,5-dideoxy- -gly-cero-
-galacto-non-2-ulopyranosylonic acid)-(2?6)-b-DD-galactopyr-
D-glycero-a-D-galacto-
D
D
a-D
D
a-
D
anosyl-(1?4)-(2-acetamido-2-deoxy-b-
D
-glucopyranosyl)-(1?2)-
-mannopyranosyl-(1?4)-
-glucopyranosyl)-(1?4)-(2-aceta-
-glucopyranosyl)}-N2-(
-seryl- -seryl- -seryl)-
L-tyrosine A
O-
O-(2-acetamido-2-deoxy-b-
mido-2-deoxy-b-
-asparaginyl-
a-D-mannopyranosyl-(1?6)]-O-b-D
D
D
L
L
L
L
Glycopeptide 16 (4.6 mg, 2.48
sodium cacodylate buffer (pH 7.4, 1400
um albumin (1.0 mg), NaN3 (2.5 mol), MnCl2 (1.4
50-diphosphogalactose (5.6 mg, 8.4
mol), alkaline phosphatase
l
mol) was dissolved in 20 mM
L) containing bovine ser-
mol), uridine-
21. Kajihara, Y.; Suzuki, Y.; Yamamoto, N.; Sasaki, K.; Sakakibara, T.; Juneja, L. R.
Chem. Eur. J. 2004, 10, 971–985.
l
l
l
22. Seko, A.; Koketsu, M.; Nishizono, M.; Enoki, Y.; Ibrahim, H. R.; Juneja, L. R.; Kim,
M.; Yamamoto, T. Biochim. Biophys. Acta 1997, 1335, 23–32.
23. Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1994, 33, 1102–1104.
24. Atherton, E.; Bury, C.; Sheppard, R. C.; Williams, B. J. Tetrahedron Lett. 1979, 32,
3041–3042.
25. Bayley, H.; Standring, D. N.; Knowles, J. R. Tetrahedron Lett. 1978, 3633–3634.
26. Green, M.; Berman, J. Tetrahedron Lett. 1990, 31, 5851–5852.
27. Mergler, M.; Dick, F.; Sax, B.; Stahelin, C.; Vorherr, T. J. Pept. Sci. 2003, 9, 518–
526.
l
(E.C. 3.1.3.1, 6 U), and GlcNAc-b-(1?4)-galactosyltransferase (E.C.
2.4.1.22, 120 mU). The reaction mixture was incubated for 24 h at
37 °C maintaining a pH value of 7.0 by addition of 1 M NaOH. After
complete reaction (TLC, 2:1 isopropanol–1 M NH4OAc) cytidine-50-
monophospho-N-acetylneuraminic acid (4.8 mg, 6.2
lmol) and b-
28. Mergler, M.; Dick, F.; Sax, B.; Weiler, P.; Vorherr, T. J. Pept. Sci. 2003, 9, 36–46.
29. Bodanszky, M.; Tolle, J. C.; Deshmane, S. S.; Bodanszky, A. Int. J. Pept. Protein Res.
1978, 12, 57–68.
30. Yamamoto, N.; Takayanagi, A.; Sakakibara, T.; Dawson, P. E.; Kajihara, Y.
Tetrahedron Lett. 2006, 47, 1341–1346.
31. Elizabeth, C.; Maljaars, P.; Halkes, K. M.; de Oude, W. L.; van der Poel, S.;
Pijnenburg, N. J. M.; Kamerling, J. P. J. Carbohydr. Chem. 2005, 24, 353–
367.
galactoside- -(2?6)-sialyltransferase (E.C. 2.4.99.1, 25 mU) were
a
added. Incubation was continued for 24 h at 37 °C (pH 7.0) followed
by another addition of cytidine-50-monophospho-N-acetylneuram-
inic acid (4.8 mg, 6.2 lmol) and b-galactoside-a-(2?6)-sialyltrans-
ferase (25 mU). After 24 h of reaction time, the precipitate was
removed by centrifugation. The supernatant was concentrated to
32. Lapatsanis, L.; Milias, G.; Froussios, K.; Kolovos, M. Synthesis 1983, 671–
673.
33. Meinjohanns, E.; Meldal, M.; Paulsen, H.; Dwek, R. A.; Bock, K. J. Chem. Soc.,
Perkin Trans. 1 1998, 549.
400
Superdex 30 (600 ꢀ 16 mm) mobile phase: 100 mM NH4HCO3, flow
rate: 750 L/min). The positive fractions were collected and lyoph-
lL and purified by gel filtration (column: Pharmacia Hi Load
l
ilized to afford A (6.2 mg, 91.1%). Rf digalactoside = 0.22 (isopropa-
nol–1 M NH4OAc 2:1); Rf A = 0.16 (isopropanol–1 M NH4OAc 2:1).
34. Carpino, L. A.; El-Faham, A. J. Org. Chem. 1994, 59, 695–698.
35. Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahedron Lett. 1994, 35, 2279–
2282.
36. Kenner, G. W.; Seely, J. H. J. Am. Chem. Soc. 1972, 94, 3259–3260.
37. Kloss, G.; Schroeder, E. Hoppe-Seyler’s Z. Physiol. Chem. 1964, 336, 248–256.
38. Barbas, C. F., III; Matos, J. R.; West, J. B.; Wong, C. H. J. Am. Chem. Soc. 1988, 110,
5162–5166.
39. Fazio, F.; Bryan, M. C.; Blixt, O.; Paulson, J. C.; Wong, C. H. J. Am. Chem. Soc. 2002,
124, 14397–14402.
Acknowledgments
This work was supported by the Deutsche Forschungsgemeins-
chaft, the Leonhard-Lorenz-Stiftung, and the Fonds der Deutschen
Chemischen Industrie.
40. Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed.
Engl. 1995, 34, 412–432.
41. Wong, C.-H.; Halcomb, R. L.; Ichikawa, Y.; Kajimoto, T. Angew. Chem., Int. Ed.
Engl. 1995, 34, 521–546.
42. Palcic, M. M. Methods Enzymol. 1994, 230, 300–316.
43. Kessler, H.; Gehrke, M.; Griesinger, C. Angew. Chem., Int. Ed. Engl. 1988, 27, 490–
536.
References
1. Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev. 2009, 109, 131–163.
2. Bennett, C. S.; Dean, S. M.; Payne, R. J.; Ficht, S.; Brik, A.; Wong, C. H. J. Am. Chem.
Soc. 2008, 130, 11945–11952.
44. Kessler, H.; Schmieder, P.; Kurz, M. J. Magn. Reson. 1989, 85, 400–405.