The (2-Phenyl-2-trimethylsilyl)ethyl-(PTMSEL)-Linker in the Synthesis of Glycopeptide Partial Structures of Complex Cell Surface Glycoproteins

Article abstract of DOI:10.1002/chem.200305304

The (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL) linker represents a novel fluoride-sensitive anchor for the solid-phase synthesis of protected peptides and glycopeptides. Its cleavage is achieved under almost neutral conditions using tetrabutylammonium fluo

Full text of DOI:10.1002/chem.200305304

FULL PAPER
The (2-Phenyl-2-trimethylsilyl)ethyl-(PTMSEL)-Linker in the Synthesis of
Glycopeptide Partial Structures of Complex Cell Surface Glycoproteins
Michael Wagner, Sebastian Dziadek, and Horst Kunz*[a]
Dedicated to Professor Reinhard Hoffmann on the occasion of his 70th birthday
Abstract: The (2-phenyl-2-trimethylsi-
lyl)ethyl-(PTMSEL) linker represents a
novel fluoride-sensitive anchor for the
solid-phase synthesis of protected pep-
tides and glycopeptides. Its cleavage is
achieved under almost neutral condi-
tions using tetrabutylammonium fluo-
ride trihydrate in dichloromethane thus
allowing the construction of complex
molecules sensitive to basic and acidic
media commonly required for the
cleavage of standard linker systems.
The advantages of the PTMSEL linker
are demonstrated in the synthesis of
glycopeptides from the liver intestine
(LI)-cadherin and the mucin MUC1,
bearing carbohydrate moieties such as
N-linked chitobiose or O-linked sialyl-
TN-residues. The synthesis of these
types of glycopeptides is difficult be-
cause they are prone to secondary
structure formation during the synthe-
sis on the solid phase as well as in the
completely deprotected form. Using
the PTMSEL linker these molecules
are accessible by automated synthesis
according to the Fmoc strategy without
frequently observed side reactions such
as aspartimide or diketopiperazine for-
mation.
Keywords:
glycopeptides
cadherins
mucins
¥
¥
¥
silicon-
based linkers ¥ solid-phase synthesis
Introduction
holds true for the synthesis of protected glycopeptides,
which shall be used in fragment condensations. Most of the
common anchors are either acid- or base-labile. Many acid-
labile anchors are based on substituted benzyl- or triphenyl-
methyl esters.[3,4] Their cleavage forms stable cations, which
may cause undesired alkylation of nucleophilic moieties.[4]
Furthermore, acid-labile protecting groups such as Boc, tert-
butyl or trityl residues, for example in the amino acid side
chains, are removed simultaneously. Base-labile linkages
often are not compatible with the fluorenylmethoxycarbonyl
(Fmoc)-strategy.[5] Under acidic and basic cleavage condi-
tions undesired side reactions can take place on the pro-
duced peptides. Among those, aspartimide formation and re-
arrangements of aspartyl peptides are difficult to prevent.[6]
Most of these side reactions can be avoided by using linker
systems, which are cleavable under neutral conditions such
as allylic anchors.[7] However, because of their insufficient
steric hindrance, the allyl ester linkers are susceptible to
aminolysis. Accordingly, they are prone to diketopiperazine
formation[3f,4] at the stage of the polymer-bound dipeptides.
Due to posttranslational modifications, proteins and pepti-
des exist in a variety of conjugated forms, most importantly
as glycoconjugates. The N- and O-glycosidically bound car-
bohydrates affect the conformation of proteins and regulate
their activity and biological half-lives. Glycoproteins and
glycopeptides, for example cadherins or mucins, play funda-
mental roles in many biological processes, such as cell adhe-
sion, regulation of cell growth and cell differentiation.[1] The
synthesis of exactly specified partial structures of these gly-
coproteins provides a feasible approach to gain more infor-
mation on structural and biological aspects of those regula-
tory processes and to detect or possibly influence pathologi-
cal deficiencies.
Such peptides and glycopeptides can efficiently be syn-
thesized applying solid-phase methodologies.[2] In all varia-
tions of solid-phase and combinatorial syntheses the linker
is of importance. It must be stable throughout the multistep
synthesis, but finally cleavable under mild conditions with-
out affecting the produced compounds.[3] This especially
Recently,
the
(2-phenyl-2-trimethylsilyl)ethyl-
(PTMSEL)-linker 1 was introduced as a fluoride-labile
linker for the solid-phase synthesis of protected peptides
and glycopeptides according to Fmoc chemistry.[8] Cleavage
of the PTMSEL-linker is achieved under almost neutral
conditions by treatment with tetrabutylammonium fluoride
trihydrate (TBAF¥3H2O) in dichloromethane. Under these
[a] Prof. Dr. H. Kunz, Dr. M. Wagner, Dipl.-Chem. S. Dziadek
Institut f¸r Organische Chemie der Universit‰t Mainz
Duesbergweg 10 14, 55128 Mainz (Germany)
Fax : (+49)6131-39-24786
E-mail: hokunz@uni-mainz.de
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/chem.200305304
Chem. Eur. J. 2003, 9, 6018 6030

6018 6030
conditions, the hydrate shells of fluoride ions are unaffected,
keeping the basicity at a low level. The increased sensitivity
towards fluoridolysis is due to the benzylic position of the
CꢀSi bond (Scheme 1).
pressed. Furthermore, the PTMSEL linker is sterically so
demanding, that even in sequences prone to diketopipera-
zine formation (Pro-Gly) this side reaction did not occur.[8]
Results and Discussion
Taking advantage of its useful
properties the (2-phenyl-2-tri-
methylsilyl)ethyl-(PTMSEL)-
linker was employed in synthe-
ses of complex glycopeptides.
On the one hand, glycopepti-
des from the LI-cadherin[11]
prone to formation of secon-
dary structures were synthe-
sized. On the other hand, gly-
copeptide sequences of the
tandem repeat region of the
mucin[12] MUC1 bearing demanding carbohydrates were
constructed.
Scheme 1. Mechanism of fluoride induced cleavage of the PTMSEL-linker.
Other fluoride-labile anchors, such as p-silylmethyl-
benzyl, p-silyloxy-benzyl and a-trimethylsilyl-benzyl (SAC)
linkers,[9] require much more basic cleavage conditions, for
example TBAF¥3H2O in dimethylformamide or tetrahydro-
furan; these conditions are not compatible for retaining the
Fmoc group. Therefore, the PTMSEL linker shows several
advantageous properties compared with linkers described
earlier: Common protecting groups (e.g. Fmoc, Boc, Z,
Aloc, tert-butyl, benzyl, allyl, trityl)[10] are stable under the
mild cleavage conditions of the PTMSEL linker and provide
the possibility of orthogonal, three dimensional protecting
group strategies. Early results showed that serious side reac-
tions, such as aspartimide formations, are decisively sup-
Synthesis of the PTMSEL-linker-system: Trimethylvinylsi-
lane 2 was treated with m-chloroperbenzoic acid to give tri-
methylsilyloxirane 3.[13] Reaction of 3 with lithium di[4-(1-
ethoxyethyloxy)phenyl]cuprate, generated in situ by lithiia-
tion of 1-(4-bromophenoxy)-1-ethoxyethane with n-butyl-
lithium and subsequent reaction with copper(i)iodide, effi-
ciently yielded 2-[4-(1-ethoxyethyloxy)phenyl]-2-trimethyl-
silyl ethanol (4) in 85% yield.[8,14] The 1-ethoxy-ethyl-(EE)-
protecting group was removed using catalytic amounts of
pyridinium-p-toluenesulfonate (PPTS).[15] The PTMSEL
linker unit, 2-(4-hydroxyphenyl)-2-trimethylsilyl-ethanol (5),
was obtained in a yield of 90% (76% from 3, Scheme 2).
Synthesis of anchor conjugates and resin loading: 2-(4-Hy-
droxyphenyl)-2-trimethylsilyl-ethanol (5) was linked to the
polymeric support via its phenolic hydroxyl group
(Scheme 2). Prior to this, the N-acyl amino acid was coupled
to the linker molecule in solution since esterification on
solid-phase is often accompanied by racemization. Accord-
ing to this strategy, 2-(4-hydroxyphenyl)-2-trimethylsilyl-eth-
anol (5) was treated with allyl chloroacetate to give allyl 4-
[2-hydroxy-1-(trimethylsilyl)ethyl]phenoxy acetate (6). Acy-
lation with the corresponding Fmoc-protected amino acid
using dicyclohexylcarbodiimide (DCC) and catalytic
amounts of 4-dimethylaminopyridine (DMAP)[16] gave com-
pounds 7a and 7b (yields: >90%) protected as allyl esters.
The allyl residue of 7a/b was removed quantitatively using
catalytic [Pd(PPh3)4] and sodium p-toluenesulfinate or other
allyl trapping reagents furnishing acids 8a and 8b, respec-
tively (yield ꢁ54% from 3).[17] Acids 8a and 8b can be con-
densed with an amino-functionalized resin using O-(benzo-
triazole-1-yl)-N,N,N’,N’-tetramethyluronium tetrafluorobo-
rate (TBTU)/N-hydroxybenzotriazole (HOBt)/diisopropyl-
ethylamine (DIPEA) in DMF/CH2Cl2.[18] Aminomethyl
polystyrene, AMPS (ACT; 200 400 mesh; loading:
1.00 mmolgꢀ1), or amino-functionalized TentaGel, NovaSyn
Tg amino resin (Novabiochem, 110 mm beads, loading:
Abstract in German: Der (2-Phenyl-2-trimethylsilyl)ethyl-
(PTMSEL)-Linker ist ein neuer fluorid-sensitiver Anker f¸r
die Festphasen-Synthese gesch¸tzter Peptide und Glycopep-
tide. Die Spaltung unter nahezu neutralen Bedingungen bei
Verwendung von Tetrabutylammoniumfluoridtrihydrat in Di-
chlormethan ermˆglicht den Aufbau komplexer Molek¸le,
die empfindlich gegen¸ber bei den Spaltungen g‰ngiger An-
kersysteme benˆtigten Basen bzw. S‰uren sind. Die Vorteile
des PTMSEL-Linkers werden gezeigt an Synthesen von Gly-
copeptiden aus dem leber-intestinalen (LI) Cadherin und aus
dem Mucin MUC1, die Kohlenhydratstrukturen wie N-ge-
bundene Chitobiose oder O-verkn¸pftes Sialyl-TN-Antigen
tragen. Aufgrund der Neigung solcher zur Ausbildung sekun-
d‰rer Strukturen sowohl w‰hrend der Synthese an fester
Phase als auch in ungesch¸tzter Form in Lˆsung, ist die Syn-
these solcher Glycopeptidstrukturen schwierig. Bei Verwen-
dung des PTMSEL-Ankers kˆnnen derartige Molek¸le in au-
tomatisierten Synthesen nach der Fmoc-Strategie hergestellt
werden, ohne dass es zu Nebenreaktion wie der h‰ufig zu
beobachtenden Aspartimid- oder Diketopiperazinbildung
kommt.
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H. Kunz et al.
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Therefore, synthetic partial
structures of this homophilic
binding region are considered
useful tools for the elucidation
of the mechanisms of LI-cad-
herin mediated cell-cell adhe-
sion.
Synthesis of the glycosylated
building block: Chitobiosyloc-
taacetate 10[21] treated with a
saturated solution of hydrogen
chloride in acetyl chloride gave
glycosyl chloride 11. Subse-
quent reaction with sodium
azide in the presence of a phase
transfer catalyst rendered chito-
biosyl azide 12.[22] Hydrogena-
tion over Raney nickel in diox-
ane/ethanol (4:1) efficiently
yielded the chitobiosyl amine
13, which was condensed via its
amino group with Fmoc-
Asp(OH)-OAll
(14)
using
TBTU,[18] HOBt and DIPEA as
coupling reagents to give the
completely protected, N-glyco-
syl asparagine derivative 15 in
76% yield. The allyl ester of 15
was cleaved by palladium(0)
catalyzed allyl transfer to
sodium p-toluenesulfinate[17] to
form the Fmoc-protected N-as-
paragine building block 16
ready for use in the solid-phase
synthesis (Scheme 3).
Scheme 2. Synthesis of the PTMSEL-linker and loading of the resin. a) mCPBA (1.0 equiv), CHCl3, RT, 60 h,
74%; b) lithium [di(p-1-ethoxyethyloxy)-phenyl]cuprate (3.0 equiv), diethyl ether, ꢀ508C, 5 h ! ꢀ208C, 14 h,
85%; c) PPTS (0.03 equiv), MeOH, RT, 2 h, 90%; d) allyl chloroacetate (1.9 equiv), K2CO3, KI, acetone, RT,
38 h, 78%; e) Fmoc-AA-OH (1.1 equiv), DCC (1.2 equiv), DMAP (0.06 equiv), CH2Cl2, 08C, 4 h, > 90%;
f) [Pd(PPh3)4] (0.06 equiv), p-toluenesulfinate (1.3 equiv), THF/MeOH, RT, 90 min, quant.; g) TBTU
(1.0 equiv), HOBt (1.0 equiv), NMM (2.0 equiv), CH2Cl2/DMF, RT, 18 h.
Solid-phase synthesis: Starting
from polymer 9a loaded with
Fmoc-Ile-OH
(loading:
0.365 mmolgꢀ1), glycododeca-
peptide Boc-Leu-Gln(Trt)-Val-
Ala-Ala-Leu-Asp(OtBu)-Ala-
Asn(bAc3GlcNAc-bAc3Glc-
0.43 mmolgꢀ1) were used as polymeric supports. Unreacted
amino groups were capped with acetic anhydride/pyridine
1:3 (Scheme 2).
NAc)-Gly-Ile-Ile-OH (17) was assembled using a peptide
synthesizer applying Fmoc strategy (Fmoc deprotection:
30% piperidine in N-methylpyrrolidone; coupling reagents:
HBTU,[23] HOBt, DIPEA) (Scheme 4). The glycosylated
building block Fmoc-Asn(bAc3GlcNAc-bAc3GlcNAc)-OH
(16) (3 equiv) was coupled manually using O-(7-aza-benzo-
triazole-1-yl)-N,N,N’,N’-tetramethyluronium-hexafluorophos-
phate (HATU)[24]/N-hydroxy-7-azabenzotriazole (HOAt).
After completion of the automated peptide synthesis, the
glycopeptide was released from the solid support by two se-
quential treatments with two equivalents of tetrabutylam-
monium fluoride trihydrate (TBAF¥3H2O) in dichloro-
methane.
Synthesis of an N-glycododecapeptide from human LI-cad-
herin: Cadherins[19] represent a class of important cell-adhe-
sion proteins. LI-cadherin is a structurally distinct, intestine
specific member of the cadherine gene superfamily. Recent
data suggest, that down-regulation of cadherin expression
parallels cellular dysregulations in human diseases.[19] There-
fore, LI-cadherin mediated cell cell adhesion could play an
important role in maintaining physiological cellular func-
tions in the intestinal mucosa.[20] It is assumed that LI-cad-
herin mediated homophilic binding proceeds exclusively via
a specific binding region in the extracellular domain EC1.
An aliquot was taken from the resin and analyzed by
HR-MAS spectroscopy. The absence of the characteristic
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Glycopeptide Synthesis
6018 6030
the described cleavage proce-
dure showed high purity with
almost no side products except
for deletion sequences. In par-
ticular, formation of an asparti-
mide (the sequence Asp-Ala is
well known to undergo this side
reaction)[6b] was not observed.
Final purification by prepara-
tive HPLC gave the protected
glycopeptide 17 in 29% yield
(based on the loading of poly-
mer 9a). The moderate yield is
mostly due to secondary struc-
ture formation during the syn-
thesis on the resin as was
shown by 2D NMR spectrosco-
Scheme 3. Synthesis of chitobiosyl asparagine building block 16. a) sat. HCl in acetyl chloride, RT, 48 h, 82%;
b) NaN3 (3.1 equiv), aliquat 336 (1.0 equiv), CHCl3/H2O, RT, 42 h, 70 %; c) H2, raney-nickel, dioxane/EtOH,
RT, 3 h, quant.; d) TBTU (1.0 equiv), HOBt (1.0 equiv), DIPEA (2.0 equiv), DMF, RT, 40 h, 76%;
e) [Pd(PPh3)4] (0.06 equiv), sodium-p-toluenesulfinate (1.3 equiv), THF/MeOH, RT, 70 min, 57%.
signal corresponding to the trimethylsilyl group gave evi-
dence for complete release of the glycopeptide from the
resin. HPLC analysis of the crude material obtained from
py (NOESY and ROESY experiments) of protected peptide
17. In addition, the poor solubility of protected glycopeptide
17 in all common solvents such as dichloromethane, acetoni-
Scheme 4. Solid-phase synthesis of glycododecapeptide 17 from LI-cadherin (human). a) 2îTBAF¥3H2O (2 equiv), CH2Cl2, RT, 25 min, 29% (based on
9a).
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H. Kunz et al.
FULL PAPER
ciated structure alterations con-
stitute a promising basis for a
selective immunological attack
at certain epithelial cancer cells.
Over the years, several TN-, T-
and sialyl-TN-antigen-containing
glycopeptides have been syn-
thesized as potential vaccines,
some of them showing interest-
ing biological effects.[2,26] The
biotinylated sialyl-TN-glycodo-
decapeptide described herein,
which is derived from the
tandem repeat unit of MUC1,
is a promising candidate for the
construction of tumor-selective
immunostimulating antigens for
the development of anticancer
vaccines. The biotinylation fa-
cilitates immunological evalua-
tion procedures. This glycocon-
jugate is also considered a chal-
lenging target molecule for a
solid-phase synthesis using the
PTMSEL-linker to demonstrate
the beneficial properties of this
novel technology even in the
case of sensitive target mole-
cules.
Scheme 5. Removal of protecting groups from 17. a) TFA/TIS/H2O (38:1:1), RT, 4 h, 97%; b) NaOMe,
MeOH, pH 10, RT, 7 h, 79%.
trile and water has to be taken into account to explain the
yield. The acid labile protecting groups of 17 were removed
by treatment with a mixture of trifluoroacetic acid, triisopro-
pylsilane and water (Scheme 5). In order to remove the O-
acetyl protecting groups from the chitobiosyl unit, glycopep-
tide 18 was treated with sodium methanolate in methanol at
pH 10 and efficiently yielded the completely deprotected
glycopeptide 19 in 79% yield (22% yield based on the pre-
loaded resin 9a). Glycopeptide 19 was analyzed by 2D
NMR spectroscopy, which showed clear evidence of a b-turn
conformation for Asp, Ala, Asn(chitobiosyl) and Gly. The
biological investigation of compound 19 and other peptides
and glycopeptides from this series are ongoing and will be
reported elesewhere.
Synthesis of the sialyl-TN-threonine building block: The
pivotal step in the preparation of the Fmoc-protected sialyl-
TN threonine building block consists of the regio- and ster-
eoselective sialylation of Fmoc-(GalNAc)-OtBu (21)[27] at
the reactive primary 6-hydroxy group.[28] (Scheme 6) This
sialylation was accomplished employing benzyl ester pro-
tected sialyl xanthate 20[28,29] activated by methyl sulfenyl
triflate[30] (generated in situ from methyl sulfenyl bromide
Synthesis of
a biotinylated O-glycododecapeptide from
MUC1: The mucin MUC1[25] is a high molecular weight inte-
gral membrane glycoprotein located on the lumenal surface
of ubiquitously distributed epithelial cells including those of
the mammary gland ducts, the bladder, and the respiratory
tract. The extracellular domain primarily consists of 20-
amino acid tandem repeats. The main function of mucins is
to provide lubrication and protection of epithelial cell surfa-
ces. In contrast to healthy cells, the expression of MUC1 in
epithelial tumors is dramatically increased. It is combined
with an incomplete glycosylation pattern occurring due to a
premature sialylation and resulting in additional exposed
peptide epitopes of the protein backbone. These tumor-asso-
Scheme 6. Synthesis of sialyl-TN-threonine building block. a) methylsul-
fenyl bromide (1.6m solution in 1,2-dichloroethane), CH3CN/CH2Cl2
(2:1), ꢀ628C, 4 h, 57%; b) pyridine/Ac2O (2:1), 4 h, 08C, 15 h, RT, 76%;
c) CH2Cl2/TFA/anisole (10:10:1), 97%.
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Glycopeptide Synthesis
6018 6030
and silver triflate). Low temper-
ature (ꢀ628C) and the use of
acetonitrile/dichloromethane
(2:1) favored the formation of
the a-sialoside 22 in a kinetical-
ly controlled reaction. The de-
sired a-sialyl-TN conjugate 22
was obtained in a yield of 57%
(Scheme 6). Apart from the b-
anomer (a/b 10:1) the glycal of
the sialic acid benzyl ester was
formed as the only by-product.
Subsequent O-acetylation (23,
76%) with acetic anhydride in
pyridine and removal of the
tert-butyl ester protecting group
using a mixture of trifluoroace-
tic acid and anisole yielded the
sialyl-TN-threonine
building
block 24 (97%) which was in-
corporated into the solid-phase
synthesis of the sialyl-TN-glyco-
dodecapeptide.
Solid-phase synthesis: Using
Fmoc-Pro-PTMSEL preloaded
NovaSyn Tg resin 9b (loading:
0.228 mmolgꢀ1) the glycodode-
capeptide biotin-Gly-Val-Thr(a-
Ac4NeuNAcCOOBn-(2!6)-a-
Ac2GalNAc)-Ser(tBu)-Ala-Pro-
Asp(OtBu)-Thr(tBu)-Arg(Pmc)-
Pro-Ala-Pro-OH (25) was pre-
pared in an automated synthe-
sis in analogy to the methodolo-
gy described above. The sialyl-
TN-threonine conjugate 24 was
introduced by a double cou-
pling
HATU,[24] HOAt and N-
methylmorpholine (NMM)
(2î1.3 equiv)
using
(Scheme 7). After N-terminal
deprotection of the resin-linked
glycopeptide, carboxy activated
biotin (HBTU/HOBt, NMM)
was attached in two consecutive
coupling steps. The glycoconju-
gate was liberated from the
Scheme 7. Solid-phase synthesis of biotinylated sialyl-TN-glycododecapeptide 27 from MUC1. a) iterative
cycles: deprotection, coupling, capping; b) TBAF¥3H2O, CH2Cl2, RT, 30 min, 42% (with respect to 9b);
c) 1. Pd/C (10%), MeOH, RT, 48 h; 2. CH2Cl2/TFA/thioanisole/1,2-ethanedithiol (10:10:1:1), RT, 2 h; d)
NaOMe/MeOH, pH 8.5 9, RT, 15 h, 20% (with respect to 25).
resin
by
treatment
with
TBAF¥3H2O in dichloromethane to furnish the selectively
deblocked derivative 25 in a yield of 42% after preparative
HPLC (based on the preloaded resin 9b). Only traces of
side products were observed, and no aspartimide formation
and rearrangements could be detected.
The overall yield of 42% is high considering not only
the introduction of a complex glycosylated amino acid and
an N-terminal biotin residue, but also that the Pro-Ala-Pro
starting sequence[4] is very prone to diketopiperazine forma-
tion. The use of a sterically less hindered resin (allyl ester
type) under identical conditions led to a high degree of di-
ketopiperazine formation with almost no peptide remaining
on the resin. The sialic acid benzyl ester of protected glyco-
peptide 25 was removed by hydrogenolysis using palladium
on activated charcoal (10%). Careful cleavage of the acid-
labile side chain protecting groups by treatment with a mix-
ture of trifluoroacetic acid, thioanisole and 1,2-ethanedithiol
and subsequent mild methanolysis of the O-acetyl groups
furnished the biotinylated MUC1 glycododecapeptide 27 in
a yield of 20% over three steps. Purification was accom-
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H. Kunz et al.
FULL PAPER
corded on a Finnigan MAT-95-spectrometer, MALDI-TOF mass spectra
were aquired on a Micromass Tofspec E spectrometer. ESI-mass spectra
were obtained on a ThemoQuest-Navigator spectrometer. HR-ESI mass
spectra were recorded on a Micromass Q-TOF Ultima spectrometer.
plished by means of preparative HPLC. Currently, the gly-
coconjugate 27 is investigated in immunological experiments
towards the induction of antibodies and the development of
anticancer-vaccines based on tumor-associated glycopeptide
antigens.
2-Trimethylsilyl oxirane (3): The title compound was synthesized accord-
ing to ref. [13] to yield a colorless liquid (8.69 g, 74%). B.p. (370 mbar):
75 798C; [a]2D5 = 1.414; 1H NMR (200 MHz, CDCl3): d=2.88 (t, 1H, b-
CH, J=5.6 Hz), 2.50 2.56 (m, 1H, b-CH), 2.17 (t, 1H, a-CH, J=4.6 Hz),
0.04 (s, 9H, Si(CH3)3).
Conclusion
2-[4-(1-Ethoxyethyloxy)phenyl]-2-trimethylsilyl-ethanol (4): The title
compound was synthesized in analogy to the procedure described for the
corresponding phenyl derivative in ref. [8b]:
The solid-phase syntheses of complex glycopeptides from
the LI-cadherin and mucin series have been accomplished
using the novel (2-phenyl-2-trimethylsilyl)ethyl-(PTMSEL)-
linker. Its cleavage under almost neutral conditions by tetra-
butylammonium fluoride trihydrate in dichloromethane has
been shown to have advantages over other common linkers:
Most of the common protecting groups (e.g., Fmoc, Boc, Z,
Aloc, tert-butyl, benzyl, allyl, trityl) are stable under the
mild cleavage conditions of the PTMSEL linker, enabling
the application of orthogonal, three dimensional protecting
group strategies. Serious side reactions such as aspartimide
formation and rearrangements can be decisively suppressed.
Base sensitive glycosidic bonds, for example, sialyl-TN-threo-
nine, remain stable under the mild cleavage conditions. As
was shown in the synthesis of the biotinylated O-glycodode-
capeptide from MUC1, the PTMSEL linker is sterically so
demanding that even in sequences prone to diketopipera-
zine formation (Pro-Ala-Pro) this side reaction does not
constitute a problem. Therefore, the novel PTMSEL-linker
is particularly useful for the solid-phase synthesis of protect-
ed sensitive and complex peptides and glycopeptides.
1) Lithiation: 1-(4-Bromophenoxy)-1-ethoxyethane (33.09 g, 135.0 mmol)
was dissolved in dry diethyl ether (150 mL). At ꢀ458C n-butyllithium
(1.6m in hexane) (84.37 mL, 135.0 mmol) was added within 30 min. The
solution was stirred at ꢀ408C for 20 min, warmed to +108C within
45 min and stirred for further 90 min at this temperature.
2) Formation of cuprate and cuprate addition: In a 500 mL three-necked
flask CuI (12.29 g, 65.5 mmol) was suspended in dry diethyl ether
(40 mL). The solution containing the lithiated 1-phenoxy-1-ethoxyethane
was transferred into a 500 mL dropping funnel (filled with argon) via a
stainless steel tube and then added dropwise to the CuII suspension at
08C within 70 min. The mixture was stirred for 30 min and then cooled to
ꢀ508C. At this temperature, 2-trimethylsilyl oxirane 3 (2.7 g, 23.2 mmol)
was added dropwise via a syringe. The reaction mixture was stirred at
ꢀ508C for 5 h and overnight at ꢀ208C. A sat. NH4Cl solution (200 mL)
was added and the resulting mixture was stirred at 08C for 20 min, dilut-
ed with ethyl acetate (300 mL) and with additional sat. NH4Cl (200 mL).
The organic phase was washed twice with
a sat. NH4Cl solution
(250 mL), dried over MgSO4 and concentrated in vacuo. The residue was
purified by flash chromatography (650 g silica gel; petrol ether/ethyl ace-
tate 7:1, addition of 0.1% triethylamine). The product containing frac-
tions were diluted with toluene and concentrated in vacuo to yield com-
pound 4 (5.57 g, 85%) as a brown oil. Rf =0.23 (petrol ether/AcOEt 6:1);
1H NMR (200 MHz, [D6]acetone): d=7.03 (d, 2H,
2 arom. H, J=
8.8 Hz), 6.89 (d, 2H, 2 arom. H, J=8.3 Hz), 5.35 (q, 1H, O-CH-O, J=
5.2 Hz), 3.92 4.07 (m, 2H, CH2-OH), 3.65 3.77 (m, 1H, OH), 3.45 3.58
(m, 2H, CH3-CH2-O), 2.31 2.38 (dd, 1H, TMSCH, J1 =3.4 Hz, J2 =
5.4 Hz), 1.39 (d, 3H, CH-CH3, J=5.4 Hz), 1.13 (t, 3H, CH2-CH3, J=
6.8 Hz), ꢀ0.04 (s, 9H, Si(CH3)3); 13C NMR (50.3 MHz, [D6]acetone, BB,
DEPT): d=155.42 (1C, Cipso,Ar-O), 136.51 (1C, Cipso,Ar-CH), 129.48 (2C, 2
arom. C), 118.02 (2C, 2 arom. C), 100.37 (1C, O-CH-O), 63.60 (1C, CH2-
OH), 61.79 (1C, CH3-CH2-O), 41.00 (1C, TMS-CH), 20.71 (1C, CH3-
CH), 15.52 (1C, CH3-CH2), ꢀ2.16 (3C, Si(CH3)3); elemental analysis
calcd (%) for C15H26O3Si: C 63.78 H 9.28; found: C 63.40 H 8.92.
Experimental Section
General: Solvents for moisture-sensitive reactions (THF, diethyl ether,
methanol, dichloromethane) were distilled and dried according to stan-
dard procedures. DMF (amine free, for peptide synthesis) was purchased
from Roth, acetic anhydride and pyridine in p.a. quality from ACROS.
Reagents were purchased at highest available commercial quality and
used without further purification unless outlined otherwise. Fmoc-pro-
tected amino acids were purchased from Novabiochem. As resins for
solid-phase synthesis aminomethyl polystyrene (Advanced ChemTech)
and aminomethylated tentagel (NovaSyn Tg amino resin, Novabiochem)
were used. Reactions were monitored by thin-layer chromatography with
precoated silica gel 60 F254 aluminium plates (Merck KGaA, Darmstadt).
Flash column chromatography was performed with silica gel (40 63 mm)
purchased form Merck KGaA, Darmstadt. Melting points were obtained
on a B¸chi-Tottoli apparatus and are uncorrected. Optical rotations [a]D
were measured with a Perkin Elmer polarimeter 241. Elemental analyses
were performed by the microanalytical laboratory of the Johannes Gu-
tenberg-Universit‰t, Mainz. RP-HPLC analyses were carried out on a
Knauer HPLC system with Eurospher C8 (250î4 mm) and Vydac Pepti-
2-(4-Hydroxyphenyl)-2-trimethylsilyl-ethanol (5): Under argon 2-[4-(1-
ethoxyethyloxy)phenyl]-2-trimethylsilyl-ethanol (4; 2.55 g, 9.01 mmol)
was dissolved in methanol (p.a.) (40 mL). A solution of pyridinium-p-tol-
uenesulfonate (PPTS) (68.1 mg, 0.27 mmol, 0.03 equiv) in methanol
(3 mL) was added and the mixture was stirred for 2 h at room tempera-
ture. The solvent was removed in vacuo and the residue purified by flash
chromatography (140 g silica gel; petrol ether/ethyl acetate 4:1) to give
the desired product 5 (1.71 g; 90%) as colorless crystals. Rf =0.18 (petrol
ether/AcOEt 3:1); m.p. 138 1418C; 1H NMR (200 MHz, [D6]acetone):
d=7.94 (brs, 1H, OH phenol), 6.95 (d, 2H, 2 arom. H, J=8.8 Hz), 6.72
(d, 2H, 2 arom. H, J=8.8 Hz), 3.86 4.07 (m, 2H, CH2-OH), 3.37 (t, 1H,
OH, J=5.4 Hz), 2.28 (dd, 1H, TMS-CH, J1 =3.4 Hz, J2 =8.8 Hz); ꢀ0.05
(s, 9H, Si(CH3)3); 13C NMR (50.3 MHz, [D6]acetone, BB, DEPT): d=
155.53 (1C, Cipso,Ar-OH), 133.74 (1C, Cipso,Ar-CH), 129.60 (2C, 2 arom. C),
115.80 (2C, 2 arom. C), 63.85 (1C, CH2-O), 41.00 (1C, TMS-CH), ꢀ2.11
de&Protein C18 columns (250î4 mm) at a pump rate of 1 mLminꢀ1
.
Preparative HPLC separations were performed on a Knauer HPLC
system with a Eurospher C8 column (250î40 mm, 7 mm) and a pump
rate of 20 mLminꢀ1. Semi-preparative HPLC separations were carried
out on a Knauer HPLC system with a Vydac Peptide&Protein column at
(3C, Si(CH3)3); FD-MS calcd for C11H18O2Si: 210.3; found: 210.4 [M]+
;
a flow rate of 10 mLminꢀ1. Water and CH3CN were used as solvents. H,
1
elemental analysis calcd for C11H18O2Si: C 62.81 H 8.63; found:C 62.79 H
8.52.
13C, and 2D NMR spectra were recorded on Bruker AC-200, AM-400,
ARX-400 or DRX-600 spectrometers. Proton chemical shifts are report-
ed in ppm relative to CHCl3 (d=7.24), DMSO (d=2.49) or acetone (d=
2.05). Multiplicities are given as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet). 13C chemical shifts are reported relative to
CDCl3 (d=77.0), DMSO (d=39.5) or acetone (d=29.84). Assignment of
proton and carbon signals was achieved by COSY, TOCSY, HSQC,
HMQC and HMBC experiments when noted. FD-mass spectra were re-
Allyl 4-[2-hydroxy-1-(trimethylsilyl)-ethyl]-phenoxyacetate (6): 2-(4-Hy-
droxyphenyl)-2-trimethylsilyl-ethanol (5; 1.55 g, 7.37 mmol) was dissolved
in acetone (p.a.) (40 mL). Under argon K2CO3 (1.33 g, 9.59 mmol), KI
(160.0 mg, 0.96 mmol) and allyl chloroacetate (1.03 mL 8.85 mmol) were
added and the mixture was stirred at room temperature for 18 h. TLC
monitoring (petrol ether/AcOEt 3:1) revealed 60% conversion. Further
addition of K2CO3 (0.80 g, 5.75 mmol), KI (96.0 mg, 0.58 mmol) and allyl
6024
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 6018 6030

Glycopeptide Synthesis
6018 6030
chloroacetate (0.62 mL, 5.32 mmol) and stirring for another 20 h resulted
in complete conversion. The mixture was filtered through Hyflo-Supercel
and concentrated in vacuo. The brown oil was purified by flash chroma-
tography (180 g silica gel, petrol ether/ethyl acetate 5:1) to afford the
title compound 6 (1.76 g, 78%) as a colorless oil, which crystallized after
drying in vacuo and storage at 48C. Rf =0.29 (petrol ether/AcOEt 3:1);
m.p. 27 288C; 1H NMR (200 MHz, CDCl3): d=7.01 (d, 2H, 2 arom. H,
J=8.3 Hz), 6.83 (d, 2H, 2 arom. H, J=8.8 Hz), 5.81 6.00 (m, 1H, CH=
CH2), 5.31 (dd, 1H, CH=CHaHb, J=16.3 Hz), 5.24 (dd, 1H, CH=
CHaHb, J=10.5 Hz), 4.69 (d, 2H, CH2-CH=CH2, J=5.5 Hz), 4.61 (s, 2H,
O-CH2-C=O), 3.89 4.12 (m, 2H, CH-CH2-OH), 2.37 (dd, 1H, TMSCH,
J1 =4.4 Hz, J2 =11.2 Hz), 1.50 (brs, 1H, OH), ꢀ0.06 (s, 9H, Si(CH3)3);
13C NMR (50.3 MHz, CDCl3; BB, DEPT): d=168.80 (1C, C=O), 155.74
(1C, Cipso,Ar-O), 133.61 (1C, Cipso,Ar-CH), 131.47 (1C, CH=CH2), 128.82 (2C,
2 arom. C), 119.02 (1C, CH=CH2), 115.03 (2C, 2 arom. C), 65.79, 65.61
(2C, CH2=CH-CH2-O, O=C-CH2-O), 63.22 (1C, TMS-CH-CH2-O), 40.81
(1C, TMS-CH), ꢀ2.11 (3C, Si(CH3)3); FD-MS: calcd for C16H24O4Si:
308.4; found: 308.4 [M]+ ; elemental analysis calcd for C16H24O4Si: C
62.30 H 7.84; found: C 62.15 H 7.64.
(2C, Car PTMSEL), 67.54 (1C, CH2-Fmoc), 66.03, 65.84, 65.51 (3C, CH2=
CH-CH2-O, O=C-CH2-O, TMS-CH-CH2-O), 59.40 (1C, a-CH Pro), 47.27
(1C, C9-Fmoc), 46.85 (1C, g-CH2 Pro), 36.59 (TMS-CH), 29.87 (b-CH2
Pro), 23.98 (1C, d-CH2 Pro), ꢀ2.65 (3C, Si(CH3)3); HR-ESI-TOF-MS:
calcd for C36H41NO7SiNa: 650.2550; found: 650.2528 [M+Na]+.
4-[2-(N-Fluorenylmethoxycarbonyl-l-isoleucyloxy)-1-(trimethyl-silyl)-
ethyl]-phenoxy-acetic acid (8a): [Pd(PPh3)4] (156.5 mg, 0.14 mmol) was
added under argon to a stirred solution of allyl 4-[2-(N-fluorenylmethoxy-
carbonyl-L-isoleucyloxy)-1-(trimethyl-silyl)-ethyl]-phenoxy-acetate (7a;
1.45 g, 2.26 mmol) in THF (p.a.) (20 mL) and a solution of sodium-p-tol-
uenesulfinate (522.9 mg, 2.94 mmol) in MeOH (p.a.) (12 mL). The reac-
tion mixture was stirred under argon at room temperature for 90 min,
concentrated in vacuo and purified by flash chromatography (CH2Cl2/
MeOH/AcOH/H2O 97.5:2.5:0.25:0.25). Compound 8a (1.36 g, quant.)
was afforded as a colorless foam. Rf =0.11 (CH2Cl2/MeOH/AcOH/H2O
97.5:2.5:0.25:0.25); [a]D24 =ꢀ6.09 (c=0.5 in CHCl3); 1H NMR (200 MHz,
CDCl3): d=8.32 (brs, 1H, COOH), 7.75 (d, 2H, H4-, H5-Fmoc, J=
7.3 Hz), 7.58 (d, 2H, H1-, H8-Fmoc, J=7.3 Hz), 7.14 7.52 (m, 4H, H2-,
H3-, H6-, H7-Fmoc), 6.95 (d, 2H, 2 arom. H PTMSEL, J=7.3 Hz), 6.80
(d, 2H, 2 arom. H PTMSEL, J=8.8 Hz), 5.28 (t, 1H, NH), 4.54 4.78 (m,
1H, a-CH Ile), 4.58 (s, 2H, O-CH2-C=O), 4.33 4.37 (m, 2H, CH2-Fmoc),
4.16 4.22 (m, 3H, TMS-CH-CH2-O, H9-Fmoc), 2.49 2.57 (m, 1H, TMS-
CH), 1.55 1.81 (m, 1H, b-CH Ile), 0.66 1.06 (m, 8H, g-CH2, g-CH3, d-
CH3 Ile), ꢀ0.01 (s, 9H, Si(CH3)3); 13C NMR (100.6 MHz, CDCl3, BB,
DEPT): d=172.09, 172.03 (2C, COOH; C=O ester Ile), 156.08, 155.67
(2C, C=O urethane, Cipso,Ar-O), 143.97, 143.81 (2C, C4a-, C4b-Fmoc),
141.31 (2C, C8a-, C9a-Fmoc), 132.20 (1C, Cipso,Ar-CH), 127.04 129.01 (6C,
2 arom. C PTMSEL, C2-, C3-, C6-, C7-Fmoc), 125.26, 125.05 (2C, C1-,
C8-Fmoc), 119.94 (2C, C4-, C5-Fmoc), 114.82 (2C, 2 arom. C PTMSEL),
67.02, 66.30, 65.25 (3C, O=C-CH2-O, CH2-Fmoc, TMS-CH-CH2-O), 58.41
(1C, a-CH Ile), 47.22 (1C, C9-Fmoc), 37.82 (1C, TMS-CH), 36.62 (1C,
b-CH Ile), 24.7 (1C, g-CH2 Ile), 15.21 (1C, g-CH3 Ile), 11.47 (1C, d-CH3
Ile), ꢀ2.72 (3C, Si(CH3)3); FD-MS: calcd for C34H41NO7Si: 603.3; found:
604.1 [M+H]+ ; elemental analysis calcd for C34H41NO7Si: C 67.63 H 6.84
N 2.32; found: C 68.12 H 7.08 N 2.01.
Allyl 4-[2-(N-fluorenylmethoxycarbonyl-l-isoleucyloxy)-1-(trimethylsil-
yl)-ethyl]-phenoxyacetate (7a): Allyl 4-[2-hydroxy-1-(trimethylsilyl)-
ethyl]-phenoxyacetate (6; 0.83 g, 2.70 mmol) was added to a solution of
Fmoc-Ile-OH (1.05 g, 2.97 mmol) in CH2Cl2 (20 mL). The solution was
stirred under argon and cooled to 08C. Successively DMAP[16] (20.0 mg,
0.16 mmol) and DCC (0.67 g, 3.26 mmol) were added. The mixture was
stirred at 08C for 3 h and filtered through Hyflo-Supercel. The filtrate
was diluted with CH2Cl2 (70 mL), washed twice with a 5% NaHCO3
(100 mL), once with water (100 mL) and brine (100 mL), dried over an-
hydrous MgSO4 and concentrated under reduced pressure. The residue
was purified by flash chromatography (130 g silica gel, petrol ether/ethyl
acetate 8.5:1) to afford the desired product 7a (1.58 g, 91%) as a color-
less oil. Rf =0.23 (petrol ether/AcOEt 5:1); [a]2D4 =ꢀ7.01 (c=1.00 in
CHCl3); 1H NMR (200 MHz, CDCl3): d=7.74 (d, 2H, H4-, H5-Fmoc,
J=7.3 Hz), 7.57 (d, 2H, H1-, H8-Fmoc, J=7.3 Hz), 7.24 7.42 (m, 4H,
H2-, H3-, H6-, H7-Fmoc), 6.93 (d, 2H, 2 arom. H PTMSEL, J=7.8 Hz),
6.78 (d, 2H, 2 arom. H PTMSEL, J=8.3 Hz), 5.81 6.00 (m, 1H, CH=
CH2), 5.17 5.31 (m, 3H, CH=CH2, NH), 4.67 (d, 2H, CH2-CH=CH2, J=
5.9 Hz), 4.57 (s, 2H, O-CH2-C=O), 4.52 4.77 (m, 1H, a-CH Ile), 4.33
4.37 (m, 2H, CH2-Fmoc), 4.04 4.22 (m, 3H, TMS-CH-CH2-O, H9-Fmoc),
2.47 2.55 (m, 1H, TMS-CH), 1.41 1.64 (m, 1H, b-CH Ile), 0.65 1.06 (m,
8H, g-CH2, g-CH3, d-CH3 Ile), ꢀ0.02 (s, 9H, Si(CH3)3); 13C NMR
(50.3 MHz, CDCl3, BB, DEPT): d=172.20 (1C, C=O ester Ile), 168.73
(1C, C=O allyl ester), 156.02, 155.74 (2C, C=O urethane, Cipso,Ar-O),
143.98, 143.84 (2C, C4a-, C4b-Fmoc), 141.32 (2C, C8a-, C9a-Fmoc),
133.37 (1C, Cipso,Ar-CH), 131.49 (1C, CH=CH2), 128.39 (2C, 2 arom. C
PTMSEL), 127.69 (2C, C3-, C6-Fmoc), 127.06 (2C, C2-, C7-Fmoc),
125.12 (2C, C1-, C8-Fmoc), 119.98 (2C, C4-, C5-Fmoc), 119.01 (1C, CH=
CH2), 114.73 (2C, 2 arom. C PTMSEL), 66.98, 66.31, 65.75, 65.59 (4C,
CH2=CH-CH2-O, O=C-CH2-O, CH2-Fmoc, TMS-CH-CH2-O), 58.33 (1C,
a-CH Ile), 47.22 (1C, C9-Fmoc), 37.85 (1C, TMS-CH), 36.52 (1C, b-CH
Ile), 24.7 (1C, g-CH2 Ile), 15.13 (1C, g-CH3 Ile), 11.56 (1C, d-CH3 Ile),
ꢀ2.11 (3C, Si(CH3)3); ESI-MS: calcd for C37H45NO7SiNa: 666.3; found:
666.4 [M+Na]+ ; elemental analysis calcd for C37H45NO7Si: C 69.02 H
7.04 N 2.18; found: C 69.53 H 6.71 N 2.01.
4-[2-(N-Fluorenylmethoxycarbonyl-l-prolyloxy)-1-(trimethyl-silyl)-ethyl]-
phenoxy-acetic acid (8b): The preparation was carried out in analogy to
that of 8a with 7b (1.50 g, 2.39 mmol), [Pd(PPh3)4] (166 mg, 0.143 mmol),
sodium-p-toluenesulfinate (553 mg, 3.11 mmol) to yield 8b as a colorless
amorphous solid (1.38 g, 98%). Rf =0.34 (CH2Cl2/MeOH/HOAc
40:1:0.05); [a]2D2 =ꢀ50.0 (c=1 in CHCl3); 1H NMR (200 MHz, CDCl3):
d=7.75 (d, 2H, H4-, H5-Fmoc, J=6.8 Hz), 7.64 7.26 (m, 6H, H1-, H8-,
H2-, H3-, H6-, H7-Fmoc), 6.98 6.63 (m, 4H, Har PTMSEL), 4.78 4.12
(m, 8H, O-CH2-C=O, TMS-CH-CH2-O, CH2-Fmoc, H9-Fmoc, a-CH
Pro), 3.53 3.36 (m, 2H, g-CH2 Pro), 2.59 2.44 (m, 1H, TMS-CH), 2.10
1.89 (m, 1H, b-CHaHb), 1.81 1.53 (m, 3H, b-CHaHb Pro, d-CH2 Pro),
ꢀ0.04 (s, 9H, Si(CH3)3); 13C NMR (50.3 MHz, CDCl3; BB, DEPT): d=
172.86 (1C, COOH), 172.53 (1C, C=O-Pro), 155.18 (1C, C=O-urethane),
154.58 (1C, Cipso,Ar-O), 144.14, 143.93 (2C, C4a-, C4b-Fmoc), 141.29 (2C,
C8a-, C9a-Fmoc), 133.85 (1C, Cipso,Ar-CH), 129.05, 128.56 (2C, Car
PTMSEL), 127.69, 127.06, 125.15, 119.95 (4C, Ctert-Fmoc), 114.64, 114.56
(2C, Car), 67.61 (1C, CH2-Fmoc), 66.09, 65.05 (2C, O=C-CH2-O, TMS-
CH-CH2-O), 59.34 (1C, a-CH Pro), 47.21 (1C, C9-Fmoc), 46.86 (1C, g-
CH2 Pro), 36.63 (1C, TMS-CH), 30.93 (1C, b-CH2 Pro), 23.97 (1C, d-
CH2 Pro), ꢀ2.72 (3C, Si(CH3)3); HR-ESI-TOF-MS: calcd for C33H36NO7-
SiNa2: 632.2056; found: 632.2037 [MꢀH+2Na]+.
Allyl 4-[2-(N-fluorenylmethoxycarbonyl-l-prolyloxy)-1-(trimethylsilyl)-
ethyl]-phenoxyacetate (7b): The preparation was carried out in analogy
to 7a, using allyl 4-[2-hydroxy-1-(trimethylsilyl)-ethyl]-phenoxyacetate
(6; 0.83 g, 2.69 mmol), Fmoc-Pro-OH (1.00 g, 2.96 mmol) to yield 7b as a
colorless oil (1.57 g, 93%). Rf =0.18 (PE/EE 5:1). 1H NMR (200 MHz,
CDCl3): d=7.75 (d, 2H, H4-, H5-Fmoc, J=6.8 Hz), 7.64 7.26 (m, 6H,
H1-, H8-, H2-, H3-, H6-, H7-Fmoc), 6.98 6.63 (m, 4H, Har PTMSEL),
6.01 5.80 (m, 1H, CH=CH2), 5.34 5.21 (m, 3H, CH=CH2), 4.73 4.15 (m,
10H, CH2-CH=CH2, O-CH2-C=O, TMS-CH-CH2-O, CH2-Fmoc, H9-
Fmoc, a-CH Pro), 3.53 3.34 (m, 2H, g-CH2 Pro), 2.85 2.43 (m, 1H,
TMS-CH), 2.09 1.86 (m, 1H, b-CHaHb Pro), 1.84 1.50 (m, 3H, b-
CHaHb Pro, d-CH2 Pro), ꢀ0.04 (s, 9H, Si(CH3)3); 13C NMR (50.3 MHz,
CDCl3; BB, DEPT): d=172.80 (1C, C=O-Pro), 168.75 (1C, C=O allyl
ester), 155.54 (1C, C=O urethane), 154.80 (1C, Cipso,Ar-O), 144.22, 143.98
(2C, C4a-, C4b-Fmoc), 141.30 (2C, C8a-, C9a-Fmoc), 133.71 (1C, Cipso,Ar-
CH), 131.48 (CH=CH2), 128.50, 128.38 (2C, Car PTMSEL), 127.67, 127.04,
125.12, 119.93 (4C, Ctert-Fmoc), 118.99 (1C, CH=CH2), 114.64, 114.55
Preparation of loaded resin 9a: In
a solid-phase synthesis reactor
(100 mL flask with a frit in the bottom) AMPS (aminomethyl polystyr-
ene) (ACT; 200 400 mesh; 1.00 mmolgꢀ1; 1.39 g, 1.39 mmol) was pre-
swollen in CH2Cl2 (20 mL) for 30 min. 4-[2-(N-fluorenylmethoxycarbon-
yl-l-isoleucyloxy)-1-(trimethylsilyl)-ethyl]-phenoxy-acetic
acid
(8a;
673.0 mg, 1.12 mmol) was dissolved in a mixture of CH2Cl2 (40 mL) and
DMF (5 mL). To this solution sequentially HOBt (171.2 mg, 1.12 mmol),
N-methylmorpholine (0.25 mL, 225.6 mg, 2.23 mmol) and TBTU
(358.0 mg, 1.12 mmol) were added. After stirring for 20 min, the solution
was transferred into the reactor containing the AMPS resin. The reaction
mixture was shaken (orbitalshaking) for 18 h at room temperature and
then filtered. The resin was washed three times with each DMF and
CH2Cl2. Pyridine/acetic anhydride (3:1; 20 mL) was added and the mix-
ture was shaken for 20 min. Subsequently, the resin was washed four
times with DMF (20 mL), once with MeOH (20 mL), CH2Cl2 (20 mL),
6025
Chem. Eur. J. 2003, 9, 6018 6030
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Kunz et al.
FULL PAPER
MeOH (20 mL), CH2Cl2 (20 mL), MeOH (20 mL), CH2Cl2 (20 mL) and
finally six times with diethyl ether (20 mL). The resin was dried in vacuo
to afford the loaded polymer 9a (2.33 g). The loading was determined by
UV absorption of the fluorenylmethyl/piperidine adduct formed by treat-
ing the loaded resin (20 mg) with piperidine. Loading: c=0.365 mmolgꢀ1
(which corresponds to a coupling efficiency of 74%).
(1C, C6’’-Glc), 64.93 (1C, CH2-CH=CH2), 61.58 (1C, C6’-Glc), 54.00
(1C, C2’’-Glc), 52.52 (1C, C2’-Glc), 50.06 (1C, a-CH Asn), 46.56 (1C,
C9-Fmoc), 36.76 (1C, b-CH2 Asn), 22.62, 22.57 (2C, 2îCH3 NHAc),
20.78, 20.46, 20.48, 20.41, 20.32 (5C, 5îCH3 OAc); ESI-MS: calcd for
C48H58N4O20Na: 1033.3; found: 1033.2 [M+Na]+.
Fmoc-Asn(b-Ac3GlcNAc-b-Ac2GlcNAc)-OH (16): Fmoc-Asn(bAc3Glc-
NAc-bAc2GlcNAc)-OAll (15; 1.62 g, 1.60 mmol) was dissolved in THF
(120 mL) and methanol (42 mL). [Pd(PPh3)4] (111.1 mg, 0.10 mmol) and
a solution of sodium-p-toluenesulfinate (371.2 mg, 2.08 mmol) in metha-
nol (21 mL) were added. The mixture was stirred for 70 min under argon
and protection from light. The solvent was evaporated in vacuo and the
residue purified by flash chromatography (120 g silica gel, CH2Cl2/
MeOH/AcOH/H2O 92:8:0.8:0.8). Compound 16 (886 mg, 57%) was af-
forded as colorless crystals. Another compound (460 mg) could be isolat-
ed, which beared one acetyl-protecting group less than the desired prod-
uct. 16: Rf =0.33 (CH2Cl2/MeOH/AcOH/H2O 90:10:1:1); [a]2D2 =3.63 (c=
0.63, DMSO); 1H NMR (400 MHz, [D6]DMSO, 1H-COSY): d=8.50 (d,
1H, w-NH Asn, J=9.0 Hz), 7.97 (d, 1H, NH, J=9.4 Hz), 7.88 (d, 2H,
H4-, H5-Fmoc, J=7.8 Hz), 7.80 (d, 1H, NH, J=9.4 Hz), 7.70 (d, 2H, H1-
, H8-Fmoc, J=7.4 Hz), 7.46 (d, 1H, NH, J=8.2 Hz), 7.30 7.42 (m, 4H,
H2-, H3-, H6-, H7-Fmoc), 5.12 (t, 1H, H3’’-Glc, J=9.8 Hz), 5.05 (t, 1H,
H1’-Glc, J=9.8 Hz), 4.94 (t, 1H, H3’-Glc, J=9.4 Hz), 4.80 (m, 1H, H4’’-
Glc), 4.65 (d, 1H, H1’’-Glc, J=8.2 Hz), 4.18 4.32 (m, 6H, a-CH Asn,
H6a’’-, H6b’’-, H6a’-Glc, CH2-Fmoc), 3.81 3.98 (m, 2H, H6b’-Glc, H9-
Fmoc), 3.71 3.81 (m, 2H, H5’’-, H2’-Glc), 3.68 (t, 1H, H4’-Glc, J=
9.4 Hz), 3.49 3.55 (m, 2H, H2’’-, H5’-Glc), 2.45 2.65 (m, 2H, b-CH2
Asn), 2.04 (s, 3H, CH3 OAc), 2.00 (s, 3H, CH3 OAc), 1.95 (s, 3H, CH3
OAc), 1.93 (s, 3H, CH3 OAc), 1.89 (s, 3H, CH3 OAc), 1.73 (s, CH3
NHAc), 1.68 (s, 3H, CH3 NHAc); 13C NMR (100.6 MHz, [D6]DMSO,
BB, DEPT, HMQC): d=172.63 (1C, COOH), 169.13 170.10 (8C, 5îC=
O OAc, 3îC=O amide: NHAc’, NHAc’’, w-Asn), 155.82 (1C, C=O ure-
thane), 143.77 (2C, C4a-, C4b-Fmoc), 140.70 (2C, C8a-, C9a-Fmoc),
127.64 (2C, C3-, C6-Fmoc), 127.08 (2C, C2-, C7-Fmoc), 125.26 (2C, C1-,
C8-Fmoc), 120.12 (2C, C4-, C5-Fmoc), 100.16 (1C, C1’’-Glc), 77.71 (1C,
C1’-Glc), 75.81 (1C, C4’-Glc), 74.13 (1C, C3’-Glc), 73.22 (1C, C5’), 72.60
(1C, C3’’-Glc), 70.59 (1C, C5’’-Glc), 68.18 (1C, C4’’-Glc), 66.18 (1C,
CH2-Fmoc), 66.04 (1C, C6’’-Glc), 61.77 (1C, C6’-Glc), 54.00 (1C, C2’’-
Glc), 52.52 (1C, C2’-Glc), 50.06 (1C, a-CH Asn), 46.73 (1C, C9-Fmoc),
36.73 (1C, b-CH2 Asn), 22.65, 22.57 (2C, 2îCH3 NHAc), 20.81, 20.51,
Preparation of resin 9b was performed in analogy to 9a using Nova-Syn-
Tg-amino-resin HL (Novabiochem; 110 mm beads, 0.43 mmolgꢀ1, 1.08 g,
0.464 mmol), 8b (217 mg, 0.369 mmol), HOBt (57 mg, 0.372 mmol), N-
methylmorpholine (75 mg, 82 mL, 0.745 mmol), TBTU (119 mg,
0.372 mmol); yield: 1.33 g loaded resin; loading: c=0.228 mmolgꢀ1
(which corresponds to a coupling yield of 81%).
bAc3GlcNAc-bAc2GlcNAc-N3 (12): Chitobiosyl chloride (bAc3GlcNAc-
aAc2GlcNAc-Cl, 11)[22] (4.90 g, 7.50 mmol) and tricaprylmethylammo-
niumchloride (aliquat 336) (3.1 g, 7.71 mmol) were dissolved in CHCl3
(100 mL).
A solution of sodium azide (1.50 g, 23.07 mmol) in H2O
(25 mL) was added and the mixture was stirred vigorously at room tem-
perature for 42 h. The organic layer was washed seven times with H2O
(25 mL), dried over anhydrous MgSO4 and concentrated in vacuo. The
residue was extracted with methanol (14 mL) and the product was pre-
cipitated into diethyl ether (60 mL). After 1 h at 08C the precipate was
filtered off and washed with cold diethyl ether. The chitobiosyl azide 12
(3.45 g, 70%) was obtained as white crystals. Rf =0.41 (CHCl3/EtOH
9:1); m.p. 1928C, lit.:[22] m.p. 1958C; [a]D22 =ꢀ49.0 (c=1.00 in CHCl3),
lit.:[22] [a]D22 =ꢀ53.1 (c=1.00 in CHCl3); ESI-MS: calcd for
C26H37N5O15Na: 682.2; found: 682.3 [M+Na]+.
bAc3GlcNAc-bAc2GlcNAc-NH2 (13): In a three necked-flask chitobiosyl
azide 12 (1.82 g, 2.73 mmol) was dissolved in dioxane (80 mL) and etha-
nol (20 mL). Under argon neutralized raney-nickel (1.00 g) was added.
Hydrogen (1 atm) was bubbled through the solution. The mixture was
stirred at room temperature for 3 h and filtered through Hyflo-Supercel.
The filter residue was washed with dioxane (80 mL) and ethanol
(100 mL). The combined filtrates were concentrated in vacuo to give the
chitobiosyl amine 13 (1.73 g, quant.) as a colorless glass which was used
without further purification and characterization for the synthesis of 15.
Rf =0.23 (CHCl3/EtOH 9:1).
Fmoc-Asn(bAc3GlcNAc-bAc2GlcNAc)-OAll) (15): Fmoc-Asp-OAll (14;
1.21 g, 3.06 mmol) was dissolved in DMF (20 mL). Sequentially
HOBt(469.0 mg, 3.06 mmol), DIPEA (1.05 mL, 6.12 mmol) and TBTU
(980.9 mg, 3.06 mmol) were added. After stirring for 10 min, a solution of
chitobiosyl amine 13 (1.76 g, 2.78 mmol) in DMF (20 mL) was added.
The mixture was stirred at room temperature under argon for 40 h. The
solvent was distilled off in vacuo, the residue diluted was with dichloro-
methane (500 mL) and washed twice with a 5% NaHCO3 (100 mL) and
once with brine (50 mL). During the washing procedure the product pre-
cipitated. The precipitate was filtered off and washed with water and di-
ethyl ether yielding the desired compound as colorless crystals (1.67 g).
The organic layer was concentrated under reduced pressure and the resi-
due extracted with water and diethyl ether to furnish further 0.46 g of 15.
In total, 2.13 g (76%) of 15 were obtained. Rf =0.18 (CH2Cl2/MeOH/
AcOH/H2O 95:5:0.5:0.5); 1H NMR (400 MHz, [D6]DMSO): d=8.54 (d,
1H, w-NH Asn, J=9.0 Hz), 7.97 (d, 1H, NH, J=9.0 Hz), 7.87 (d, 2H,
H4-, H5-Fmoc, J=7.9 Hz), 7.81 (d, 1H, NH, J=9.4 Hz), 7.67 (d, 2H, H1-
, H8-Fmoc, J=7.4 Hz), 7.24 7.41 (m, 4H, H2-, H3-, H6-, H7-Fmoc),
5.80 5.89 (m, 1H, CH=CH2), 5.06 5.27 (m, 3H, CH=CH2, H3’’-Glc), 5.04
(t, 1H, H1’-Glc, J=9.5 Hz), 4.93 (t, 1H, H3’-Glc, J=9.4 Hz), 4.80 (t, 1H,
H4’’-Glc, J=9.8 Hz), 4.65 (d, 1H, H1’’-Glc, J=8.2 Hz), 4.42 4.57 (m, 3H,
a-CH Asn, CH2-CH=CH2), 4.18 4.32 (m, 5H, H6a’’-, H6b’’-, H6a’-Glc,
CH2-Fmoc), 3.81 3.96 (m, 2H, H6b’-Glc, H9-Fmoc), 3.66 3.78 (m, 3H,
H5’’-, H2’-, H4’-Glc), 3.43 3.55 (m, 2H, H2’’-, H5’-Glc), 2.46 2.67 (m,
2H, b-CH2 Asn), 2.03 (s, 3H, CH3 OAc), 2.00 (s, 3H, CH3 OAc), 1.94 (s,
3H, CH3 OAc), 1.93 (s, 3H, CH3 OAc), 1.89 (s, 3H, CH3 OAc), 1.73 (s,
3H, CH3 NHAc), 1.68 (s, 3H, CH3 NHAc); 13C NMR (100.6 MHz,
[D6]DMSO, BB, DEPT): d=171.09 (1C, COOAll), 169.13 170.10 (8C,
5îC=O OAc, 3îC=O amide: NHAc’, NHAc’’, w-Asn), 155.82 (1C, C=
O urethane), 143.74 (2C, C4a-, C4b-Fmoc), 140.71 (2C, C8a-, C9a-
Fmoc), 132.31 (1C, CH=CH2), 127.64 (2C, C3-, C6-Fmoc), 127.08 (2C,
C2-, C7-Fmoc), 125.26 (2C, C1-, C8-Fmoc), 120.12 (2C, C4-, C5-Fmoc),
117.45 (1C, CH=CH2), 100.16 (1C, C1’’-Glc), 77.71 (1C, C1’-Glc), 75.81
(1C, C4’-Glc), 74.13 (1C, C3’-Glc), 73.22 (1C, C5’), 72.60 (1C, C3’’-Glc),
70.59 (1C, C5’-Glc), 68.18 (1C, C4’’-Glc), 66.18 (1C, CH2-Fmoc), 66.04
20.48, 20.42, 20.34 (5C, 5îCH3 Ac); ESI-MS: calcd for C45H54N4O20
:
970.3; found: 993.2 [M+Na]+, 1009.2 [M+K]+, 1015.1 [M+2Na]+.
Boc-Leu-Gln(Trt)-Val-Ala-Ala-Leu-Asp(OtBu)-Ala-Asn(bAc3GlcNAc-
bAc2GlcNAc)-Gly-Ile-Ile-OH (17): Starting from polymer 9a loaded
with Fmoc-Ile-OH (274.1 mg,0.1 mmol; loading: 0.365 mmolgꢀ1), glyco-
dodecapeptide
Boc-Leu-Gln(Trt)-Val-Ala-Ala-Leu-Asp(OtBu)-Ala-
Asn(bAc3GlcNAc-bAc3GlcNAc)-Gly-Ile-Ile-OH (17) was assembled
using a peptide synthesizer (Perkin Elmer ABI 433 A). Fmoc deprotec-
tions were carried out by up to five four minute treatments with 30% pi-
peridine in NMP. Peptide couplings were performed by reaction with
10 equiv Fmoc or Boc amino acids and coupling reagents (10 equiv
HBTU, 10 equiv HOBt and 20 equiv DIPEA) within 35 min. The glyco-
sylated building block Fmoc-Asn(bAc3GlcNAc-bAc3GlcNAc)-OH
(291.2 mg, 3 equiv) was coupled manually using 3 equiv HATU/3 equiv
HOAt and 6 equiv DIPEA (reaction time: 2 h). After each coupling step
unreacted amino groups were capped with acetic anhydride/DIPEA/
HOBt. After completion of the peptide synthesis, the resin was exten-
sively washed with NMP (6î2 min) and CH2Cl2 (4î2 min), transferred
to a reactor and dried in vacuo. The resin was washed three times with
CH2Cl2 (10 mL). For the cleavage procedure the resin was treated with a
solution of tetrabutylammonium fluoride trihydrate (63.1 mg, 0.2 mmol)
in dry CH2Cl2 (10 mL) and shaken at room temperature for 25 min.
After filtration, the resin was washed four times with CH2Cl2 (10 mL).
The cleavage procedure was repeated once with
a solution of
TBAF¥3H2O (63.1 mg, 0.2 mmol) in CH2Cl2 (10 mL). The collected cleav-
age and washing filtrates of each cleavage were separately shaken with
water (20 mL) which led to the precipitation of the target compound.
The dichloromethane was distilled off and the remaining water was re-
moved with a pasteur pipette. In this way, two product fractions were ob-
tained: 1) cleavage: 78 mg colorless crystals, 2) cleavage: 41 mg colorless
crystals. Both product fractions were analyzed by HPLC and identified to
be of identical composition. Final purification was achieved by prepara-
6026
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 6018 6030

Glycopeptide Synthesis
6018 6030
tive RP-HPLC (30% MeCN in H2O ! 100% MeCN in 100 min). After
lyophilysation the target compound (64.2 mg, 29%) was obtained as a
colorless, amorphous solid. Rf =0.30 (CH2Cl2/MeOH/AcOH/H2O
90:10:1:1); HPLC: tR =27.50 min (Eurosphere C8, 30% MeCN in H2O
! 100% MeCN in 40 min); [a]2D3 =ꢀ14.3 (c=0.38 in DMSO); 1H NMR
(600 MHz, [D6]DMSO, 300 K, 1H-COSY, NOESY, TOCSY): d=8.55 (s,
1H, NH w-NH Gln), 8.52 (m, 1H, w-NH Asn), 8.20 (m, 2H, NH Asn,
Asp), 8.12 (d, 1H, NH Gln, J=5.6 Hz), 7.75 8.05 (m, 8H, NH 2îAla
(8.20, 7.83), Gly (7.83), Leu (7.84), 2îIle (7.83, 7.79), NHAc’ (7.78),
NHAc’’ (7.97)), 7.62 (d, 2H, NH Val, Ala, J=8.2 Hz), 7.14 7.27 (m, 15H,
Trt), 6.92 (d, 1H, NH LeuN-terminal, J=8.2 Hz), 5.14 (t, 1H, H3’’-Glc, J=
10.0 Hz), 5.05 (t, 1H, H1’-Glc, J=9.4 Hz), 4.96 (t, 1H, H3’-Glc, J=
9.4 Hz), 4.81 (t, 1H, H4’’-Glc, J=9.7 Hz), 4.65 (d, 1H, H1’’-Glc, J=
8.2 Hz), 4.52 4.55 (m, 2H, a-CH Asp, Asn), 4.15 4.32 (m, 9H, 7îa-CH:
3îAla (4.17, 4.24, 4.25), Ile (4.25), Leu (4.25), Val (4.15), Gln (4.25),
H6a’-Glc (4.27), H6a’’-Glc (4.27)), 4.08 (m, 1H, a-CH Ile), 3.92 3.97 (m,
2H, a-CH LeuN-terminal, H6b’-Glc), 3.88 (d, 1H, H6b’’-Glc, J=10.6 Hz),
3.67 3.84 (m, 5H, H5’’-Glc (3.81), H2’-Glc (3.77), a-CH2 Gly (3.68), H4’-
Glc (3.68)), 3.51 3.55 (m, 2H, H5’-Glc (3.51), H2’’-Glc (3.53)), 2.40 2.70
(m, 4H, b-CHaHb Asp (2.64, 2.40), Asn (2.70, 2.44)), 2.31 (t, 2H, g-CH2
Gln, J=7.6 Hz), 1.87 2.05 (m, 16H, 5îCH3 OAc, b-CH Val), 1.65 1.81
(m, 10H, 2îCH3 NHAc, b-CH 2îIle, b-CH2 Gln), 1.57 (m, 2H, g-CH
2îLeu), 1.33 1.45 (m, 24H, 3îCH3 tBu, 3îCH3 Boc, g-CHaHb 2îIle,
b-CH2 2îLeu), 1.14 1.24 (m, 10H, b-CH3 3îAla; g-CHaHb Ile), 1.04
(m, 1H, g-CHaHb Ile), 0.73 0.88 (m, 30H, g-CH3 2îIle, d-CH3 2îIle, g-
CH3 2îVal, d-CH3 4îLeu); 13C NMR (150.9 MHz, [D6]DMSO, 300 K,
HSQC): d=128.47 (3C, Cpara-Trt), 127.23 (6C, Cortho-Trt), 126.15 (6C,
Cmeta-Trt), 100.24 (1C, C1’’-Glc), 77.76 (1C, C1’-Glc), 75.82 (1C, C4’-
Glc), 74.40 (1C, C3’-Glc), 73.62 (1C, C5’-Glc), 72.33 (1C, C3’’-Glc), 70.52
(1C, C5’’-Glc), 68.19 (1C, C4’’-Glc), 62.64 (1C, C6’-Glc), 61.82 (1C, C6’’-
Glc), 57.34 (1C, a-CH Val), 56.69 (1C, a-CH Ile), 56.43 (1C, a-CH Ile),
53.85 (1C, C2’’-Glc), 53.07 (1C, a-CH Leu), 52.42 (1C, C2’-Glc), 52.30
(1C, a-CH Leu), 51.07 (1C, a-CH Gln), 49.46 (2C, a-CH Asn, Asp),
48.82, 48.42, 48.16 (3C, a-CH 3îAla), 42.35 (1C, a-CH2 Gly), 41.14,
40.68 (2C, b-CH2 2îLeu), 37.21 (1C, b-CH2 Asn), 36.98 (1C, b-CH Ile),
36.91 (1C, b-CH2 Asp), 36.30 (1C, b-CH Ile), 32.68 (1C, g-CH2 Gln),
30.83 (1C, b-CH Val), 28.21 (1C, b-CH2 Gln), 27.98 (6C, 6îCH3 tBu,
Boc), 24.91 (1C, g-CH2 Ile), 24.29 (2C, g-CH 2îLeu), 24.21 (1C,g-CH2
Ile), 22.90 (2C, d-CH3 2îLeu), 22.67 (2C, 2îCH3 NHAc), 20.98, 20.44,
20.28 (5C, 5îCH3 OAc), 19.34 (2C, d-CH3 2 îLeu), 18.13 (3C, b-CH3
3îAla), 17.67 (2C, g-CH3 2îVal), 15.43, 15.36 (2C, g-CH3 2îIle), 11.21,
solid. HPLC: tR =23.48 min (Eurospher C8, 1% MeCN in H2O ! 60%
1
MeCN in H2O + 0.1% TFA in 40 min): H NMR (600 MHz,[D6]DMSO,
300 K, 1H-COSY, TOCSY, NOESY, ROESY): d=8.64 (d, 1H, NH Gln,
J=7.6 Hz), 8.18 8.20 (m, 2H, NH Asp (8.19), w-Asn (8.21)), 8.12 (d, 1H,
NH Asn, J=5.9 Hz), 8.07 (d, 1H, NH AlaVal-Ala-Ala, J=7.0 Hz), 8.03 (d,
1H, NH Ile), 7.93 7.96 (m, 2H, NH Ala (7.94), Val (7.93), 7.87 7.89 (d,
2H, NH Leu (7.89), NHAc’ (7.87), J=7.6 Hz), 7.69 7.80 (m, 4H, NH
Ala (7.75), Ile (7.78), Gly (7.74), NHAc’’ (7.72)), 7.27 (brs, 1H, w-
NHaHb Gln), 6.80 (brs, 1H, w-NHaHb Gln), 5.09 (d, 1H, OH4’’-Glc,
J=5.3 Hz), 4.99 (d, 1H, OH3’’-Glc, J=5.6 Hz), 4.82 (t, 1H, H1’-Glc, J=
9.4 Hz), 4.75 (brs, 1H, OH), 4.70 (t, 1H, OH6’’-Glc), 4.59 (t, 1H, OH6’-
Glc), 4.48 4.50 (m, 2H, a-CH Asp (4.48), Asn (4.50), 4.37 (m, 1H, a-CH
Gln), 4.33 (d, H1’’-Glc, J=8.2 Hz), 4.15 4.29 (m, 6H, a-CH: 3îAla
(4.29, 2î4.26), Ile (4.29), Leu (4.25), Val (4.16)), 4.09 (t, 1H, a-CH Ile,
J=6.5 Hz), 3.62 3.79 (m, 4H, a-CH LeuN-terminal (3.75), a-CH2 Gly (3.71),
H6a’’H6b’’-Glc (3.71)), 3.56 3.57 (d, 2H, H2’-Glc (3.57), H6a’H6b’-Glc
(3.56)), 3.21 3.50 (m, 6H, H6a’H6b’-Glc (3.41), H6a’’H6b’’-Glc (3.35),
H3’-Glc (3.48), H4’-Glc (3.28), H2’’-Glc (3.42), H3’’-Glc (3.26)), 3.17 (m,
1H, H5’’-Glc), 3.11 (m, 2H, H3’-, H5’-Glc), 3.00 (m, 1H, H4’’-Glc), 2.67
(m, 1H, b-CHaHb Asp), 2.40 2.61 (m, 3H, b-CHaHb Asp (2.50), b-CH2
Asn (2.40, 2.58), 2.09 2.18 (m, 2H, g-CH2 Gln (2.09, 2.16)), 1.96 (m, 1H,
b-CH Val), 1.67 1.87 (m, 10H, CH3 2îNHAc (1.78, 1.81), b-CH2 Gln
(1.76, 1.83), b-CH 2îIle (1.68, 1.75)), 1.36 1.64 (m, 8H, b-CH2 2îLeu
(1.41, 1.54; 1.47, 1.51), g-CH 2îLeu (1.53, 1.59), g-CHaHb 2îIle (1.39,
1.40)), 1.12 1.20 (m, 10H, b-CH3 3îAla (1.17), g-CHaHb Ile (1.15)),
1.03 (m, 1H, g-CHaHb Ile), 0.75 0.90 (m, 3H, g-CH3 2îIle, d-CH3 2î
Ile, g-CH3 2îVal, d-CH3 4îLeu); 13C NMR (150.9 MHz, [D6]DMSO,
300 K, HSQC): d=102.22 (1C, C1’’-Glc), 81.46 (1C, C4’-Glc), 78.81 (1C,
C1’-Glc), 77.09 (1C, C5’’-Glc), 76.90 (1C, C5’-Glc), 74.13 (1C, C3’’-Glc),
73.04 (1C, C3’-Glc), 70.91 (1C, C4’’-Glc), 61.32 (1C, C6’’-Glc), 60.04 (1C,
C6’-Glc), 57.62 (1C, a-CH Val), 56.47, 56.58 (2C, a-CH 2îIle), 55.61
(1C, C2’’-Glc), 54.08 (1C, C2’-Glc), 52.44 (1C, a-CH Gln), 50.88 (2C, a-
CH 2îLeu), 49.47 (2C, a-CH Asn, Asp), 48.26 (1C, a-CH Ala), 48.21
(2C, a-CH 2îAla), 42.30 (1C, a-CH2 Gly), 41.11 (1C, b-CH2 Leu), 40.48
(1C, b-CH2 Leu), 37.04 (1C, b-CH Ile), 36.08 (1C, b-CH Ile), 36.95 (1C,
b-CH2 Asp), 35.76 (1C, b-CH2 Asn), 31.44 (1C, g-CH2 Gln), 30.72 (1C,
b-CH Val), 28.01 (1C, b-CH2 Gln), 24.40, 24.81 (2C, g-CH2 2îIle), 23.53,
24.25 (2C, g-CH 2îLeu), 22.89, 23.21 (2C, CH3 2îNHAc), 21.77, 22.49
(4C, d-CH3 4îLeu), 19.25 (2C, g-CH3 2îVal), 18.17 (3C, b-CH3 3î
Ala), 15.30, 15.61 (2C, g-CH3 2îIle), 11.14, 11.54 (2C, d-CH3 2îIle);
MALDI-TOF-MS: calcd for C69H118O27N16
: 1603.77; found: 1605.4
[M+H]+, 1627.5 [M+Na]+, 1649.4 [M+2Na]+, 1671.4 [M+3Na]+ ; ESI-
MS found: 824.8 ([M+2Na]+/2), 835.8 ([M+3Na]+/2), 843.7
([M+2Na+K]+/2), 846.9 ([M+4Na]+/2).
11.05 (2C, d-CH3 2îIle), MALDI-TOF-MS: calcd for C107H158N16O34
:
2211.1; found: 2235.0 [M+Na+]+, 2256.9 [M+2Na]+ ; ESI-MS found:
1129.1 ([M+2Na]+/2), 1137.2 ([M+Na+K]+/2), 1140.1 ([M+3Na]+/2),
1148.1 ([M+2Na+K]+/2), 1050.9 ([M+2NaꢀBocꢀtBu]+/2), 1059.0
([M+Na+KꢀBocꢀtBu]+/2), 1062.0 ([M+3NaꢀBocꢀtBu]+/2).
N-(9H-Fluoren-9-yl)-methoxycarbonyl-O-{2-acetamido-2-deoxy-6-O-
[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-glycero-d-gal-
acto-2-nonulopyranosyl)onat]-a-d-galactopyranosyl}-l-threonine-tert-bu-
tylester (22): Fmoc-Thr(a-GalNAc)-OtBu (21)[27] (1.50 g, 2.50 mmol) and
a-NeuAc4NAcCOOBnXan (20)[28] (4.00 g, 5.95 mmol) were dissolved in
a mixture of dry acetonitrile (50 mL) and dry dichloromethane (25 mL).
The solution was stirred for 1 h in the presence of flame-dried molecular
sieves (6.00 g, powder, 3 ä) under an argon atmosphere and the exclu-
sion of moisture. Subsequently, dried silver triflate (1.53 g, 5.95 mmol)
was added and the mixture was cooled to ꢀ658C. A pre-cooled (08C)
solution (1.6m) of methylsulfenyl bromide[30] in 1,2-dichloroethane
(3.72 mL, 5.95 mmol) was added dropwise over a period of 25 minutes.
The suspension was stirred for 4 h at ꢀ658C and was, after treatment
with diisopropylamine (1.67 mL, 11.9 mmol), allowed to slowely warm to
108C. After dilution with dichloromethane (200 mL), the reaction mix-
ture was filtered through Hyflo-Supercel. The filtrate was concentrated
in vacuo and the crude residue was purified by flash chromatography
(silica gel; ethyl acetate/ethanol 60:1 ! 50:1 ! 40:1) to yield the disac-
TFA¥H-Leu-Gln-Val-Ala-Ala-Leu-Asp-Ala-Asn(bAc3GlcNAc-bAc2Glc-
NAc)-Gly-Ile-Ile-OH) (18): Glycopeptide 17 (25.0 mg, 0.011 mmol) was
stirred at room temperature for 4 h in a mixture containing trifluoroace-
tic acid (20 mL), triisopropylsilane (0.5 mL) and water (0.5 mL). Toluene
(80 mL) was added and the solvent was removed under vacuo. The resi-
due was extracted three times with diethyl ether (20 mL) and the ether
solution was removed with
a capillary. A colorless crystalline solid
(20.5 mg, 97%) was isolated. The product was analyzed by analytical
HPLC and shown to be almost pure. HPLC: tR =28.90 min (Eurosphere
C8, 1% MeCN in H2O ! 60% MeCN in H2O+0.1% TFA in 40 min);
tR =19.73 min (Eurosphere C8, 1% MeCN in H2O
!
100%
MeCN+0.1% TFA in 40 min); ESI-MS: calcd for C79H128O32N16: 1814.00;
found: 929.9 ([M+2Na]+/2), 937.8 ([M+Na+K]+/2), 945.8 ([M+2K]+/2),
948.8 ([M+2Na+K]+/2), 952.0 ([M+4Na]+/2).
TFA¥H-Leu-Gln-Val-Ala-Ala-Leu-Asp-Ala-Asn(bGlcNAc-bGlcNAc)-
Gly-Ile-Ile-OH) (19): Glycopeptide 18 (20.0 mg, 0.01 mmol) was dis-
solved in dry methanol (14 mL). A 0.1 molar solution of sodium metha-
nolate in methanol (4 mL) was added (pH ꢁ10). The mixture was stirred
for 7 h at room temperature. After completion of the deacetylation reac-
tion (HPLC-MS analysis), the mixture was acidified by addition of con-
centrated acetic acid (pH ꢁ4) and the solvent was removed in vacuo.
The residue was dissolved in water (20 mL) and lyophilized. The crude
product was purified by preparative RP-HPLC (10% MeCN in H2O !
40% MeCN in H2O + 0.1% TFA in 100 min). After lyophilisation the
title compound 19 (14 mg, 79%) was obtained as a colorless, amorphous
charide 22 (1.64 g, 57%) as
a colorless amorphous solid. Rf =0.22
(AcOEt/EtOH 20:1); [a]2D2 =11.8 (c=1.00 in CHCl3), 1H NMR
(400 MHz, CDCl3): d=7.74 (d, 2H, H4-, H5-Fmoc, J=7.4 Hz), 7.59 (d,
2H, H1-, H8-Fmoc, J=7.3 Hz), 7.40 7.26 (m, 9H, H2-, H3-, H6-, H7-
Fmoc, Harom.-Bn (5H)), 6.68 (d, 1H, NH-GalNAc, J=7.4 Hz), 5.48 (d,
1H, NH-Thr, J=9.4 Hz), 5.36 5.27 (m, 2H, H7’, H8’), 5.23 (d, 1H,
OCH2a-Bn, J=12.1 Hz), 5.15 (d, 1H, OCH2b-Bn, J=12.1 Hz), 4.87 4.79
(m, 1H, H4’), 4.77 (d, 1H, H1-Gal, J=3.5 Hz), 4.45 (d, 2H, CH2-Fmoc,
J=6.7 Hz), 4.36 4.15 (m, 4H, H9’a H2-Gal, H9-Fmoc, a-CH Thr), 4.11
6027
Chem. Eur. J. 2003, 9, 6018 6030
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Kunz et al.
FULL PAPER
4.00 (m, 4H, H6’,b-CH Thr, H5’, H9’b), 3.90 (dd, 1H, H6a-Gal, J1 =
7.0 Hz, J2 =10.2 Hz), 3.73 3.60 (m, 3H, H5-, H3-, H4-Gal), 3.49 (dd, 1H,
H6b-Gal, J1 =4.3 Hz, J2 =10.2 Hz), 2.60 (dd, 1H, H3’e, J1 =12.9 Hz, J2 =
4.7 Hz), 2.10, 2.09, 2.07, 1.99, (s, 15H, NHAc, OAc), 1.93 (t, 1H, H3’’eq),
1.84 (s, 3H, OAc), 1.43 (s, 9H, C(CH3)3), 1.25 (d, 3H, g-CH3 Thr, J=
6.3 Hz); 13C NMR (50.3 MHz, CDCl3): d=170.94, 170.79, 170.42, 170.35,
170.11 (C=O OAc, NHAc), 167.52 (C1’), 156.57 (C=O urethane), 143.71
(C4a-, C4b-Fmoc), 141.35 (C8a-, C9a-Fmoc), 134.82 (Cipso-Bn), 128.90,
128.81, 128.58, 127.80, 127.13 (C-Bn, C2-, C3-, C6-, C7-Fmoc), 125.06
(C1-, C8-Fmoc), 120.04 (C4-, C5-Fmoc), 99.49 (C1-Gal), 98.80 (C2’),
83.24 (C(CH3)3), 72.90, 69.45, 69.33, 68.98, 68.43, 67.89, 67.60, 67.22 (C3-,
C4-, C5-Gal, C4’, C6’, C7’, C8’, CH2-Fmoc, OCH2-Bn), 62.55 (C6-Gal),
59.11 (a-CH Thr), 49.30 (C5’), 47.91 (C2-Gal), 47.22 (C9-Fmoc), 37.44
(C3’), 28.12 (C(CH3)3), 23.13, 22.86, 21.10, 20.85, 20.78 (CH3CO), 18.98
(g-CH3 Thr); ESI-MS: calcd for C57H71N3O22: 1149.45, found: 1172.5
[M+Na]+, 1116.5 [MꢀtBu+Na]+, 775.3 [MꢀFmoc-Thr-OtBuꢀH+Na]+.
N-(9H-Fluoren-9-yl)-methoxycarbonyl-O-{2-acetamido-3,4-di-O-acetyl-2-
deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-
glycero-d-galacto-2-nonulopyranosyl)onat]-a-d-galactopyranosyl}-l-
threonine-tert-butylester (23): Fmoc-Thr(a-NeuAc4NAcCOOBn-(2!6)-
a-GalNAc)-OtBu (22; 1.24 g, 1.08 mmol) was dissolved in pyridine
(10 mL) and acetic anhydride (5 mL) at 08C. After stirring at 08C for
4 h, the solution was allowed to warm and kept at room temperature for
15 h. The mixture was diluted with dichloromethane (100 mL) and excess
acetic anhydride was hydrolyzed by the addition of ice. The organic layer
was washed with a sat. aq. NaHCO3 solution (4î120 mL) and brine
(150 mL), dried over MgSO4, filtered and concentrated in vacuo. Purifi-
cation of the resulting residue was performed by flash chromatography
(silica gel; ethyl acetate) to furnish disaccharide 23 (1.00 g, 76%) as a
colorless amorphous solid. [a]2D2 =30.2 (c=1.00 in CHCl3); Rf =0.33
(AcOEt); 1H NMR (200 MHz, CDCl3): d=7.77 (d, 2H, H4-, H5-Fmoc,
J=7.3 Hz), 7.63 (d, 2H, H1-, H8-Fmoc, J=7.3 Hz), 7.43 7.26 (m, 9H,
H2-, H3-, H6-, H7-Fmoc, H2-, H3-, H4-, H5-, H6-Bn), 5.99 (d, 1H, NH-
urethane, J=8.1 Hz), 5.63 (d, 1H, NH-GalNAc, J=9.3 Hz), 5.32 5.21 (m,
3H, H7’, H8’, OCH2-Bna), 5.16 5.07 (m, 2H, H4, OCH2-Bnb), 4.96 (dd,
1H, H3-Gal, J=2.9 Hz), 4.87 4.71 (m, 2H, H1-Gal, OCH2-Bna), 4.82 (d,
1H, H4’, J=3.4 Hz), 4.59 4.46 (m, 3H, CH2-Fmocb, H2-Gal, b-CH Thr),
4.27 4.14 (m, 4H, H6’, H9’a, H9-Fmoc, a-CH Thr), 4.11 3.97 (m, 3H,
H5-Gal, H5’, H9’b), 3.83 (dd, 1H, H6a-Gal, J1 =7.6 Hz, J2 =10.1 Hz),
3.10 (dd, 1H, H6b, J1 =4.4 Hz, J2 =10.0 Hz), 2.54 (dd, 1H, H3’e, J1 =
12.7 Hz, J2 =4.4 Hz), 2.10, 2.02, 2.00, 1.98, 1.85 (s, 25H, NHAc, OAc, H-
3’a), 1.43 (s, 9H, C(CH3)3), 1.23 (d, 3H, g-CH Thr, J=7.3 Hz); 13C NMR
(100.6 MHz, CDCl3, BB, DEPT): d=173.51, 173.38, 172.36, 172.14,
171.72, 170.24, 170.14, 169.68 (C=O OAc, C=O NHAc), 167.36 (C1’),
156.87 (C=O urethane), 143.74 (C4a-, C4b-Fmoc), 141.35 (C8a-, C9a-
Fmoc), 134.73 (Cipso-Bn), 128.87, 128.71, 127.80, 127.15 (C-Bn, C2-, C3-,
C6-, C7-Fmoc), 125.07 (C1-, C8-Fmoc), 120.06 (C4-, C5-Fmoc), 100.03
(C1-Gal), 98.61 (C2’), 83.09 (C(CH3)3), 76.69 (b-CH Thr), 72.63, 68.91,
68.55, 68.10, 67.89, 67.62, 67.27, 67.14 (C3-, C4-, C5-Gal, C4’, C6’, C7’,
C8’, CH2-Fmoc, OCH2-Bn), 63.90, 62.40 (C6-Gal, C9’), 59.16 (a-CH Thr),
49.20 (C5’), 47.38 (C2-Gal), 47.26 (C9-Fmoc), 37.31 (C3’), 28.11
(C(CH3)3), 23.19, 21.02, 20.82, 20.72, 20.65 (CH3CO), 18.61 (g-CH3 Thr);
ESI-MS: calcd for C61H75N3O24: 1233.47, found: 1256.6 [M+Na]+, 1200.5
[MꢀtBu+Na]+ ; HR-ESI-TOF-MS: calcd for C61H75N3O24Na: 1256.4638,
found: 1256.4634 [M+Na]+.
3.1 Hz), 4.90 4.83 (m, 2H, H1, H4’), 4.63 (dd, J1 =6.24, J2 =10.6 Hz, 1H,
OCH2-Fmoca), 4.48 4.37 (m, 2H, OCH2-Fmocb, b-CH Thr), 4.35 4.18 (m,
5H, H-2, H-6’, H-9’a, H9-Fmoc, a-CH Thr), 4.13 3.97 (m, 3H, H5, H5’,
H-9’b), 3.83 (dd, 1H, H6a, J1 =7.4 Hz, J2 =10.0 Hz), 3.15 (dd, 1H, H6b,
J1 =4.7 Hz, J2 =10.4 Hz), 2.68 (dd, 1H, H3’e, J1 =12.7 Hz, J2 =4.3 Hz),
2.14, 2.12, 2.03, 2.02, 2.00, 1.98, 1.87 (s, 25H, NHAc, OAc), 1.43 (s, 9H,
C(CH3)3), 1.22 (d, g-CH3, 3H, J=6.2 Hz); 13C NMR (100.6 MHz CDCl3):
d=173.57, 173.11, 172.24, 171.96, 171.75, 171.62, 171.40 (C=O OAc,
NHAc), 168.68 (C1’), 159.04 (C=O urethane), 145.50, 145.18 (C4a-, C4b-
Fmoc), 142.71 (C8a-, C9a-Fmoc), 136.60 (Cipso-Bn), 129.99, 129.88,
128.85, 128.21 (C-Bn, C2-, C3-, C6-, C7-Fmoc), 126.20, 126.08 (C1-, C8-
Fmoc), 120.96 (C4-, C5-Fmoc), 100.11 (C1-Gal), 99.91 (C2’), 77.75 (b-CH
Thr), 73.37, 70.66, 70.04, 69.20, 68.96, 68.58, 67.57 (C3-, C4-, C5-Gal, C4’,
C6’, C7’, C8’, CH2-Fmoc, OCH2-Bn), 65.10, 63.53 (C6-Gal, C9’), 61.24 (a-
CH Thr), 49.20 (C5’), 38.89 (C3’), 28.11 (C(CH3)3), 23.05, 22.69, 21.42,
21.25, 20.95, 20.72 (CH3CO), 19.35 (g-CH3 Thr); ESI-MS: calcd for
C57H67N3O24: 1177.41, found: 1200.6 [M+Na]+, 1216.7 [M+K]+, 1222.7
[MꢀH+2Na]+, 1238.7 [MꢀH+Na+K]+
; HR-ESI-TOF-MS: calcd for
C57H66N3O24Na2: 1222.3831, found: 1222.3821 [MꢀH+2Na]+.
Biotinyl-Gly-Val-Thr(a-NeuAc4NAcCOOBn-(2!6)-a-GalAc2NAc)-
Ser(tBu)-Ala-Pro-Asp(OtBu)-Thr(tBu)-Arg(Pmc)-Pro-Ala-Pro-OH
(25): The solid-phase glycopeptide synthesis was carried out according to
the standard protocol described above employing a Perkin Elmer A433
peptide synthesizer. Starting from Fmoc-Pro-PTMSEL preloaded Nova-
Syn Tg resin 9b (439 mg, 0.1 mmol, loading: 0.228 mmolgꢀ1) the protect-
ed fragment Fmoc-Ser(tBu)-Ala-Pro-Asp(tBu)-Thr(tBu)-Arg(Pmc)-Pro-
Ala-Pro-PTMSEL-NovaSyn Tg was prepared by iterative coupling of the
corresponding amino acids. Upon N-terminal deprotection by treatment
with piperidine in NMP, the glycosylated building block 24 was intro-
duced in
a manual double coupling. To this end, a solution of
Fmoc-Thr(a-NeuAc4NAcCOOBn-(2!6)-a-GalAc2NAc)-OH (165.0 mg,
0.14 mmol, 1.4 equiv), HATU (57.0 mg, 0.15 mmol, 1.5 equiv), HOAt
(20.4 mg, 0.15 mmol, 1.5 equiv) and NMM (30.4 mg, 33.4 mL, 0.3 mmol,
3 equiv) in NMP (3 mL) was added twice to the resin which was then vor-
texed for 1 h. Resuming the standard protocol, the concluding two amino
acids were attached. The resin was thoroughly washed with NMP and di-
chloromethane, removed from the reaction vessel and dried in vacuo to
furnish 592 mg of loaded resin. The terminating biotinylation was carried
out in the peptide synthesizer using 296 mg (max. 0.05 mmol) of the
resin. After N-terminal deprotection (treatment with 30% piperidine in
NMP), biotin was attached to the N-terminus by a manual double cou-
pling. The resin was treated twice with
a solution of d-(+)-biotin
(122.0 mg, 0.5 mmol, 10 equiv), HBTU (208.5 mg, 0.55 mmol, 11 equiv),
HOBt (84.2 mg, 0.55 mmol, 11 equiv) and NMM (111.2 mg, 120.94 mL,
1.1 mmol, 22 equiv) in NMP (4 mL; 1 h vortex each time). After filtra-
tion, the polymeric support was washed with NMP and dichloromethane.
For the cleavage procedure the resin was placed into a reactor and treat-
ed with a solution of tetrabutylammonium fluoride trihydrate (31.5 mg,
0.1 mmol, 2 equiv) in CH2Cl2 (10 mL) and shaken for 30 min at room
temperature. The mixture was filtered and the resin was washed four
times with CH2Cl2 (10 mL). The cleavage process was repeated with a
solution of TBAF¥3H2O (11.5 mg, 0.035 mmol) in dichloromethane
(10 mL). Filtrates and washing solutions of both cleavage procedures
were separately washed with water (3î20 mL), dried over anhydrous
MgSO4 and liberated from the solvents in vacuo to yield 69.1 mg and
9.1 mg, respectively, of a colorless crystalline solid. Both fractions were
combined and purified by semi-preparative HPLC (Vydac Protein&Pep-
tide C18, 1% MeCN in H2O ! 100% MeCN in H2O in 100 min). Lyo-
philisation yielded the protected glycopeptide 25 as a colorless amor-
phous solid (56 mg, 42%). HPLC: tR =26.9 min (Phenomenex Luna C18,
5% MeCN in H2O ! 100% MeCN in H2O in 42 min); 1H NMR
(400 MHz, CDCl3,1H-COSY): d=7.57 7.89 (m, 4H, 4îNH), 7.42 7.55
(m, 2H, 2îNH), 7.35 (s, 5H, arom. H Bn), 7.01 7.17 (m, 2H, 2îNH),
5.12 5.32 (m, 2H, H7’, H8’), 5.27 (s, 2H, OCH2 Bn), 5.03 5.11 (m, 1H,
H4’), 4.76 4.91 (m, 2H, H1-Gal, a-CH Asp), 4.57 4.71 (m, 4H, a-CH
Arg, Thr2, H2-Biot., Alaa), 4.22 4.55 (m, 9H, a-CH Alab, Ser, H5-Biot.,
3îPro, Thr1, Val, b-CH Thr2), 3.93 4.10 (m, 3H, b-CH Thr1, H6’, H2-
Gal), 3.14 3.84 (m, 19H, H3-Gal, H4-Gal, H5-Gal, H6a,b-Gal, H5’, H9a,b’,
a-CH2 Gly, b-CH2 Ser, 3îd-CH2 Pro, H4-Biot.), 2.98 3.06 (m, 2H, d-
CH2 Arg), 2.80 2.96 (m, 3H, H3a-Biot., b-CH2 Asp), 2.73 (d, 1H, H3b-
Biot., J=12.5 Hz), 2.54 2.64 (m, 3H, CH2-Pmc, H3’e), 2.52 (s, 6H, o,o’-
N-(9H-Fluoren-9-yl)-methoxycarbonyl-O-{2-acetamido-3,4-di-O-acetyl-2-
deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-a-
glycero-d-galacto-2-nonulopyranosyl) onat]-a-d-galactopyranosyl}-l-threo-
nine (24): A solution of Fmoc-Thr(a-NeuAc4NAcCOOBn-(2!6)-a-Ac2-
GalNAc)-OtBu (23; 0.983 g, 0.796 mmol) in dichloromethane (22 mL)
was sequentially treated with anisole (2.2 mL) and trifluoroacetic acid
(22 mL). The reaction mixture was stirred at room temperature for 3 h,
diluted with toluene (75 mL) and concentrated in vacuo. The resulting
residue was coevaporated with toluene (3î30 mL) and dichloromethane
(3î30 mL). The crude product was purified by flash chromatography
(silica gel; ethyl acetate/methanol 2:1) to give a colorless amorphous
solid (0.91 g, 97%). Rf =0.22 (AcOEt/MeOH 2:1); [a]2D2 =38.3 (c=1.00 in
CHCl3); 1H NMR (400 MHz, CD3OD): d=7.88 7.81 (m, 2H, H4-, H5-
Fmoc), 7.75 7.68 (m, 2H, H1-, H8-Fmoc), 7.48 7.33 (m, 9H, H2-, H3-,
H6-, H7-Fmoc, Harom.-Bn), 5.42 5.30 (m, 3H, H7’, H8’, OCH2-Bna), 5.25
5.15 (m, 2H, H-4, OCH2-Bnb), 5.05 (dd, 1H, H-3, J1 =8.9 Hz, J2 =
6028
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 6018 6030

Glycopeptide Synthesis
6018 6030
CH3-Pmc), 2.36 (m, 2H, H9a,b-Biot.), 1.88 2.32 (m, 9H, 3îb-CHaHb Pro,
3îg-CHaHb Pro, b-CH Val, CH2-Pmc), 1.98, 2.00, 2.06, 2.07, 2.08, 2.10
(s, 27H, 2îNHAc, 6îOAc, m-CH3-Pmc), 1.49 1.84 (m, 12H, 3îg-
CHaHb Pro, b-CH2 Arg, g-CH2 Arg, H3’a, H8a,b-Biot., H6a,b-Biot.), 1.11,
1.17, 1.29, 1.39 (s, 33H, 4îC(CH3)3, C(CH3)2-Pmc), 1.35 1.08 (m, 10H,
H7a,b-Biot., g-CH3 Thr2, b-CH3 2îAla), 1.04 (d, 3H, g-CH3 Thr1, J=
6.2 Hz), 0.95 (d, 6H, 2îg-CH3 Val, J=6.6 Hz); MALDI-TOF-MS: calcd
for C123H185N19O42S2: 2664.24, found: 2666.4 [M+H]+, 2688.3 [M+Na]+,
2704.5 [M+K]+, 2710.2 [MꢀH+2Na]+, 2726.6 [MꢀH+Na+K]+, 2400.1
[MꢀPmc+2îH]+, 2422.0 [MꢀPmc+H+Na]+, 2438.0 [MꢀPmc+H+K]+.
Acknowledgement
This work was supported by the Deutsche Forschungsgemeinschaft, the
Volkswagen-Stiftung and by the Stiftung Rheinland-Pfalz f¸r Innovation.
We thank H. Kolshorn for NMR spectroscopic assistance. M.W. is grate-
ful for a doctoral fellowship sponsored by the Fonds der Chemischen In-
dustrie. S.D. wishes to thank the Fonds der Chemischen Industrie and the
Bundesministerium f¸r Bildung und Forschung for a postgraduate fellow-
ship.
Biotinyl-Gly-Val-Thr(a-NeuAc4NAcCOOH-(2!6)-a-GalAc2NAc)-Ser-
Ala-Pro-Asp-Thr-Arg-Pro-Ala-Pro-OH (26): Catalytic palladium on acti-
vated charcoal (10%; 0.1 mg) was added to a solution of biotinyl-Gly-
Val-Thr(a-NeuAc4NAcCOOBn-(2!6)-a-GalAc2NAc)-Ser(tBu)-Ala-Pro-
[1] a) J. Montreuil, Adv. Carbohydr. Chem. Biochem. 1980, 37, 157;
b) H. Kis, N. Sharon, Eur. J. Biochem. 1993, 218, 1; c) P. M. Rudd,
R. J. Woods, M. R. Wormald, G. Opdenakker, A. K. Downing, I. D.
Campbell, R. A. Derek, Biochim. Biophys. Acta 1995, 1248, 1;
d) R. P. McEver, K. L. Moore, R. D. Cummins, J. Biol. Chem. 1995,
270, 11025; e) R. J. Wieser, F. Oesch, Trends Glycosci. Glycotechnol.
1992, 4, 160.
[2] a) J. Kihlberg, M. Elofsson, Curr. Med. Chem. 1997, 4, 85;
b) H. M. I. Osborn, T. H. Khan, Tetrahedron 1999, 55, 1807; c) C.
Brocke, H. Kunz, Bioorg. Med. Chem. 2002, 10, 3085; d) H. Herz-
ner, T. Reipen, M. Schultz, H. Kunz, Chem. Rev. 2000, 100, 4495.
[3] a) R. Warras in Combinatorial Chemistry (Ed.: G. Jung), Wiley-
VCH, Weinheim, 1999, pp. 167 228; b) B. A. Bunin, The Combina-
torial Index, Academic Press, San Diego, 1998; c) N. Sewald, H. D.
Jakubke, Peptides: Chemistry and Biology, Wiley-VCH, Weinheim,
2002, pp. 212 223; d) S. S. Wang, J. Am. Chem. Soc. 1973, 95, 1328;
e) M. Mergler, R. Tanner, J. Gosteli, P. Grogg, Tetrahedron Lett.
1988, 29, 4005; f) K. Barlos, O. Chatzi, D. Gatos, G. Stavropoulos,
Int. J. Pept. Protein Res. 1991, 37, 513.
Asp(OtBu)-Thr(tBu)-Arg(Pmc)-Pro-Ala-Pro-OH
(25;
56 mg,
0.021 mmol) in methanol (15 mL) under argon. The reaction flask was
purged with hydrogen and the solution was stirred under a hydrogen at-
mosphere for 48 h. The mixture was filtered through Hyflo-Supercel
which was washed with methanol (3î25 mL) afterwards. After removal
of the solvent in vacuo, the resulting colorless solid was dissolved in a
mixture of dichloromethane/trifluoroacetic acid/thioanisole/1,2-ethanedi-
thiol (5 mL, 10:10:1:1) and stirred for 2 h. The reaction mixture was con-
centrated in vacuo and coevaporated with toluene (3î25 mL). By addi-
tion of precooled (08C) diethyl ether (20 mL) the product 26 was precipi-
tated as a colorless crystalline solid, washed with diethyl ether (3î
20 mL), and dissolved in water (25 mL). Lyophilisation afforded 36 mg
(crude: 80%) of glycopeptide 26 which was further deprotected without
additional purification. HPLC: tR =30.0 min (Phenomenex Luna C18,
214 nm, 10% MeCN in H2O ! 30% MeCN in H2O+0.1% TFA in
40 min); MALDI-TOF-MS: calcd for C90H137N19O39S: 2139.90; found:
2232.0 [Mꢀ3H+4Na]+, 2247.9 [Mꢀ3H+3Na+K]+, 1682.8 [MꢀNeuAc4-
NAcCOOH+2H]+, 1747.5 [MꢀNeuAc4NAcCOOHꢀH+3Na]+.
[4] W. C. Chan, P. D. White, Fmoc Solid Phase Synthesis, Oxford Uni-
versity Press, 2000, p. 50.
Biotinyl-Gly-Val-Thr(a-NeuNAcCOOH-(2!6)-a-GalNAc)-Ser-Ala-Pro-
Asp-Thr-Arg-Pro-Ala-Pro-OH (27): A solution of sodium methanolate
in methanol (0.1m) was added dropwise until pH 8.5 9 was reached to a
solution of crude glycopeptide 26 in methanol (15 mL). After stirring for
15 h the reaction mixture was neutralized by adding Amberlyst IR 120.
The mixture was filtered and concentrated under reduced pressure. The
residue was dissolved in water (15 mL), lyophilized and the crude prod-
uct was purified by preparative RP-HPLC (Vydac Protein&Peptide C18,
1% MeCN in H2O ! 20% MeCN in H2O+0.1% TFA in 110 min).
Completely deprotected glycopeptide 27 was obtained as a colorless lyo-
philisate (8 mg, 20% over three steps). HPLC: tR =13.2 min (Vydac Pro-
tein&Peptide C18, 10% MeCN in H2O ! 30% MeCN in H2O+0.1%
TFA in 40 min); [a]2D2 =ꢀ58.7 (c=0.71, CHCl3); 1H NMR (600 MHz,
D2O, 1H-COSY, TOCSY, HMQC): d=4.82 (d, 1H, H1- Gal, J=3.8 Hz),
4.62 (t, 1H, a-CH Asp, J=6.6 Hz), 4.49 4.56 (m, 3H, a-CH Arg (4.54},
Thr2 (4.52), H2-Biot.(4.50)), 4.43 4.48 (q, 1H, a-CH Alaa, J=7.0 Hz),
4.40 4.34 (m, 2H, a-CH Alab (4.37), Ser (4.36)), 4.26 4.34 (m, 4H, H5-
Biot. (4.32), a-CH 3îPro (4.30)), 4.16 4.23 (m, 3H, a-CH Thr1 (4.22),
Val (4.18), b-CH Thr2 (4.19)), 4.08 4.13 (m, 1H, b-CH Thr1), 4.00 4.04
(m, 1H, H6’), 3.96 (dd, 1H, H2-Gal, J1 =3.5 Hz, J2 =11.0 Hz), 3.82 3.89
(m, 3H, H4-Gal (3.87), H6a-Gal, H5’), 3.47 3.79 (m, 17H, H3-Gal (3.77),
H8’ (3.77), a-CH2 Gly (3.75), b-CHaHb Ser (3.73), b-CHaHb Ser (3.67),
H4’ (3.60), d-CH2 3îPro (3.56, 3.50), H6b-Gal (3.53), H5-Gal, H9a,b’),
3.43 3.47 (m, 1H, H7’), 3.18 3.25 (m, 1H, H4-Biot.), 3.04 3.15 (m, 2H,
d-CH2 Arg), 2.84 2.90 (m, 2H, H3a-Biot. {2.87}, b-CHaHb Asp (2.85)),
2.77 2.83 (m, 1H, b-CHaHb Asp), 2.65 (d, 1H, H3b-Biot., J=12.9 Hz),
[5] N. Sewald, H. D. Jakubke, Peptides: Chemistry and Biology, Wiley-
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2.59 (dd, 1H, H3’e, J1 =12.5 Hz, J2 =4.7 Hz), 2.13 2.29 (m, 5H, H9a,b
-
Biot. (2.24), b-CHaHb 3îPro (2.18)), 1.87 1.98 (m, 10H, b-CH Val
(1.94), 2îCH3 NHAc (1.92), g-CHaHb 3îPro (1.91)), 1.70 1.87 (m, 4H,
g-CHaHb 3îPro (1.79), b-CHaHb Arg (1.73)), 1.48 1.67 (m, 8H, b-
CHaHb Arg, g-CH2 Arg (1.63), H3’a (1.61), H8a,b-Biot. (1.56), H6a,b-Biot.
(1.53)); 1.36 1.43 (m, 1H, H7a-Biot.), 1.29 1.34 (m, 1H, H7b-Biot.), 1.24
1.29 (m, 6H, b-CH3 2îAla), 1.20 (d, 3H, g-CH3 Thr2, J=6.2 Hz), 1.08 (d,
3H, g-CH3 Thr1, J=6.5 Hz), 0.85 (d, 6H, g-CH3 Val, J=6.8 Hz);
MALDI-TOF-MS: calcd for C78H125N19O33S: 1887.84, found: 1890.4
[M+H]+, 1912.3 [M+Na]+, 1933.8 [MꢀH+2Na]+, 1956.3 [Mꢀ2îH+3Na]+,
1599.4 (MꢀNeuNAcCOOHꢀ+2H]+, 1637.7 [MꢀNeuNAcCOOH+H+K]+,
1659.6 [MꢀNeuNAcCOOH+Na+K]+.
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6029
Chem. Eur. J. 2003, 9, 6018 6030
www.chemeurj.org
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

H. Kunz et al.
FULL PAPER
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treatment with acetic anhydride and concentrated sulphuric acid
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Received: July 7, 2003 [F5304]
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Chem. Eur. J. 2003, 9, 6018 6030