Glycocluster synthesis by native chemical ligation

Article abstract of DOI:10.1055/s-0030-1258157

Serine-and mannoside-derived thioesters and a mannosidic cysteine derivative were prepared to test native chemical ligation (NCL) for protecting-group-free synthesis of small glycopeptide clusters, which are valuable tools in the glycosciences. The glycopeptides and glycopeptide clusters, respectively, which were obtained via NCL, bear the potential for dimerization and other derivatization of the thiol functionality. Georg Thieme Verlag Stuttgart - New York.

Full text of DOI:10.1055/s-0030-1258157

3070
PAPER
Glycocluster Synthesis by Native Chemical Ligation
G
lycopept
o
ide Clusters
hby NCL annes W. Wehner, Thisbe K. Lindhorst*
Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, Otto-Hahn-Platz 3/4, 24098 Kiel, Germany
Fax +49(431)8807410; E-mail: tklind@oc.uni-kiel.de
Received 21 April 2010; revised 25 May 2010
Abstract: Serine- and mannoside-derived thioesters and a manno-
sidic cysteine derivative were prepared to test native chemical liga-
tion (NCL) for protecting-group-free synthesis of small
glycopeptide clusters, which are valuable tools in the glycosciences.
The glycopeptides and glycopeptide clusters, respectively, which
were obtained via NCL, bear the potential for dimerization and oth-
er derivatization of the thiol functionality.
O
H2N
N
H
O
peptide 1
peptide 2
HS
SR
+
CYSTEINE
derivative
THIOESTER
Key words: carbohydrates, glycopeptides, glycoclusters, native
transthioesterification
chemical ligation, protecting-group-free synthesis
O
H2N
Thioesters are important key intermediates of biosynthet-
ic pathways and precursors in the synthesis of amide
bonds.1 In preparative chemistry, especially in protein
synthesis, they became popular since Kent and co-work-
ers showed that thioesters can be used for the chemoselec-
tive ligation of large peptide fragments.2 This approach is
referred to as ‘Native Chemical Ligation’ (NCL) and has
been developed into a standard procedure for ligation of
unprotected peptides as well as glycopeptides.3
N
H
O
S
rearrangement
HS
O
H
N
N
Classical NCL requires a thioester, a N- and S-unprotect-
ed cysteine derivative, thiol catalysis and neutral aqueous
reaction conditions. The mechanism of NCL typically in-
volves transthioesterification of a peptide thioester with
another peptide that carries a cysteine residue at its N-ter-
minus (Figure 1). After the new thioester with the cysteine
mercapto function is formed, the cysteine amino group
initiates rearrangement to form a ‘native’ peptide bond, as
first reported by Wieland et al. in 1953.4
H
O
'native'
LIGATION
product
Figure 1 Mechanism of native chemical ligation, which is typically
used for protein synthesis, employing a polypeptide thioester and a
second peptide carrying an N-terminal cysteine residue. Transthio-
esterification is followed by rearrangement to form the ligated native
peptide.
NCL has been employed for alternative purposes, such as
for the synthesis of peptide-based non-symmetric
dendrimers5 or for the preparation of multivalent peptide
ligands and assemblies.6 Here, we report on a new appli-
cation of this effective ligation reaction. Because NCL has
the intriguing advantage that it requires no protecting
groups, it became our goal to test it for the synthesis of
multivalent glycomimetics, which are important tools in
the glycosciences.7 We planned to react a functionalized
thioester with a glycosylated cysteine derivative under the
conditions of NCL to furnish a glycopeptide thiol as de-
picted in Figure 2. This thiol could be further processed,
for example by oxidation to form the respective dimerized
disulfide, or by radical addition to alkenes.8 In addition, li-
gation of glycosidic thioesters should allow direct synthe-
sis of cluster glycosides.
The target molecules that are required for glycocluster
synthesis are considerably smaller and often less polar
than the molecules that are used in standard NCL, which
typically proceeds in aqueous media. Here, starting mate-
rials of different polarity were investigated and reaction
conditions were tuned to enable NCL with small, less hy-
drophilic reaction partners. Thus, protected as well as
deprotected glycocysteine derivatives were employed to-
gether with thioesters of different polarity. Four types of
thioesters were selected to test for compatibility with the
reaction conditions required for NCL (Figure 3). The pro-
tected serine thioesters of type 1 are the least polar com-
pounds in this series, followed by OH-unprotected serine
thioesters of type 2. Thioesters of types 3 and 4 are clearly
more polar and complex at the same time. However, syn-
thesis and purification of these hydroxylated thioesters
was anticipated to be critical due to the lability of
thioesters under protic conditions. Nevertheless, the car-
bohydrate-modified thioesters are attractive target mole-
SYNTHESIS 2010, No. 18, pp 3070–3082
x
x.
x
x
.
2
0
1
0
Advanced online publication: 07.07.2010
DOI: 10.1055/s-0030-1258157; Art ID: T09310SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Glycopeptide Clusters by NCL
3071
HS
O
O
H
L
N
L
H2N
HS
NCL
FG
N
O
O
O
N
H
H
O
FG
SR
+
O
O
(OH)n
(OH)n
thioester
glycocysteine
derivative
glycopeptide
thiol
O
H
N
FG
N
L
H
O
O
S
S
O
(OH)n
O
H
oxidation
L
N
FG
O
N
H
O
O
(OH)n
Figure 2 Use of NCL for glycocluster synthesis by coupling glycocysteine derivatives with small functionalized thioesters to achieve gly-
copeptide thiols in a protecting-group-free reaction. These can be easily oxidized to the respective dimers giving rise to functional glycoclusters.
FG: functional group, L: linker.
cules as they allow the construction of rather complex
glycoclusters when reacted with glycocysteine derivatives
O
via NCL.
O
FmocHN
For the synthesis of the required thioesters, a method was
required that avoids epimerization at C-a. For activation
of the carboxylic acid component, carbodiimides together
S
2
a)
with 1-hydroxybenzotriazole (HOBt) have often been em-
O
ployed,9 however, many other activation conditions have
O
b)
c)
been investigated.10 In our study, O-benzotriazole-
O
O
FmocHN
FmocHN
N,N,N¢,N¢-tetramethyluronium
hexafluorophosphate
S
OH
(HBTU) worked best for the synthesis of type 1 thioesters
(Figure 3) according to the literature.11 Hence, reaction of
the serine carboxylic acid Fmoc-L-Ser(t-Bu)-OH (1) with
ethanethiol in a tetrahydrofuran–N,N-dimethylformamide
solvent mixture furnished thioester 2 in 86% yield
(Scheme 1) and thioester 3 was obtained in almost quan-
titative yield upon HBTU-mediated reaction of 1 with
benzyl mercaptan. Likewise, the analogous conversion of
1 with thiophenol proceeded as a spot-to-spot reaction ac-
cording to TLC. Purification of the reactive phenyl
thioester 4, however, led to a diminished yield of 67%.
Thioesters 2, 3 and 4 display graded reactivity, the ethyl
thioester 2 being the most stable, 4 being the least stable.
1
3
O
O
FmocHN
S
4
Scheme 1 Synthesis of type 1 thioesters. Reagents and conditions:
(a) EtSH, HBTU, DIPEA, anhydrous THF–DMF (3:1), r.t., overnight,
86%; (b) BnSH, HBTU, DIPEA, anhydrous THF–DMF (3:1), r.t.,
5 d, 98%; (c) PhSH, HBTU, DIPEA, anhydrous THF–DMF (2:1), r.t.,
4 d, 67%.
It is essential that no epimerisation is allowed to occur
during the preparation of the amino acid thioesters. This
epimerisation was monitored by using 2D NMR spectros-
OH
OH
O
HO
HO
OH
OH
OH
OH
O
O
OH
O
O
O
O
HO
HO
HO
HO
O
R
O
R
O
N
O
N
H
H
O
S
O
S
S
O
O
R
S
S
N
H
R
type 1
type 2
type 3
type 4
O
Figure 3 Four types of low molecular weight thioesters were selected for NCL, which vary in polarity and complexity; type 1 thioesters have
the lowest, type 4 thioesters the highest polarity. R = typically Ph, Bn or Et.
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

3072
J. W. Wehner, T. K. Lindhorst
PAPER
copy and the shift reagent Eu(hfc)3 (see Supporting Infor- at room temperature. This reaction required 2.2 hours
mation). According to this study, no epimerization with thiophenol to yield 11 and 17 hours with the less re-
occurred during thioester synthesis with HBTU.
active thioethanol to furnish 12.
Type 2 thioesters 5 and 6 (Figure 4) have previously been Workup and purification of thioesters 11 and 12 was com-
reported in the literature and were prepared from Boc-L- plicated because they easily hydrolyzed in protic solvents.
Ser(OH)-OH. Synthesis of 5 was reported to proceed in This hinders reverse-phase (RP) silica gel-chromatogra-
DMF,12 whereas that of 6 proceeded in ethyl acetate.13 In phy as well as gel permeation chromatography (GPC).
our experiments, HBTU in the THF–DMF mixture de- Hydrolysis experiments with phenyl thioester 11 in wet
scribed above was employed for preparation of 5 and 6.
methanol revealed 12% hydrolysis after two hours and
30% after 26 hours according to 1H NMR analysis. HPLC
analysis of 11 in aqueous phosphate buffer (0.2 M, pH
7.0) showed complete hydrolysis to the carboxylic acid 10
within three hours. The ethyl thioester 12 on the other
hand was significantly more stable, as expected.
OH
OH
O
O
O
O
O
N
O
N
H
H
S
S
5
6
Purification of thioesters 11 and 12 was successfully im-
plemented by following a two-step purification protocol.
Flash chromatography on normal-phase silica using ethyl
acetate and distilled methanol as eluents was followed by
MPLC on reverse-phase material employing an acetoni-
trile–water gradient and immediate lyophilisation of the
product fractions.
Figure 4 Known thioesters 5 and 6 of type 2
For the synthesis of the a-D-mannosidic type 3 thioesters
(Figure 3), the known carboxylic acid mannoside 1014
was required as starting material. Differing from the pro-
cedure reported in the literature, glycosylation of methyl
p-hydroxyphenylacetate was achieved by employing the
mannosyl trichloroacetimidate 715 to prepare mannoside 8
(Scheme 2). The required unprotected acid 10 was ob-
tained after cleavage of the ester protecting groups in a
high-yielding, two-step sequence. For the synthesis of
thioesters 11 and 12 a 20-fold excess of the respective thi-
ol was applied to avoid esterification with one of the hy-
droxy groups. For both reactions, the conditions
optimized in the synthesis of type 1 thioesters (Scheme 1)
were suitable. Thus, the combination of HBTU and diiso-
propylethylamine (DIPEA) in THF–DMF (3:1) was used
Preparation of the bivalent a-D-mannosidic thioesters of
type 4 (Figure 3) was first tried starting from the thioesters
5 and 6. However, all our attempts to connect the required
sugar units to these thioesters either failed or gave very
poor yields owing to the lability of the thioester function-
OH
OH
O
HO
HO
OH
OH
O
O
HO
HO
13
a)
+
O
S
HS
OAc
OAc
O
OR1
OR1
15
O
O
H2N
a)
HCl⋅H2N
O
AcO
AcO
R1O
R1O
O
O
CCl3
NH
O
O
14
O
OH
OH
OR2
O
7
HO
HO
b)
8
9
R1 = Ac, R2 = Me
R1 = H, R2 = Me
OH
OH
O
b)
c)
10
O
HO
HO
10 R1 = H, R2 = H
CO2H
O
OH
O
OH
OH
OH
d)
e)
S
O
O
R
OH
OH
O
OH
OH
N
H
O
O
HO
HO
HO
HO
16 R = OEt
17 R = OH
c)
O
O
O
O
11
d)
d)
12
18 R = SEt
19 R = SPh
S
S
Scheme 2 Synthesis of type 3 thioesters. Reagents and conditions:
(a) methyl p-hydroxyphenylacetate, 4 Å MS, TMSOTf, anhydrous
CH2Cl2, r.t., overnight, 78%; (b) NaOMe, anhydrous MeOH, r.t.,
overnight, 95%; (c) LiOH, H2O–THF (1:1), r.t., overnight, 89%; (d)
PhSH, HBTU, DIPEA, anhydrous THF–DMF (3:1), r.t., 2.2 h, 49%;
(e) EtSH, HBTU, DIPEA, anhydrous THF–DMF (3:1), r.t., 17 h,
62%.
Scheme 3 Synthesis of dimannosidic cysteine 17 as a precursor for
thioesters 18 and 19. Reagents and conditions: (a) AIBN, anhydrous
dioxane, 65 °C, 4 h, 95%; (b) HATU, DIPEA, anhydrous DMF, 0 °C
→ r.t., overnight, 59%; (c) LiOH, H2O–THF, r.t., overnight, quant.;
(d) HBTU, DIPEA, anhydrous THF–DMF (1:1), r.t., EtSH and 2.5 h
for 18, crude product; PhSH and 22 h for 19, crude product.
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

PAPER
Glycopeptide Clusters by NCL
3073
ality. Therefore, synthesis of type 4 thioesters was based (Scheme 3). However, isolation of the products in pure
on the L-cysteine ethylester 14, which was added to the form was once more complicated, and purification led to
double bond of the allylmannoside 1316 in a radical reac- almost complete hydrolysis of 19 leaving a yield of 3%
tion (Scheme 3). This high-yielding, chemoselective reac- (2 mg). Purification of the more stable thioester 18 was
tion led to the formation of glycoamino acid 15, which more feasible, however, in this case impurities also re-
carries a free amino group for peptide coupling with the mained after chromatography and therefore, 18 was pre-
mannosidic carboxylic acid 10. Peptide coupling required pared and used in situ.
the use of a combination of 2-(7-aza-1H-benzotriazole-1-
Besides the prepared thioesters, an unprotected glycocys-
yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
teine amide was needed to test NCL for glycocluster syn-
thesis. The fully protected mannosyloxyethyl cysteine
amide 20 was considered to be a suitable starting material
for the required synthesis. It could be prepared from
Fmoc-Cys(Trt)-OH and 2-azidoethyl a-D-mannoside by a
(HATU) and N,N-diisopropylethylamine (DIPEA) to pro-
vide the dimannosidic L-cysteine ethylester 16, which
could be saponified with lithium hydroxide to the respec-
tive acid 17 in a quantitative reaction.
Conversion of the glycoamino acid 17 into the desired Staudinger ligation as previously reported (Scheme 4).17
thioesters was the last step in this synthetic route to type 4 Deprotection of the Fmoc-protected amino group led to 21
thioesters. Indeed, HBTU-mediated thioesterification of in an almost quantitative reaction.
17 to form the desired thioesters 18 and 19 proceeded well
according to TLC and MALDI-TOF-MS analysis
During the next two deprotection steps, removal of the tri-
tyl protecting group and de-O-acetylation, partial disul-
fide formation occurred. Two product fractions were
isolated, one containing the pure disulfide 22 and another
fraction containing a mixture of 22 and thiol 23. The com-
OAc
OAc
O
AcO
AcO
bined fractions, a mixture of thiol and disulfide, were sub-
jected to deprotection to give solely the disulfide 24 after
oxidation upon standing in air. Thus, 24 was obtained
O
O
NHR
N
H
from 20 in 88% overall yield. Disulfides such as 24 are
STrt
well suited for NCL because the common protocols for
20 R = Fmoc
a)
21 R = H
OAc
OAc
OAc
OAc
a)
O
O
AcO
AcO
AcO
AcO
OAc
OAc
b)
O
OH
O
O
AcO
AcO
O
O
22
25
H
N
OAc
OAc
NH2
S
N
O
N
NHBoc
O
H
O
H
AcO
AcO
O
+
NH2
S
S
N
SH
H
H
O
OH
N
O
S
H2N
22
23
NH2
SH
+
H
O
O
N
O
N
NHBoc
H
H2N
OAc
S
O
O
AcO
OAc
O
6
OAc
OAc
OAc
O
OAc
AcO
OH
OH
OH
OH
O
b)
O
OH
HO
HO
OH
O
HO
HO
HO
HO
c)
O
O
O
O
O
24
26
H
N
O
O
NH2
N
NHFmoc
N
H
NH2
S
H
O
N
+
H
S
S
SH
S
H
N
O
H
H2N
N
H2N
O
O
O
NHFmoc
O
O
OH
OH
S
O
OH
OH
4
O
OH
24
HO
OH
HO
Scheme 5 Conditions for native chemical ligations employing reac-
tion partners of differing polarity. Reagents and conditions: (a)
TCEP, phosphate buffer (0.2 M, pH 7.0), pH 7.0, r.t., 47 h, 57%; (b)
TCEP, phosphate buffer (0.2 M, pH 7.6)–MeCN (1:1), acetone,
DMF, pH 7.0, r.t., 18 h; then PhSH, r.t., 70 h, 57%.
Scheme 4 Synthesis of disulfide 24 after three deprotection steps.
Reagents and conditions: (a) morpholine, anhydrous DMF, r.t., 1.5 h,
92%; (b) TFA, TES, anhydrous CH2Cl2, r.t., 1.5 h, 98% (mixture of
22 and 23); (c) NaOMe, anhydrous MeOH, r.t., overnight, 98%.
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

3074
J. W. Wehner, T. K. Lindhorst
PAPER
native chemical ligation include application of the reduc- effected the reduction of the disulfide to the respective
ing agent tris(2-carboxyethyl)phosphane (TCEP), which glycocysteine thiol in situ, which underwent transthio-
reduces disulfides to the respective thiols.
esterification with 12, followed by rearrangement to form
the native ligation product 27 (Scheme 6). For purifica-
tion, it was important to subject the crude product 27 to
methanol extraction, followed by evaporation of the sol-
vent and rapid MP-GPC using methanol as eluent. This
procedure allowed most of the buffer salts to be removed.
Because during this chromatographic step some disulfide
formation can occur by air oxidation, subsequent addition
of TCEP is an option to ensure reduction to the respective
thiol. A combination of RP-MPLC and GPC finally deliv-
ered pure 27 in 47% yield. Air oxidation converted 27 into
the known tetravalent cluster mannoside 2818 in a quanti-
tative reaction.
With cysteine disulfides 22 and 24 and a series of
thioesters of different polarity in hand, NCL could then be
tested. First, the O-acetylated mannosylcysteine disulfide
22 and the OH-free serine thioester were selected and re-
acted in phosphate buffer, in which both reaction partners
were soluble (Scheme 5). This reaction furnished glyco-
dipeptide 25 in good yield. The protected thioester 4, on
the other hand, was not soluble in pure aqueous media,
and the highly polar disulfide 24 was insufficiently solu-
ble in apolar solvents. Thus, organic co-solvents were
used for the NCL of 4 and 24, as reported in the litera-
ture.11 Here, the reaction was carried out in a buffer–
acetonitrile–acetone–DMF solvent mixture with addition By analogy to the synthesis of the tetravalent glycocluster
of a small amount (150 mL) of thiophenol as catalyst. This 28, preparation of the hexavalent cluster mannoside 30
allowed isolation of the orthogonally protected OH-free was possible by NCL of disulfide 24 and thioester 18,
mannosyl dipeptide 26 in 57% yield, with 21% recovery which was employed as the crude product (Scheme 7).
of thioester 4.
Buffer salts were removed from the intermediate product
29, which was in turn allowed to oxidize to form the target
molecule 30 as a colourless syrup. The yield, however,
Encouraged by these results, the mannosidic thioester 12
was also ligated with disulfide 22 under the conditions of
NCL in buffer solution. The added reducing agent TCEP
OH
OH
O
HO
HO
O
12
O
OH
OH
O
+
S
HO
HO
OH
OH
O
O
O
H
N
N
H
a)
HO
HO
O
O
O
O
HS
OH
OH
NH2
S
24
N
O
H
27
OH
S
HO
H
N
H2N
O
O
OH
OH
O
b)
OH
HO
OH
OH
O
HO
HO
O
O
H
N
N
OH
H
OH
O
O
O
S
S
HO
HO
OH
OH
O
O
O
OH
H
HO
N
N
H
O
O
OH
OH
O
28
OH
HO
Scheme 6 Cluster glycopeptides by NCL. Reagents and conditions: (a) TCEP, phosphate buffer (0.2 M, pH 8.4), pH 7.0, r.t., 21.5 h, 47%;
(b) MeOH, r.t., 6 d, quant.
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PAPER
Glycopeptide Clusters by NCL
3075
OH
OH
O
HO
HO
O
OH
O
OH
OH
OH
OH
OH
O
S
O
HO
HO
O
S
N
H
O
OH
OH
O
18
O
HO
HO
+
O
OH
OH
S
O
H
a)
O
N
OH
HO
HO
N
O
H
OH
HN
O
O
O
O
HS
O
OH
OH
NH2
S
N
29
H
24
S
H
N
H2N
O
O
OH
OH
O
b)
OH
HO
OH
OH
O
HO
HO
OH
OH
O
O
HO
HO
O
S
O
H
N
OH
OH
OH
N
O
H
OH
HN
O
O
HO
HO
O
S
S
O
OH
OH
O
O
NH
S
H
OH
O
N
N
OH
H
O
O
O
OH
OH
O
OH
OH
O
OH
HO
30
Scheme 7 Cluster glycopeptides by NCL. Reagents and conditions: (a) TCEP, phosphate buffer (0.2 M, pH 8.5), pH 7.3, BnSH, PhSH, r.t.,
47 h; (b) MeOH, r.t., 6 d, 19% based on 24 over two steps.
was low, due to the extensive purification that was re- to make branched glycopeptides of considerable com-
quired.
plexity and, thus, NCL is an interesting alternative to clas-
sical peptide coupling sequences for the preparation of
peptide-based glycoclusters.18 In addition, glycocysteine
glycoclusters can easily be oxidized to the respective di-
sulfide dimers, which were shown to serve well in stan-
dard biological testing, when no special reducing
conditions are employed.18
In conclusion, it was shown that NCL is an intriguing
option for the protecting-group-free synthesis of cluster
glycosides, employing glycocysteine derivatives and
thioesters of varying complexity as the starting materials.
Optimisation of solvent mixtures was necessary to extend
the scope of NCL to a variety of ligation partners. Howev-
er, removal of buffer salts has often complicated purifica-
tion of the products and this can be a disadvantage of this
new application of native chemical ligation. On the other
hand, readily available starting materials were sufficient
Commercially available starting materials and reagents were used
without further purification unless otherwise noted. Anhydrous
DMF was purchased, other solvents were dried for reactions or dis-
tilled for chromatography. Air- and/or moisture-sensitive reactions
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

3076
J. W. Wehner, T. K. Lindhorst
PAPER
were carried out under an atmosphere of nitrogen. Compounds 5,12
6,13 1316 and 2017 were prepared according to the literature. Thin
layer chromatography was performed on silica gel plates (GF 254,
Merck). Detection was effected by UV irradiation and subsequent
charring with 10% sulfuric acid in EtOH followed by heat treat-
ment. Flash chromatography was performed on silica gel 60 (230–
400 mesh, particle size 0.040–0.063 mm, Merck). Preparative
MPLC was performed on a Büchi apparatus using a LiChroprep
RP-18 (40–60 mm, Merck) column for reverse-phase and a LiChro-
prep Si 60 (40–60 mm, Merck) column for normal-phase silica gel
9.1 Hz, 1 H, H-bbSer), 2.90 (q, J = 7.4 Hz, 2 H, SCH2CH3), 1.23 (t,
J = 7.4 Hz, 3 H, SCH2CH3), 1.17 (s, 9 H, CH3-tBu).
13C NMR (125 MHz, CDCl3): d = 200.3 (COS), 156.0 (COO-
Fmoc), 144.0, 141.3 (Fmoc-aryl-C), 127.7, 127.1, 125.4, 120.0
(Fmoc-aryl-CH), 73.6 (CCH3-tBu), 67.3 (CH2CH-Fmoc), 61.9 (C-
b
Ser), 61.0 (C-aSer), 47.2 (CH2CH-Fmoc), 27.3 (CCH3-tBu), 23.5
(SCH2CH3), 14.5 (SCH2CH3).
MS (MALDI-TOF, CCA): m/z = 466.7 [M + K]+, 450.7 [M + Na]+.
HRMS (ESI): m/z [M + Na]+ calcd for C24H29NNaO4S: 450.1710;
1
chromatography. H and 13C NMR spectra were recorded with
found: 450.1757.
Bruker DRX-500 and AV-600 spectrometers. 2D NMR experi-
Anal. Calcd for C24H29NO4S: C, 67.42; H, 6.84; N, 3.18; S, 6.84.
Found: C, 67.05; H, 7.16; N, 2.86; S, 6.69.
1
1
1
ments (1H–1H COSY, H–1H TOCSY, H–13C HSQC and H–13C
HMBC) were performed for full assignment of the spectra. Chemi-
cal shifts are reported relative to the following shifts: TMS (1H: d =
0.00 ppm); CHCl3 (13C: d = 77.00 ppm), MeOH (1H: d = 3.31 ppm;
13C: d = 49.05 ppm), or H2O (d = 4.65 ppm). ESI-MS measurements
were recorded with a Mariner ESI-TOF 5280 (Applied Biosystems)
instrument and MALDI-MS measurements with a MALDI-TOF-
MS-Biflex III (Bruker) instrument using 2,5-dihydroxybenzoic acid
(DHB) and a-cyano-4-hydroxycinnamic acid (CCA) as matrices.
IR spectroscopic measurements were recorded with a Paragon 1000
(Perkin–Elmer) FT-IR-spectrometer. For sample preparation, a
Golden Gate-diamond-ATR-unit with a saphire stamp was used.
Optical rotations were measured with a Perkin–Elmer 241 polarim-
eter (Na-D-line: 589 nm, length of cell 1 dm). Elemental analyses
were measured with a Euro-EA elemental analyzer (EuroVector) at
the Institute of Inorganic Chemistry (Christiana Albertina Universi-
ty of Kiel). For assignment of NMR spectra, different mannosidic
residues were primed or double-primed, respectively, according to
a convention explained in the Supporting Information.
N-(Fluoren-9-ylmethoxycarbonyl)-O-(tert-butyl)-L-serine S-
Benzyl Ester (3)
According to the general procedure, Fmoc-Ser(tBu)-OH (1;
150 mg, 390 mmol), HBTU (562 mg, 1.56 mmol, 4 equiv), benzyl
mercaptan (180 mL, 1.56 mmol, 4 equiv), and DIPEA (530 mL,
3.12 mmol, 8 equiv) were reacted in an anhydrous THF/DMF mix-
ture (3:1, 20 mL) for 5 d. After removal of the solvents, the crude
product was purified by two consecutive chromatographic steps on
silica gel (EtOH–Cy, 1:6 → 1:10; then Cy–CH2Cl2, 1:1 → CH2Cl2)
to yield the title compound.
Yield: 198 mg (407 mmol, 98%); colourless solid; Rf = 0.33 (Cy–
EtOH, 6:1); mp 92 °C; [a]D23 –12.1 (c = 0.75, CHCl3).
IR (ATR): 3395, 2968, 1721, 1680, 1505, 1450, 1360, 1330, 1214,
1172, 1098, 1071, 1050, 964, 926, 874, 806, 737, 697, 574, 537, 469
cm–1.
1H NMR (600 MHz, CDCl3): d = 7.75 (d, 3J = 7.5 Hz, 2 H, Fmoc-
aryl-H), 7.59 (dd~t, 3J = 7.8 Hz, 2 H, Fmoc-aryl-H), 7.38 (mc, 2 H,
Fmoc-aryl-H), 7.30–7.21 (m, 7 H, 5 × SCH2Ph, Fmoc-aryl-H), 5.73
(d, 3JH–aSer,N–H = 9.0 Hz, 1 H, N-H), 4.52 (mc, 2 H, H-aSer, CHHCH-
Fmoc), 4.35 (dd, J = 7.3, 10.7 Hz, 1 H, CHHCH-Fmoc), 4.26 (dd~t,
J = 7.1, 7.1 Hz, 1 H, CH2CH-Fmoc), 4.17 (d, J = 13.9 Hz, 1 H,
Thioester Synthesis; General Procedure
The carboxylic acid (1 equiv) and HBTU (3.8–4.5 equiv) were
dried in vacuo for 2 h and then dissolved under a nitrogen atmo-
sphere in a mixture of anhydrous THF and anhydrous DMF
(3:1 to 1:1 depending on polarity of the employed carboxylic acid).
The desired thiol (4 equiv or 20–22 equiv) was added, DIPEA (8–
9 equiv) was added dropwise and the reaction mixture was stirred at
r.t. for between 2.2 h and 5 d. The solvent was removed under re-
duced pressure and the residue was purified by chromatography on
normal-phase silica gel, on reverse-phase (RP-18), or by gel-
permeation chromatography (GPC).
SCHH), 4.09 (d, J = 13.8 Hz, 1 H, SCHH), 3.93 (dd, 3JH–aSer,H–baSer
2.5 Hz, 2JH–baSer,H–bbSer = 9.0 Hz, 1 H, H-ba), 3.51 (dd, 3JH–aSer,H–bbSer
=
=
3.4 Hz, 2JbaSer,H–bbSer = 9.0 Hz, 1 H, H-bb), 1.13 (s, 9 H, CH3-tBu).
13C NMR (150 MHz, CDCl3): d = 199.8 (COS), 155.0 (COO-
Fmoc), 143.5, 141.4 (Fmoc-aryl-C), 137.1 (C-Ar-1-benzyl), 128.9
(CH-Ar-2-benzyl), 128.6 (CH-Ar-3-benzyl), 127.8 (Fmoc-aryl-
CH), 127.3 (CH-Ar-4-benzyl), 127.1, 125.1, 120.0 (Fmoc-aryl-
CH), 74.1 (CCH3-tBu), 67.3 (CH2-Fmoc), 62.0 (C-bSer), 61.0
(C-aSer), 47.2 (CH-Fmoc), 33.4 (SCH2Ph), 27.3 (CCH3-tBu).
N-(Fluoren-9-ylmethoxycarbonyl)-O-(tert-butyl)-L-serine S-
Ethyl Ester (2)
Following the general procedure, Fmoc-Ser(tBu)-OH (1; 150 mg,
390 mmol), HBTU (562 mg, 1.56 mmol, 4 equiv), ethanethiol
(120 mL, 1.56 mmol, 4 equiv), and DIPEA (530 mL, 3.12 mmol,
8 equiv) were reacted in an anhydrous THF/DMF mixture (3:1, 20
mL) for 24 h. After removal of the solvents, the crude product was
purified by silica gel chromatography (EtOAc–Cy, 1:6→1:3).
MS (MALDI-TOF, CCA): m/z = 529.0 [M + K]+, 513.0 [M + Na]+.
HRMS (ESI): m/z [M + Na]+ calcd for C29H31NNaO4S: 512.1866;
found: 512.1854.
Anal. Calcd for C29H31NO4S: C, 71.14; H, 6.38; N, 2.86; S, 6.55.
Found: C, 71.52; H, 6.52; N, 2.99; S, 6.49.
Yield: 144 mg (337 mmol, 86%); colourless solid; Rf = 0.32 (Cy–
EtOAc, 5:1); mp 75–81 °C; [a]D23 –8.0 (c = 0.8, CHCl3).
N-(Fluoren-9-ylmethoxycarbonyl)-O-(tert-butyl)-L-serine S-
Phenyl Ester (4)
IR (ATR): 3337, 2973, 2873, 1719, 1671, 1506, 1448, 1358, 1269,
1216, 1195, 1114, 1076, 1049, 982, 934, 887, 801, 755, 735, 696,
635, 583, 543, 503, 463 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.78 (d, 3J = 7.5 Hz, 2 H, Fmoc-
According to the general procedure, Fmoc-Ser(tBu)-OH (1;
300 mg, 782 mmol), HBTU (1.12 g, 2.95 mmol, 3.8 equiv),
thiophenol (320 mL, 3.12 mmol, 4 equiv), and DIPEA (1.10 mL,
6.24 mmol, 8 equiv) were reacted in an anhydrous THF/DMF mix-
ture (3:2, 25 mL) for 4 d. The solvent was removed and the crude
product was purified by two consecutive chromatographic steps on
silica gel (EtOAc–Cy, 1:7 → 1:5; then Cy–CH2Cl2, 1:1 → CH2Cl2)
to yield the title compound.
3
3
aryl-H), 7.66 (dd, J = 11.1 Hz, J = 7.6 Hz, 2 H, Fmoc-aryl-H),
3
3
7.41 (t, J = 7.5 Hz, 2 H, Fmoc-aryl-H), 7.33 (t, J = 7.4 Hz, 2 H,
Fmoc-aryl-H), 5.75 (d, JH–aSer,N–H = 8.7 Hz, 1 H, N-H), 4.54 (dd,
J = 6.9, 10.5 Hz, 1 H, CHHCH-Fmoc), 4.48 (td, JH–aSer,N–H
8.9 Hz, JH–aSer,H–bSer = 3.1 Hz, 1 H, H-aSer), 4.36 (dd, J = 7.4,
10.5 Hz, 1 H, CHHCH-Fmoc), 4.29 (t, J = 7.0 Hz, 1 H, CH2CH-
Fmoc), 3.92 (dd, JH–aSer,H–baSer = 2.7 Hz, JH–baSer,H–bbSer = 9.1 Hz,
1 H, H-baSer), 3.56 (dd, JH–aSer,H–bbSer = 3.6 Hz, JH–baSer,H–bbSer
3
3
=
3
Yield: 249 mg (524 mmol, 67%); pale-yellow solid; Rf = 0.21 (Cy–
EtOAc, 7:1); mp 84–87 °C; [a]D23 –23.8 (c = 0.8, CHCl3).
3
2
3
2
=
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

PAPER
Glycopeptide Clusters by NCL
3077
2
IR (ATR): 3381, 2971, 1714, 1682, 1505, 1473, 1446, 1361, 1314,
1275, 1218, 1110, 1062, 1022, 965, 921, 880, 755, 726, 689, 646,
606, 549, 463 cm–1.
3.67 (dd~d, J6a,6b = 11.6 Hz, 1 H, H-6b), 3.65 (s, 3 H,
CH2CO2CH3), 3.63 (mc, 1 H, H-5), 3.51 (s, 2 H, CH2CO2CH3).
13C NMR (125 MHz, CDCl3): d = 172.5 (CO2CH3), 155.1, 130.4,
128.0, 116.5 (Ar-C), 98.2 (C-1), 72.9 (C-2), 71.4 (C-3), 70.9 (C-5),
66.1 (C-4), 60.9 (C-6), 52.0 (CO2CH3), 40.1 (CH2CO2CH3).
HRMS (ESI): m/z [M + Na]+ calcd for C15H20NaO8: 351.1050;
found: 351.1013.
1H NMR (500 MHz, CDCl3): d = 7.70 (d, 3J = 7.4 Hz, 2 H, Fmoc-
aryl-H), 7.59 (dd~t, 3J = 8.2 Hz, 2 H, Fmoc-aryl-H), 7.35–7.27 (m,
3
7 H, 5 × SPh, Fmoc-aryl-H), 7.25 (dd~t, J = 7.4 Hz, 2 H, Fmoc-
3
aryl-H), 5.75 (d, JH–aSer,N–H = 9.0 Hz, 1 H, N-H), 4.56–4.51 (m,
1 H, H-aSer), 4.52 (dd, J = 7.0, 10.5 Hz, 1 H, CHHCH-Fmoc), 4.31
(dd, J = 7.3, 10.5 Hz, 1 H, CHHCH-Fmoc), 4.23 (dd~t, J = 7.1 Hz,
4-(Carboxymethyl)phenyl-a-D-mannopyranoside (10)
1 H, CH2CH-Fmoc), 3.88 (dd, 3JH–aSer,H–baSer = 2.3 Hz, 2JH–baSer,H–bbSer
9.0 Hz, 1 H, H-baSer), 3.51 (dd, 3JH–aSer,H–bbSer = 3.5 Hz, 2JH–baSer,H–bbSer
9.0 Hz, 1 H, H-bbSer), 1.12 (s, 9 H, CH3-tBu).
=
=
The methyl ester 9 (1.00 g, 3.05 mmol) was dissolved in a mixture
of THF–H2O (1:1, 16 mL), LiOH (104 mg, 4.36 mmol, 1.3 equiv)
was added and the reaction mixture was stirred at r.t. overnight. Af-
ter neutralisation with acidic ion-exchange resin (Amberlite IR-
120 H+), the mixture was filtered, and the filtrate was concentrated
under reduced pressure to yield the title compound.
13C NMR (125 MHz, CDCl3): d = 199.0 (COS), 156.0 (COO-
Fmoc), 143.9, 141.3 (Fmoc-aryl-C), 134.7 (CH-Ar-2-phenyl),
129.4 (CH-Ar-3-phenyl), 129.2 (CH-Ar-4-phenyl), 127.8 (Fmoc-
aryl-CH), 127.5 (C-Ar-1-phenyl), 127.1, 125.2, 120.0 (Fmoc-aryl-
CH), 73.7 (CCH3-tBu), 67.4 (CH2CH-Fmoc), 61.9 (C-bSer), 61.1
(C-aSer), 47.2 (CH2CH-Fmoc), 27.4 (CCH3-tBu).
Yield: 853 mg (2.71 mmol, 89%); colourless lyophilisate after pu-
rification (RP-18; MeOH–H2O, 1:1); Rf = 0.80; mp 49–51 °C;
[a]D23 +108.0 (c = 0.45, MeOH).
HRMS (ESI): m/z [M + Na]+ calcd for C28H29NNaO4S: 498.1710;
found: 498.1738.
IR (ATR): 3284, 1495, 1611, 1509, 1222, 1102, 1005, 666, 574,
515, 460 cm–1.
1H NMR (500 MHz, D2O): d = 7.28 (d, J = 8.6 Hz, 2 H, ArH), 7.14
4-(Methoxycarbonylmethyl)phenyl-2,3,4,6-tetra-O-acetyl-a-D-
mannopyranoside (8)
3
(d, J = 8.6 Hz, 2 H, ArH), 5.62 (d, J1,2 = 1.7 Hz, 1 H, H-1), 4.20
(dd, 3J1,2 = 1.8 Hz, 3J2,3 = 3.4 Hz, 1 H, H-2), 4.08 (dd, 3J2,3 = 3.4 Hz,
3J3,4 = 9.3 Hz, 1 H, H-3), 3.84–3.74 (m, 4 H, H-6a, H-4, H-6b, H-5),
3.53 (s, 2 H, CH2CO2H).
13C NMR (125 MHz, D2O): d = 183.8 (CO2H), 156.0, 133.9, 132.5,
119.3 (Ar-C), 100.5 (C-1), 75.4 (C-5), 72.5 (C-3), 72.0 (C-2), 68.7
(C-4), 62.7 (C-6), 45.3 (CH2CO2H).
Methyl p-hydroxyphenylacetate (886 mg, 5.34 mmol) and the man-
nosyl donor 7 (3.00 g, 8.01 mmol) were dried for 5 h in vacuo. Mo-
lecular sieves (4 Å, 2.00 g, dried) and anhydrous CH2Cl2 (10 mL)
were added and the mixture was cooled to 0 °C under an argon at-
mosphere. TMSOTf (30 mL) was added and the reaction mixture
was stirred at r.t. overnight, then diluted with CH2Cl2 (30 mL) and
NaHCO3 (~50 mg) was added. After filtration, the solution was
concentrated under reduced pressure and the crude product was pu-
rified by silica gel chromatography (toluene–EtOAc, 5:1 → 1:1) to
yield the title mannoside 8.
MS (ESI): m/z [M + Na]+ calcd for C14H18NaO8: 337.09; found:
337.09.
2-[4-(a-D-Mannopyranosyloxy)phenyl]thioaceticAcidS-Phenyl
Ester (11)
Yield: 2.06 g (4.15 mmol, 78%); colourless, amorphous solid;
Rf = 0.11 (toluene–EtOAc, 5:1).
According to the general procedure, acid 10 (80.0 mg, 255 mmol,
1 equiv), HBTU (387 mg, 1.02 mmol, 4 equiv), thiophenol
(520 mL, 5.10 mmol, 20 equiv) and DIPEA (350 mL, 2.04 mmol,
8 equiv) were reacted in an anhydrous THF–DMF mixture (3:1, 6
mL) at r.t. for 2.2 h. The solution was concentrated under reduced
pressure and the crude product was purified by silica gel chroma-
tography (EtOAc–MeOH, 6:1 → 3:1) followed by MPLC (RP-18;
A = H2O, B = MeCN, 15→80% B, 100 min) yielding the title
thioester.
1H NMR (500 MHz, CDCl3): d = 7.21 (d, J = 8.9 Hz, 2 H, Ar-H),
3
7.04 (d, J = 8.7 Hz, 2 H, Ar-H), 5.56 (dd, J2,3 = 3.5 Hz,
3
3J3,4 = 10.0 Hz, 1 H, H-3), 5.50 (d, J1,2 = 1.8 Hz, 1 H, H-1), 5.43
3
3
(dd, J1,2 = 1.8 Hz, J2,3 = 3.5 Hz, 1 H, H-2), 5.36 (dd~t,
3J3,4 = 9.9 Hz, J4,5 = 9.9 Hz, 1 H, H-4), 4.28 (dd, J5,6a = 5.7 Hz,
2J6a,6b = 12.5 Hz, 1 H, H-6a), 4.14–4.03 (m, 2 H, H-5, H-6b), 3.68
(s, 3 H, CH2CO2CH3), 3.57 (s, 2 H, CH2CO2CH3), 2.19, 2.05, 2.03,
2.03 (4 × s, 4 × 3 H, 4 × COCH3).
3
3
23
13C NMR (125 MHz, CDCl3): d = 172.0 (CO2CH3), 170.5, 169.9,
169.9, 196.7 (4 × COCH3), 154.8, 130.5, 128.6, 116.6 (aryl-C), 95.9
(C-1), 69.4 (C-2), 69.2 (C-5), 68.9 (C-3), 66.0 (C-4), 62.1 (C-6),
52.0 (CH2CO2CH3), 40.3 (CH2CO2CH3), 21.4, 20.8, 20.7, 20.6 (4 ×
COCH3).
Yield: 51.0 mg (125 mmol, 49%); colourless lyophilisate; [a]D
+119.0 (c = 0.25, MeOH).
IR (ATR): 3337, 1694, 1506, 1225, 971, 668 cm–1.
1H NMR (500 MHz, CD3OD): d = 7.45–7.39 (m, 5 H, SPhH), 7.30
(d, J = 8.5 Hz, 2 H, manOPhH), 7.15 (d, J = 8.6 Hz, 2 H,
3
manOPhH), 5.52 (d, J1,2 = 1.8 Hz, 1 H, H-1), 4.04 (dd,
4-(Methoxycarbonylmethyl)phenyl-a-D-mannopyranoside (9)
The acetyl-protected mannoside 8 (1.84 g, 3.70 mmol) was dis-
solved in anhydrous MeOH (20 mL), solid NaOMe (100 mg, 1.85
mmol) was added and the reaction mixture was stirred at r.t. over-
night. After neutralization with ion-exchange resin (Amberlite IR-
120 H+), the mixture was filtered and the filtrate was concentrated
under reduced pressure. The crude product was purified by silica gel
chromatography (MeOH–EtOAc, 1:9) to give the title mannoside.
3
3
3J1,2 = 1.8 Hz, J2,3 = 3.4 Hz, 1 H, H-2), 3.94 (dd, J2,3 = 3.4 Hz,
3J3,4 = 9.4 Hz, 1 H, H-3), 3.82–3.73 (m, 5 H, CH2COS, H-4, H-6a,
H-6b), 3.65 (ddd, 3J5,6b = 2.7 Hz, 3J5,6a = 5.2 Hz, 3J4,5 = 9.8 Hz, 1 H,
H-5).
13C NMR (125 MHz, CD3OD): d = 197.4 (COS), 157.4 (manOPh-
C), 135.6 (SPh-C), 131.9 (manOPh-C), 131.4 (manOPh-C), 130.5,
130.2 (SPh-C), 128.8 (SPh-C), 117.9 (manOPh-C), 100.3 (C-1),
75.4 (C-5), 72.4 (C-3), 72.0 (C-2), 68.3 (C-4), 62.7 (C-6), 49.9
[CH2C(O)S].
HRMS (ESI): m/z [M + Na]+ calcd for C20H22NaO7S: 429.0978;
found: 429.0985.
Yield: 1.15 g (3.51 mmol, 95%); colourless, amorphous solid;
Rf = 0.19 (EtOAc–MeOH, 9:1).
1H NMR (500 MHz, CDCl3): d = 7.10 (d, J = 8.7 Hz, 2 H, 2 × ArH),
6.94 (d, J = 8.7 Hz, 2 H, 2 × ArH), 5.54 (s, 1 H, H-1), 4.17–4.05 (m,
3 H, H-2, H-3, H-4), 3.92 (dd~d, 2J6a,6b = 11.2 Hz, 1 H, H-6a), 3.75–
Anal. Calcd for C20H22O7S·0.5CH3OH: C, 58.28; H, 5.73; S, 7.59.
Found: C, 58.31; H, 5.46; S, 7.26.
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

3078
J. W. Wehner, T. K. Lindhorst
PAPER
2-[4-(a-D-Mannopyranosyloxy)phenyl]thioacetic Acid S-Ethyl
Ester (12)
HRMS (ESI): m/z [M + Na]+ calcd for C14H27NNaO8S: 392.1350;
found: 392.1261.
According to the general procedure, acid 10 (200 mg, 636 mmol,
1 equiv), HBTU (1.15 mg, 3.03 mmol, 4 equiv), ethanethiol
(940 mL, 12.7 mmol, 20 equiv) and DIPEA (870 mL, 5.09 mmol,
8 equiv) were reacted in an anhydrous THF–DMF mixture (3:1, 12
mL) at r.t. for 17 h. The solution was concentrated under reduced
pressure and the crude product was purified by two consecutive
MPLC runs (I: normal-phase silica gel; A = EtOAc, B = MeOH,
15 → 85% B, 60 min; II: RP-18; A = H2O, B = MeCN,
20→70% B, 50 min) yielding the title thioester.
N-{[4-(a-D-Mannopyranosyloxy)phenyl]acetyl}-S-[3-(a-D-man-
nopyranosyloxy)propyl]-L-cysteine Ethyl Ester (16)
Carboxylic acid 10 (100 mg, 318 mmol), amine 15 (176 mg,
477 mmol, 1.5 equiv) and HATU (145 mg, 382 mmol, 1.2 equiv)
were dried in vacuo for 1 h. Under nitrogen, anhydrous DMF (7
mL) was added, the reaction mixture was cooled to 0 °C, DIPEA
(65.0 mL, 382 mmol, 1.2 equiv) was added and the reaction was
stirred at r.t. overnight. The solution was concentrated in vacuo and
the crude product was purified by silica gel chromatography
(MeOH–EtOAc–Cy, 1:4:1 → 2:4:1) to yield the divalent title gly-
coamino acid.
Yield: 140 mg (391 mmol, 62%); colourless lyophilisate; Rf = 0.53
(MeOH–EtOAc, 1:3); [a]D23 +150.3 (c = 0.75, MeOH).
IR (ATR): 3327, 2927, 1679, 1610, 1506, 1412, 1226, 1103, 1008,
974, 835, 668, 504 cm–1.
Yield: 125 mg (188 mmol, 59%); colourless foam; Rf = 0.23
(MeOH–EtOAc–Cy, 2:4:1); [a]D23 +54.9 (c = 0.55, MeOH).
IR (ATR): 2921, 2013, 1679, 1416, 1211, 630, 531, 499 cm–1.
1H NMR (500 MHz, CD3OD): d = 7.09 (d, J = 8.8 Hz, 2 H, ArH),
3
6.97 (d, J = 8.7 Hz, 2 H, ArH), 5.37 (d, J1,2 = 1.8 Hz, 1 H, H-1),
3
3
3.91 (dd, J1,2 = 1.8 Hz, J2,3 = 3.4 Hz, 1 H, H-2), 3.81 (dd,
1H NMR (500 MHz, CD3OD): d = 7.29 (d, J = 8.7 Hz, 2 H, ArH),
3J2,3 = 3.4 Hz, J3,4 = 9.5 Hz, 1 H, H-3), 3.68–3.60 (m, 5 H,
7.11 (d, J = 8.7 Hz, 2 H, ArH), 5.50 (d, J1¢,2¢ = 1.6 Hz, 1 H, H-1¢),
3
3
3
CH2COS, H-4, H-6a, H-6b), 3.53 (ddd, J4,5 = 9.8 Hz,
4.77 (d, 3J1,2 = 1.4 Hz, 1 H, H-1), 4.63 (dd, 3JH–aCys,H–baCys = 5.0 Hz,
3JH–aCys,H–bbCys = 8.1 Hz, 1 H, H-aCys), 4.22 (q, J = 7.1 Hz, 2 H,
3J5,6a = 5.0 Hz, J5,6b = 2.6 Hz, 1 H, H-5), 2.73 (q, J = 7.4 Hz, 2 H,
3
3
3
SCH2CH3), 1.09 (t, J = 7.4 Hz, 3 H, SCH2CH3).
OCH2CH3), 4.04 (dd, J1¢,2¢ = 1.7 Hz, J2¢,3¢ = 3.3 Hz, 1 H, H-2¢),
3 3
3.94 (dd, J2¢,3¢ = 3.4 Hz, J3¢,4¢ = 9.4 Hz, 1 H, H-3¢), 3.90–3.70 (m,
8 H, H-6a, H-2, OCHHCH2CH2, H-4¢, H-6b, H-6a¢, H-6b¢, H-3),
13C NMR (125 MHz, CD3OD): d = 199.6 (COS), 157.3, 131.8,
131.6, 117.9 (ArC), 100.3 (C-1), 75.4 (C-5), 72.5 (C-3), 72.1 (C-2),
68.4 (C-4), 62.7 (C-6), 50.4 [CH2C(O)S], 24.5 (SCH2CH3), 15.1
(SCH2CH3).
HRMS (ESI): m/z [M + Na]+ calcd for C16H22NaO7S: 381.0978;
found: 381.1008.
3
3
3.65 (dd~t, J3,4 = 9.6 Hz, J4,5 = 9.6 Hz, 1 H, H-4), 3.65–3.61 (m,
1 H, H-5¢), 3.57 (s, 2 H, CH2CO2Et), 3.56–3.54 (m, 1 H, H-5), 3.51
3
(mc, 1 H, OCHHCH2CH2S), 3.04 (dd, JH–aCys,H–baCys = 5.0 Hz,
2JH–baCys,H–bbCys = 13.9 Hz, 1 H, H-baCys), 2.89 (dd, 3JH–aCys,H–bbCys
=
2
8.2 Hz, JH–baCys,H–bbCys = 13.9 Hz, 1 H, H-bbCys), 2.64 (t,
J = 7.2 Hz, 2 H, OCH2CH2CH2S), 1.85 (mc, 2 H, OCH2CH2CH2S),
1.29 (t, J = 7.1 Hz, 3 H, OCH2CH3).
S-[3-(a-D-Mannopyranosyloxy)propyl]-L-cysteine Ethyl Ester
(15)
13C NMR (125 MHz, CD3OD): d = 174.2 (CONH), 172.1 (COC-aCys),
157.0, 131.4, 130.5, 117.9 (Ar-C), 101.6 (C-1), 100.3 (C-1¢), 75.3
(C-5¢), 74.7 (C-5), 72.6 (C-3), 72.4 (C-3¢), 72.2 (C-2), 72.0 (C-2¢),
68.6 (C-4), 68.3 (C-4¢), 66.8 (OCH2CH2CH2S), 62.9 (C-6), 62.7
(OCH2CH3), 62.6 (C-6¢), 54.0 (C-aCys), 42.7 (CH2CONH), 34.3
(C-bCys), 30.5 (OCH2CH2CH2S), 30.0 (OCH2CH2CH2S), 14.5
(OCH2CH3).
Mannoside 13 (500 mg, 2.27 mmol) and L-cysteine ethyl ester hy-
drochloride (14; 2.12 g, 11.4 mmol, 5 equiv) were dried in vacuo
for 1 h. A catalytic amount of azobisisobutyronitrile (AIBN) and
anhydrous dioxane (20 mL) were added under nitrogen and the ob-
tained solution was stirred for 4 h at 65 °C. After concentration un-
der reduced pressure, the residue was dissolved in H2O (5 mL) and
the aqueous phase was washed with EtOAc (3 × 5 mL) and concen-
trated in vacuo. The crude product was purified by silica gel chro-
matography (EtOH–EtOAc, 3:1 → 1:1) to give the title compound.
MS (MALDI-TOF, DHB): m/z = 704.1 [M + K]+, 688.2 [M + Na]+.
HRMS (ESI): m/z [M + Na]+ calcd for C28H43NNaO15S: 688.2246;
Yield: 795 mg (2.15 mmol, 95%); colourless syrup; Rf = 0.35
found: 688.2233.
(MeOH–EtOAc, 5:6).
N-{[4-(a-D-Mannopyranosyloxy)phenyl]acetyl}-S-[3-(a-D-man-
nopyranosyloxy)propyl]-L-cysteine (17)
IR (ATR): 2978, 1728, 1444, 1368, 1195, 1090, 1018, 856, 675,
576, 465 cm–1.
1H NMR (500 MHz, CD3OD): d = 4.79 (d, 3J1,2 = 1.5 Hz, 1 H, H-1),
Ethyl ester 16 (50.0 mg, 75.1 mmol) was dissolved in a mixture of
THF and H2O (1:1, 4 mL), LiOH (2.30 mg, 97.6 mmol, 1.3 equiv)
was added and the reaction mixture was stirred at r.t. overnight. The
solution was neutralised with acidic ion-exchange resin (Amberlite
IR-120 H+), filtered and washed with H2O (5 × 5 mL). The solution
was concentrated under reduced pressure to yield the title carboxy-
lic acid.
3
4.35 (q, J = 7.1 Hz, 2 H, OCH2CH3), 4.29 (dd, JH–aCys,H–baCys
=
3
4.7 Hz, JH–aCys,H–bbCys = 7.1 Hz, 1 H, H-aCys), 3.89–3.84 (m, 2 H,
OCHHCH2CH2S, H-6a), 3.83 (dd, J1,2 = 1.6 Hz, J2,3 = 3.3 Hz,
3
3
3
2
1 H, H-2), 3.76 (dd, J5,6b = 5.8 Hz, J6a,6b = 11.9 Hz, 1 H, H-6b),
3.72 (mc, 1 H, H-3), 3.65 (dd, 3J3,4 = 8.4 Hz, 3J4,5 = 10.5 Hz, 1 H, H-4),
3.59–3.53 (m, 2 H, H-5, OCHHCH2CH2S), 3.18 (dd, 3JH–aCys,H–baCys
=
Yield: 48.0 mg (75.1 mmol, quant.); colourless foam; Rf = 0.12
(MeOH–EtOAc–Cy, 1:5:1); [a]D23 +34.5 (c = 0.50, MeOH).
2
4.7 Hz, JH–baCys,H–bbCys = 14.6 Hz, 1 H, H-baCys), 3.09 (dd,
3JH–aCys,H–bbCys = 7.1 Hz, JH–baCys,H–bbCys = 14.6 Hz, 1 H, H-bbCys),
2
2.74 (mc, 2 H, OCH2CH2CH2), 1.93 (dt~t, J = 6.4 Hz, 2 H,
OCH2CH2CH2), 1.37 (t, J = 7.1 Hz, 3 H, OCH2CH3).
IR (ATR): 3244, 1642, 1508, 1414, 1228, 1055, 813, 658, 577, 518,
470 cm–1.
13C NMR (125 MHz, CD3OD): d = 169.8 (COC-aCys), 101.6 (C-1),
74.7 (C-5), 72.7 (C-3), 72.2 (C-2), 68.6 (C-4), 66.8
(OCH2CH2CH2), 64.0 (OCH2CH3), 62.9 (C-6), 53.8 (C-aCys), 33.3
(C-bCys), 30.4 (OCH2CH2CH2), 30.3 (OCH2CH2CH2), 14.5
(OCH2CH3).
1H NMR (500 MHz, CD3OD): d = 7.30 (d, J = 8.7 Hz, 2 H, Ar-H),
7.10 (d, J = 8.7 Hz, 2 H, Ar-H), 5.49 (d, 3J1¢,2¢ = 1.7 Hz, 1 H, H-1¢),
4.76 (d, 3J1,2 = 1.6 Hz, 1 H, H-1), 4.63 (dd, 3JH–aCys,H–baCys = 4.7 Hz,
3JH–aCys,H–bbCys = 8.0 Hz, 1 H, H-aCys), 4.03 (dd, 3J1¢,2¢ = 1.8 Hz,
3J2¢,3¢ = 3.4 Hz, 1 H, H-2¢), 3.93 (dd, 3J2¢,3¢ = 3.4 Hz, 3J3¢,4¢ = 9.5 Hz,
3
2
1 H, H-3¢), 3.87 (dd, J5,6a = 2.3 Hz, J6a,6b = 11.8 Hz, 1 H, H-6a),
MS (MALDI-TOF, DHB): m/z = 408.7 [M + K]+, 391.8 [M + Na]+,
3
3
3.83 (dd, J1,2 = 1.7 Hz, J2,3 = 3.3 Hz, 1 H, H-2), 3.81–3.75 (m,
5 H, OCHHCH2CH2, H-4¢, H-6b, H-6a¢, H-6b¢), 3.73 (dd,
369.8 [M + H]+.
3
3
3J2,3 = 3.4 Hz, J3,4 = 9.4 Hz, 1 H, H-3), 3.64 (dd, J4,5 = 9.4 Hz,
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

PAPER
Glycopeptide Clusters by NCL
3079
3J3,4 = 9.8 Hz, 1 H, H-4), 3.62 (mc, 1 H, H-5¢), 3.59–3.56 (m, 1 H,
H-5), 3.57 (s, 2 H, CH2CONH), 3.49 (td, J = 5.9, 9.9 Hz, 1 H,
80 → 60% B, 120 min) to yield the crude title compound (25.0 mg).
Further purification by GPC (Sephadex LH-20, H2O or MeOH) led
to hydrolysis of 19 and methyl ester formation, leaving the pure title
compound.
OCHHCH2CH2S), 3.06 (dd, 3JH–aCys,H–baCys = 4.7 Hz, 2JH–baCys,H–bbCys
=
3
13.9 Hz, 1 H, H-baCys), 2.90 (dd, JH–aCys,H–bbCys = 8.0 Hz,
2JH–baCys,H–bbCys = 13.9 Hz, 1 H, H-bbCys), 2.63 (t, J = 7.2 Hz, 2 H,
OCH2CH2CH2S), 1.84 (mc, 2 H, OCH2CH2CH2S).
Yield: 2.00 mg (2.74 mmol, 3%).
MS (ESI): m/z [M + Na]+ calcd for C32H43NNaO14S2: 752.20;
found: 752.20.
MS (MALDI-TOF, DHB): m/z = 752.5 [M + Na]+.
13C NMR (125 MHz, CD3OD): d = 174.2 (CONH), 172.6 (COC-
a
Cys), 157.0, 131.4, 130.4, 117.9 (Ar-C), 101.6 (C-1), 100.3 (C-1¢),
75.3 (C-5¢), 74.7 (C-5), 72.6 (C-3), 72.4 (C-3¢), 72.2 (C-2), 72.0 (C-
2¢), 68.6 (C-4), 68.3 (C-4¢), 66.8 (OCH2CH2CH2S), 62.9 (C-6), 62.6
(C-6¢), 53.8 (C-aCys), 42.6 (CH2CONH), 34.3 (C-bCys), 30.5
(OCH2CH2CH2S), 29.9 (OCH2CH2CH2S).
S-(Triphenylmethyl)-L-cysteine-[2-(2,3,4,6-tetra-O-acetyl-a-D-
mannopyranosyloxy)ethyl]amide (21)
The protected mannosidic L-cysteine derivative 20 (570 mg,
594 mmol) was dissolved in anhydrous DMF (5 mL) under a nitro-
gen atmosphere and morpholine (310 mL, 3.56 mmol, 6 equiv) was
added. The reaction mixture was stirred at r.t. for 1.5 h and then
concentrated under reduced pressure. The remaining solid was dis-
solved in EtOAc (15 mL) and the formed precipitate was filtered
off. The solution was concentrated under reduced pressure and the
crude product was purified by silica gel chromatography
(EtOAc → EtOAc–MeOH, 9:1) to yield the unprotected amide 21.
MS (MALDI-TOF, DHB): m/z [M + Li]+ calcd for C26H39NLiO15S:
644.2; found: 644.0, 660.00 [M + Na]+, 666.03 [M + Li + Na]+,
681.9 [M + K]+.
HRMS (ESI): m/z [M + Na]+ calcd for C26H39NNaO15S: 660.1933;
found: 660.1961.
N-{[4-(a-D-Mannopyranosyloxy)phenyl]acetyl}-S-[3-(a-D-man-
nopyranosyloxy)propyl]-L-cysteine S-Ethyl Ester (18)
According to the general procedure, acid 17 (25.0 mg, 39.2 mmol)
and HBTU (67.0 mg, 176 mmol, 4.5 equiv) were dissolved in a mix-
ture of anhydrous THF–DMF (1:1, 6 mL), ethanethiol (65.0 mL,
878 mmol, 22 equiv) and DIPEA (60.0 mL, 351 mmol, 9 equiv) were
added and the solution was stirred at r.t. for 2.5 h. After removal of
the solvents the crude product was purified by GPC on Sephadex
LH-20 (DMF) to yield the crude (slightly contaminated according
to NMR analysis) title thioester as a colourless, amorphous solid
(15.2 mg), which was used without further purification.
Yield: 402 mg (546 mmol, 92%); colourless foam; Rf = 0.19
(EtOAc); mp 86–91 °C; [a]D23 +27.5 (c = 0.6, CHCl3).
IR (ATR): 3453, 2013, 1742, 1694, 1366, 1217, 1136, 1045, 743,
699, 600, 517 cm–1.
1H NMR (500 MHz, CDCl3): d = 7.44 (d, 3J = 7.6 Hz, 6 H, Trt-H),
7.31–7.27 (m, 6 H, Trt-H), 7.22 (mc, 3 H, Trt-H), 5.31 (dd,
3
3
3J2,3 = 3.4 Hz, J3,4 = 9.9 Hz, 1 H, H-3), 5.26 (dd, J3,4 = 9.9 Hz,
3J4,5 = 9.9 Hz, 1 H, H-4), 5.23 (mc, 1 H, H-2), 4.79 (d, 3J1,2 = 1.6 Hz,
1 H, H-1), 4.25 (dd, J5,6a = 5.5 Hz, J6a,6b = 12.2 Hz, 1 H, H-6a),
3
2
Rf = 0.08 (MeOH–EtOAc, 1:3).
3
2
4.08 (dd, J5,6b = 2.5 Hz, J6a,6b = 12.2 Hz, 1 H, H-6b), 3.95 (ddd,
1H NMR (500 MHz, CD3OD): d = 7.15 (d, J = 8.6 Hz, 2 H, m-Ar-
3
3
3J5,6b = 2.3 Hz, J5,6a = 5.5 Hz, J4,5 = 9.9 Hz, 1 H, H-5), 3.70 (mc,
1 H, OCH2CH2), 3.53–3.49 (m, 1 H, OCH2CH2), 3.47–3.42 (m,
1 H, OCH2CHH), 3.37–3.30 (m, 1 H, OCH2CHH), 3.06 (dd,
3JH–aCys,H–baCys = 4.3 Hz, 3JH–aCys,H–bbCys = 8.3 Hz, 1 H, H-aCys), 2.74
(dd, 3JH–aCys,H–baCys = 4.3 Hz, 2JH–baCys,H–bbCys = 13.1 Hz, 1 H, H-baCys),
H), 6.97 (d, J = 8.6 Hz, 2 H, o-Ar-H), 5.35 (d, 3J1¢,2¢ = 1.5 Hz, 1 H,
3
3
H-1¢), 4.63 (d, J1,2 = 1.3 Hz, 1 H, H-1), 4.54 (dd, JH–aCys,H–baCys
=
4.3 Hz, 3JH–aCys,H–bbCys = 9.3 Hz, 1 H, H-aCys), 3.90 (dd,
3
3J1¢,2¢ = 1.4 Hz, J2¢,3¢ = 3.3 Hz, 1 H, H-2¢), 3.94 (dd, 3J2¢,3¢ = 3.3 Hz,
3J3¢,4¢ = 9.1 Hz, 1 H, H-3¢), 3.74–3.58 (m, 8 H, H-6a, H-2,
OCHHCH2CH2S, H-4¢, H-6b, H-6a¢, H-6b¢, H-3), 3.52 (dd~t,
3
2
2.64 (dd, JH–aCys,H–bbCys = 8.3 Hz, JH–baCys,H–bbCys = 13.2 Hz, 1 H,
H-bbCys), 2.14, 2.07, 2.03, 1.99 (4 × s, 4 × 3 H, 4 × COCH3).
3
3J3,4 = 8.9 Hz, J4,5 = 9.4 Hz, 1 H, H-4), 3.51–3.47 (m, 1 H, H-5¢),
13C NMR (125 MHz, CDCl3): d = 172.3 (COC-a), 170.7, 170.1,
170.0, 169.7 (4 × COCH3), 144.5 (Trt-C), 129.6, 128.1, 126.9 (Trt-
C), 97.6 (C-1), 69.4 (C-2), 69.0 (C-3), 68.7 (C-5), 67.2 (CPh3), 67.1
(OCH2CH2), 66.1 (C-4), 62.5 (C-6), 53.7 (C-aCys), 38.7
(OCH2CH2), 36.5 (C-bCys), 20.9, 20.9, 20.8, 20.7 (4 × COCH3).
3.47 (s, 2 H, CH2COS), 3.44–3.40 (m, 1 H, H-5), 3.36 (mc, 1 H,
OCHHCH2CH2S), 2.93 (dd, 3JH–aCys,H–baCys = 4.5 Hz, 2JH–baCys,H–bbCys
=
14.0 Hz, 1 H, H-baCys), 2.75 (q, J = 7.4 Hz, 2 H, SCH2CH3), 2.67
3
2
(dd, JH–aCys,H–bbCys = 9.5 Hz, JH–baCys,H–bbCys = 13.9 Hz, 1 H, H-
bbCys), 2.47 (mc, 2 H, OCH2CH2CH2S), 1.85 (t, J = 6.6 Hz, 2 H,
OCH2CH2CH2S), 1.11 (t, J = 7.4 Hz, 3 H, SCH2CH3).
MS (MALDI-TOF, CCA): m/z [M
+
Na]+ calcd for
C38H44N2NaO11S: 759.3; found: 759.7 [M + Ma]+, 774.2 [M + K]+.
13C NMR (125 MHz, CD3OD): d = 201.0 (COS), 174.5 (CONH),
157.0 (OCCHCHC), 131.5 (OCCHCHC), 130.2 (OCCHCHC),
117.9 (OCCHCHC), 101.6 (C-1), 100.3 (C-1¢), 75.3 (C-5¢), 74.6 (C-
5), 72.6 (C-3), 72.4 (C-3¢), 72.2 (C-2), 72.0 (C-2¢), 68.6 (C-4), 68.3
(C-4¢), 66.8 (OCH2CH2CH2S), 62.8 (C-6), 62.5 (C-6¢), 60.4 (C-
HRMS (ESI): m/z [M + H]+ calcd for C38H45N2O11S: 737.2744;
found: 737.2739.
L-Cystine-di-[2-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl-
oxy)ethyl]amide (22) and L-Cysteine-[2-(2,3,4,6-tetra-O-acetyl-
a-D-mannopyranosyloxy)ethyl]amide (23)
a
Cys), 42.8 (CH2COS), 34.6 (C-bCys), 30.4 (OCH2CH2CH2S), 29.8
(OCH2CH2CH2S), 24.2 (SCH2CH3), 14.9 (SCH2CH3).
MS (ESI): m/z [M + Na]+ calcd for C28H43NNaO14S2: 704.20;
found: 704.20.
MS (MALDI-TOF, DHB): m/z = 704.2 [M + Na]+.
The S-protected mannosidic L-cysteine 21 (405 mg, 550 mmol) was
dissolved in anhydrous DMF (5 mL) under a nitrogen atmosphere,
TES (450 mL, 2.75 mmol, 5 equiv) and TFA (400 mL, 5.50 mmol,
10 equiv) were added and the reaction mixture was stirred at r.t. for
1.5 h. After concentration under reduced pressure, the residue was
codistilled with toluene (3 × 15 mL). The crude product was puri-
fied by reverse-phase MPLC (RP-18; A = H2O, B = EtOH,
5 → 80% B, 60 min) to yield a first fraction containing a mixture of
22 and 23, and a second fraction containing pure 22.
N-{[4-(a-D-Mannopyranosyloxy)phenyl]acetyl}-S-[3-(a-D-man-
nopyranosyloxy)propyl]-L-cysteine S-Phenyl Ester (19)
According to the general procedure, carboxylic acid 17 (58.0 mg,
91.6 mmol) and HBTU (139 mg, 366 mmol, 4 equiv) were dissolved
in a mixture of anhydrous THF–DMF (1:1, 20 mL). Thiophenol
(140 mL, 1.83 mmol, 20 equiv) and DIPEA (130 mL, 733 mmol,
8 equiv) were added and the solution was stirred at r.t. overnight.
After concentration under reduced pressure, the crude product was
purified by normal-phase MPLC (A = MeOH, B = EtOAc,
Overall yield: 267 mg (540 mmol, 98%, calculated based on the mo-
lecular weight of 23).
MS (MALDI-TOF, DHB): m/z = 1025.35 [22 + K]+, 1009.37 [22 +
Na]+, 987.39 [22 + H]+, 517.37 [23 + Na]+.
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

3080
J. W. Wehner, T. K. Lindhorst
PAPER
3
2
First fraction: Disulfide 22 and thiol 23.
(dd, JH–aCys,H–bbCys = 7.9 Hz, JH–baCys,H–bbCys = 14.6 Hz, 2 H, H-
bbCys, H-bb¢Cys).
Yield: 160 mg (324 mmol, 59% calculated based on the molecular
weight of 23).
13C NMR (125 MHz, CD3OD): d = 167.9, 167.9 (COC-aCys, COC-
a¢Cys), 101.6, 101.6 (C-1, C- 1¢), 74.9, 74.9 (C-5, C-5¢), 72.5, 72.5
(C-3, C-3¢), 72.0, 72.0 (C-2, C-2¢), 68.8, 68.8 (C-4, C-4¢), 67.0, 67.0
NMR data for 23:
1H NMR (600 MHz, CD3OD): d = 5.35–5.31 (m, 1 H, H-3), 5.30
(dd, 3J1,2 = 1.8 Hz, 3J2,3 = 3.4 Hz, 1 H, H-2), 5.28–5.25 (m, 1 H, H-
4), 4.91 (d, 3J1,2 = 1.8 Hz, 1 H, H-1), 4.25 (mc, 1 H, H-6a), 4.13 (dd,
(OCH2CH2, OCH2CH2¢), 63.1, 63.1 (C-6, C-6¢), 53.1, 53.1 (C-aCys
,
C-a¢Cys), 40.9, 40.9 (OCH2CH2, OCH2CH2¢), 40.0, 40.0 (C-bCys, C-
b¢Cys).
2
3J5,6a = 2.4 Hz, J6a,6b = 12.2 Hz, 1 H, H-6a), 4.06 (mc, 1 H, H-5),
MS (MALDI-TOF, DHB): m/z = 689.7 [M + K]+, 673.8 [M + Na]+,
3
3
4.01 (dd, JH–aCys,H–baCys = 4.0 Hz, JH–aCys,H–bbCys = 5.7 Hz, 1 H, H-
Cys), 3.81 (mc, 1 H, OCHHCH2), 3.62 (mc, 1 H, OCHHCH2), 3.49–
651.7 [M + H]+.
a
3.40 (m, 2 H, OCHHCH2), 3.04 (mc, 1 H, H-baCys), 2.98 (mc, 1 H,
H-bbCys), 2.16, 2.07, 2.04, 1.98 (4 × s, 4 × 3 H, 4 × COCH3).
HRMS (ESI): m/z [M + H]+ calcd for C22H43N4O14S2: 651.2212;
found: 651.2348.
13C NMR (150 MHz, CD3OD): d = 172.6 (COC-aCys), 172.0, 171.8,
171.7, 171.4 (4 × COCH3), 98.7 (C-1), 70.8 (C-2), 70.6 (C-3), 69.9
(C-5), 67.4 (OCH2CH2), 67.2 (C-4), 63.6 (C-6), 56.2 (C-aCys), 40.1
(OCH2CH2), 26.4 (C-bCys), 20.7, 20.6, 20.6, 20.6 (4 × COCH3).
N-(tert-Butyloxycarbonyl)-L-serinyl-L-cysteine-[2-(2,3,4,6-tet-
ra-O-acetyl-a-D-mannopyranosyloxy)ethyl]amide (25)
Thioester Boc-Ser(OH)-SPh (6; 9.20 mg, 30.7 mmol), acetylated
disulfide 22 (30.3 mg, 122.6 mmol) and TCEP (35.0 mg, 122 mmol)
were dissolved in degassed phosphate buffer (6 mL, 0.2 M, pH 7.0)
and the pH was adjusted to 7.0 with 1 M NaOH solution. The reac-
tion mixture was stirred at r.t. for 47 h; progress of the reaction was
monitored by TLC (RP-18, MeCN–H2O, 1:1). After the reaction
was complete, the mixture was concentrated under reduced pressure
and the crude product was purified by reverse-phase MPLC (RP-18;
A = H2O, B = MeCN, 10 → 90% B, 100 min). The crude product
was dissolved in H2O (10 mL) and stirred at r.t. for 30 min with
TCEP (35.0 mg, 122 mmol). Purification by reverse-phase MPLC
(RP-18; A = H2O, B = MeCN, 10 → 90% B, 100 min) yielded the
pure ligation product.
Second fraction: Disulfide 22.
Yield: 107 mg (108 mmol, 39%); Rf = 0.47 (MeOH); mp 52–54 °C;
[a]D23 +30.6 (c = 0.55, MeOH).
IR (ATR): 2967, 2012, 1737, 1669, 1368, 1218, 1131, 1042, 835,
657, 598, 517, 459 cm–1.
1H NMR (600 MHz, CD3OD): d = 5.31–5.24 (m, 4 H, H-3, H-3¢, H-
3
3
2, H-2¢), 5.24 (dd, J3,4 = 9.3 Hz, J4,5 = 9.9 Hz, 3J3¢,4¢ = 9.3 Hz,
3J4¢,5¢ = 9.9 Hz, 2 H, H-4, H-4¢), 4.89 (d~s, 2 H, H-1, H-1¢), 4.28–
3
4.25 (m, 2 H, H-aCys
,
H-a¢Cys), 4.25 (dd, J5,6a = 4.9 Hz,
2J6a,6b = 12.2 Hz, J5¢,6a¢ = 4.9 Hz, J6a¢,6b¢ = 12.2 Hz, 2 H, H-6a, H-
3
2
6a¢), 4.13 (dd, J5,6a = 2.3 Hz, J6a,6b = 12.2 Hz, 3J5¢,6a¢ = 2.3 Hz,
2J6a¢,6b¢ = 12.2 Hz, 2 H, H-6b, H-6b¢), 4.06 (mc, 2 H, H-5, H-5¢), 3.81
(mc, 2 H, OCHHCH2, OCHHCH2¢), 3.61 (mc, 2 H, OCHHCH2,
OCHHCH2¢), 3.49 (mc, 4 H, OCHHCH2, OCHHCH2¢), 3.12 (dd~td,
3
2
Yield: 12.0 mg (17.6 mmol, 57%); colourless, amorphous solid;
Rf = 0.21 (RP-18; MeCN–H2O, 2:1).
1H NMR (500 MHz, CD3OD): d = 5.33–5.20 (m, 3 H, H-2, H-3, H-
4), 4.86 (s, 1 H, H-1), 4.54 (dd, 3JH–aCys,H–baCys = 3.1 Hz, 3JH–aCys,H–bbCys
=
3JH–aCys,H–baCys = 5.6 Hz, JH–baCys,H–bbCys = 13.4 Hz, JH–a¢Cys,H–ba¢Cys
=
2
3
6.0 Hz, 1 H, H-aCys), 4.25 (dd, 3J5,6a = 5.1 Hz, 2J6a,6b = 12.2 Hz, 1 H,
2
5.6 Hz, JH–ba¢Cys,H–bb¢Cys = 13.4 Hz, 2 H, H-baCys, H-ba¢Cys), 2.89
(mc, 2 H, H-bbCys, H-bb¢Cys), 2.14, 2.07, 2.05, 2.03 (4 × s, 4 × 3 H,
8 × COCH3).
3
3
H-6a), 4.18 (dd, JH–aSer,H–baSer = 5.6 Hz, JH–aSer,H–bbSer = 5.6 Hz,
1 H, H-aSer), 4.11 (dd, 3J5,6b = 2.6 Hz, 2J6a,6b = 12.2 Hz, 1 H, H-6b),
4.06 (mc, 1 H, H-5), 3.85–3.73 (m, 3 H, H-baSer, OCHHCH2, H-bbSer),
3.59 (mc, 1 H, OCHHCH2), 3.45–3.32 (m, 2 H, OCH2CH2), 2.93
(mc, 2 H, H-bCys), 2.14, 2.06, 2.05, 1.96 (4 × s, 4 × 3 H, 4 × COCH3),
1.46 (s, 9 H, Boc-CH3).
13C NMR (150 MHz, CD3OD): d = 175.9 (COC-aCys, COC-a¢Cys),
172.4, 171.6, 171.5, 171.4 (8 × COCH3), 98.9 (C-1, C-1¢), 70.7 (C-
2, C-2¢), 70.6 (C-3, C-3¢), 69.9 (C-5, C-5¢), 67.6 (OCH2CH2,
OCH2CH2¢), 67.2 (C-4, C-4¢), 63.6 (C-6, C-6¢), 55.3 (C-aCys, C-
a¢Cys), 44.7 (C-bCys), 44.5 (C-b¢Cys), 40.1 (OCH2CH2, OCH2CH2¢),
20.7, 20.7, 20.6, 20.5 (8 × COCH3).
13C NMR (125 MHz, CD3OD): d = 172.5 (COC-aSer), 172.3 (COC-
a
Cys), 171.6, 171.4, 171.2, 170.6 (4 × COCH3), 99.0 (C-1), 80.8
[Boc-C(CH3)3], 70.7 (C-2), 70.4 (C-3), 69.9 (C-5), 67.5
(OCH2CH2), 67.2 (C-4), 63.5 (C-6), 63.4 (C-bSer), 57.9 (C-aSer),
56.9 (C-aCys), 40.5 (OCH2CH2), 28.6 (Boc-CH3), 26.5 (C-bCys),
20.8, 20.8, 20.7, 20.5 (4 × COCH3).
MS (MALDI-TOF, DHB): m/z = 1009.3 [M + Na]+, 987.3 [M +
H]+.
HRMS (ESI): m/z [M + Na]+ calcd for C38H58N4NaO22S2:
1009.2876; found: 1009.2739.
MS (MALDI-TOF, DHB): m/z [M
+
Na]+ calcd for
C27H43N3NaO15S: 704.23; found: 704.67 [M + Na]+, 720.6 [M + K]+.
L-Cystine-di[2-(a-D-mannopyranosyloxy)ethyl]amide (24)
A mixture of 22 and 23 (840 mg, 1.70 mmol based on monomer 23)
was dissolved in anhydrous MeOH (10 mL) under a nitrogen atmo-
sphere and NaOMe (54.0 mg, 1.00 mmol) was added. The reaction
mixture was stirred at r.t. overnight, then neutralized with acidic
ion-exchange resin (Amberlite IR-120 H+) and filtered. The filtrate
was concentrated under reduced pressure to yield the title com-
pound.
N-(Fluoren-9-ylmethoxycarbonyl)-O-(tert-butyl)-L-serinyl-L-
cysteine-[2-(a-D-mannopyranosyloxy)ethyl]amide (26)
Thioester Fmoc-Ser(tBu)-SPh (4; 19.2 mg, 40.4 mmol) was dis-
solved in degassed MeCN (6.5 mL), and disulfide 24 (19.2 mg,
29.5 mmol) and TCEP (36.0 mg, 126 mmol; 0.2 M solution in de-
gassed phosphate buffer, 4 mL, pH 7.6) were added. The pH was ad-
justed to 7.0 by adding 1 M NaOH solution. Acetone (2 mL) and
DMF (2 mL) were added and the reaction mixture was stirred for
18 h at r.t. under a nitrogen atmosphere. Thiophenol (1% v/v,
150 mL) and additional TCEP (70.0 mg, 244 mmol) were added and
the reaction mixture was stirred at r.t. for another 70 h. The solution
was concentrated under reduced pressure and the crude product was
purified by reverse-phase MPLC (RP-18; A = H2O, B = MeCN,
20 → 70% B, 50 min) to yield the desired ligation product together
with recovered 4 (4 mg, 8.41 mmol, 21%).
Yield: 543 mg (834 mmol, 98% calculated based on the molecular
weight of 23); colourless foam; Rf = 0.74 (RP-18, MeOH); mp 64–
69 °C; [a]D23 +14.8 (c = 0.6, MeOH).
1H NMR (500 MHz, CD3OD): d = 4.83 (br s, 2 H, H-1, H-1¢), 4.23
3
3
(dd, JH–aCys,H–baCys = 5.3 Hz, JH–aCys,H–bbCys = 7.9 Hz, 2 H, H-aCys
,
H-a¢Cys), 3.92–3.92 (m, 6 H, H-6a, H-6a¢, H-2, H-2¢, OCHHCH2,
OCHHCH2¢), 3.77–3.70 (m, 4 H, H-3, H-3¢, H-6b, H-6b¢), 3.53–
3.50 (m, 6 H, OCHHCH2, OCHHCH2¢, H-4, H-4¢, H-5, H- 5¢),
3.50–3.45 (m, 6 H, OCH2CH2, OCH2CH2¢, H-baCys, H-ba¢Cys), 3.17
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

PAPER
Glycopeptide Clusters by NCL
3081
Yield: 16.0 mg (23.1 mmol, 57%); colourless lyophilisate; Rf = 0.51
(MeOH–EtOAc, 1:1); [a]D23 +16.3 (c = 0.6, CHCl3).
N,N¢-{[4-(a-D-Mannopyranosyloxy)phenyl]acetyl}-L-cystine-
di[2-(a-D-mannopyranosyloxy)ethyl]amide (28)
Thiol 27 (11.5.0 mg, 18.5 mmol) was dissolved in MeOH (5 mL)
and kept under an oxygen-containing atmosphere for 6 d at r.t. The
solution was concentrated under reduced pressure to yield disulfide
28 (11.5 mg, 9.25 mmol, quant.) as a colourless syrup. Analytical
data of the product were in perfect accordance with the literature.18
1H NMR (600 MHz, CD3OD): d = 7.84 (d, 3J = 7.7 Hz, 2 H, Fmoc-
3
aryl-H), 7.71 (mc, 2 H, Fmoc-aryl-H), 7.43 (t, J = 7.8 Hz, 2 H,
4
3
Fmoc-aryl-H), 7.36 (dt, J = 1.1 Hz, J = 7.4 Hz, 2 H, Fmoc-aryl-
3
H), 4.80 (br s, 1 H, H-1), 4.55 (dd~t, JH–aCys,H–baCys = 6.1 Hz,
3JH–aCys,H–bbCys = 6.1 Hz, 1 H, H-aCys), 4.45 (mc, 2 H, CHH-Fmoc),
4.28 (dd~t, 3JH–aSer,H–baSer = 6.7 Hz, 3JH–aSer,H–bbSer = 6.7 Hz, 1 H, H-
N,N¢-(N-{[4-(a-D-Mannopyranosyloxy)phenyl]acetyl}-S-[3-(a-
D-mannopyranosyloxy)propyl]-l-cysteinyl)-L-cystine-di[2-(a-D-
mannopyranosyloxy)ethyl]amide (30)
3
a
Ser), 4.27–4.24 (m, 1 H, CHCH2-Fmoc), 3.87 (dd, J5,6a = 2.3 Hz,
2J6a,6b = 11.8 Hz, 1 H, H-6a), 3.84 (dd, 3J1,2 = 1.8 Hz, 3J2,3 = 3.4 Hz,
3
1 H, H-2), 3.80 (mc, 1 H, OCHHCH2), 3.74 (dd, J5,6b = 5.6 Hz,
Disulfide 24 (8.60 mg, 13.2 mmol) and TCEP (38.0 mg, 133 mmol)
were dissolved in degassed phosphate buffer (0.2 M, pH 8.5, 12
mL), the pH was adjusted to 7.3 by addition of 1 M NaOH solution
and the slightly contaminated divalent thioester 18 (9.00 mg, ob-
tained from 23.2 mmol 17) was added under a nitrogen atmosphere.
Benzyl mercaptan (260 mL) and thiophenol (520 mL) were added
and the reaction mixture was stirred for 47 h at r.t. The solution was
extracted with Et2O (3 × 10 mL) and the aqueous phase was lyophi-
lised. The solid residue was pre-purified by GPC (LH-20, DMF)
yielding a mixture of the target disulfide 30 and the corresponding
thiol 29. Fractions containing 29 or 30 were combined and oxidized
by standing in air for 6 d. Subsequent purification by GPC (LH-20,
H2O) led to a white lyophilisate, which was dissolved in MeOH
(500 mL). The formed precipitate was filtered off and the filtrate was
concentrated to yield the pure disulfide 30.
2J6a,6b = 11.8 Hz, 1 H, H-6b), 3.73 (dd, 3J2,3 = 3.4 Hz, 3J3,4 = 9.1 Hz,
3
1 H, H-3), 3.68–3.65 (m, 2 H, H-bSer), 3.64 (dd~t, J3,4 = 9.6 Hz,
3J4,5 = 9.6 Hz, 1 H, H-4), 3.60–3.52 (m, 2 H, H-5, OCHHCH2),
3.52–3.47 (m, 1 H, OCH2CHH), 3.37–3.32 (m, 1 H, OCH2CHH),
1.24 (s, 9 H, Boc-CH3).
13C NMR (151 MHz, CD3OD): d = 173.0 (COC-aSer), 172.0 (COC-
a
Cys), 158.7 (COO-Fmoc), 145.3, 142.6 (Fmoc-aryl-C), 128.8,
128.2, 126.2, 120.9 (Fmoc-aryl-CH), 101.7 (C-1), 75.0 [Boc-
C(CH3)3], 74.7 (C-5), 72.5 (C-3), 72.0 (C-2), 68.7 (C-4), 68.2 (CH2-
Fmoc), 66.9 (OCH2CH2), 62.9 (C-6), 62.7 (C-bSer), 57.3 (C-aCys),
57.0 (C-aSer), 48.4 (CHCH2-Fmoc), 40.5 (OCH2CH2), 27.7 (Boc-
CH3), 26.9 (C-bCys).
HRMS (ESI): m/z [M + Na]+ calcd for C33H45N3NaO11S: 714.2667;
found: 714.2623.
Yield: 4.70 mg (2.48 mmol, 19% based on 24, 11% based on 17); co-
N-{[4-(a-D-Mannopyranosyloxy)phenyl]acetyl}-L-cysteine-[2-
(a-D-mannopyranosyloxy)ethyl]amide (27)
lourless syrup.
1H NMR (600 MHz, CD3OD): d = 7.28–7.22 (m, 4 H, Ar-H), 7.07
(mc, 4 H, Ar-H), 5.46 (br s, 2 H, H-1¢), 4.78–4.72 (m, 4 H, H-1, H-
1¢¢), 4.65 (mc, 2 H, H-a¢Cys), 4.62–4.52 (m, 1 H, H-a¢¢Cys), 4.46
Disulfide 24 (29.0 mg, 44.6 mmol) and TCEP (115 mg, 400 mmol)
were dissolved in degassed phosphate buffer (0.2 M, pH 8.4, 10
mL), the pH was adjusted to 7.0 by addition of 1 M NaOH solution,
and thioester 12 (14.0 mg, 39.0 mol) was added. Benzyl mercaptan
(800 mL) and thiophenol (800 mL) were added and the reaction mix-
ture was stirred for 21.5 h at r.t. under a nitrogen atmosphere. The
solution was extracted with Et2O (3 × 10 mL) and the aqueous
phase was lyophilised. MeOH (10 mL) was added to the lyophili-
sate followed by ultrasound treatment to facilitate dissolution of thi-
ol 27. After filtration, this procedure was repeated twice. Then the
filtrate was concentrated under reduced pressure and the crude
product was purified by MPLC-GPC (Sephadex LH-20; MeOH)
and RP-MPLC (RP-18; A = MeOH, B = MeCN, 95 → 50% B,
50 min) followed by GPC on Sephadex LH-20 (MeOH) to yield 27.
3
3
(dd~t, JH–aCys,H–baCys = 7.1 Hz, JH–aCys,H–bbCys = 7.1 Hz, 1 H, H-
Cys), 4.00 (br s, 2 H, H-2¢), 3.92–3.88 (m, 2 H, H-3¢), 3.86–3.79 (m,
a
8 H, H-6a, H-6¢¢a, H-2, H-2¢¢), 3.78–3.67 (m, 18 H,
OCHHCH2CH2S, OCHHCH2N, H-4¢, H-6b, H-6b¢¢, H-6a¢, H-6b¢,
H-3, H-3¢¢), 3.64–3.45 (m, 18 H, H-4, H-4¢¢, H-5¢, CH2CONH, H-5,
H-5¢¢, OCHHCH2CH2S, OCHHCH2N), 3.43–3.18 (m, 6 H,
OCH2CH2N, H-ba¢Cys), 3.05–2.87 (m, 4 H, H-baCys, H-bb¢Cys), 2.82
3
2
(dd, JH–aCys,H–bbCys = 8.1 Hz, JH–baCys,H–bbCys = 13.5 Hz, 2 H, H-
bbCys), 2.63 (mc, 4 H, OCH2CH2CH2S), 1.84 (mc, 4 H,
OCH2CH2CH2S).
13C–1H HSQC NMR (600 MHz, CD3OD): d = 131.2/7.26 (Ar-CH),
117.8/7.07 (Ar-CH), 101.1/4.76 (CH-1, CH-1¢¢), 100.1/5.46 (CH-
1¢), 74.9/3.61 (CH-5¢), 74.5/3.54 (CH-5, CH-5¢¢), 72.3/3.72 (CH-3,
CH-3¢¢), 72.1/3.90 (CH-3¢), 71.9/3.82 (CH-2, CH-2¢¢), 71.7/4.00
(CH-2¢), 68.6/3.61 (CH-4, CH-4¢¢), 68.2/3.75 (CH-4¢), 66.7/3.78–
Yield: 11.5 mg (18.5 mmol, 47%); colourless syrup.
1H NMR (500 MHz, CD3OD): d = 7.25 (d, 3J = 8.7 Hz, 2 H, Ar-H),
7.08 (d, 3J = 8.7 Hz, 2 H, Ar-H), 5.46 (d, 3J1¢,2¢ = 1.9 Hz, 1 H, H-1¢),
4.76 (d, 3J1,2 = 1.6 Hz, 1 H, H-1), 4.44 (dd, 3JH–aCys,H–baCys = 5.9 Hz,
3JH–aCys,H–bbCys = 7.0 Hz, 1 H, H-aCys), 4.00 (dd, 3J1¢,2¢ = 1.8 Hz,
3J2¢,3¢ = 3.4 Hz, 1 H, H-2¢), 3.89 (dd, 3J2¢,3¢ = 3.4 Hz, 3J3¢,4¢ = 9.5 Hz,
3.72
(OCHHCH2CH2S,
OCHHCH2N),
66.7/3.53–3.48
(OCHHCH2CH2S, OCHHCH2N), 62.8/3.84 (CH-6a, CH-6¢¢a),
62.6/3.74–3.71 (CH-6b, CH-6b¢¢, CH2–6¢), 54.9/4.46 (CH-a¢Cys),
54.4/4.57 (CH-a¢¢Cys), 54.0/4.64 (CH-aCys), 42.5/3.56 (CH2CONH),
41.1/3.29–3.18 (CHH-b¢Cys), 41.1/2.95 (CHH-b¢Cys), 40.3/3.35–
3.32 (OCH2CH2N), 34.1/2.98 (CHH-bCys), 34.1/2.83 (CHH-bCys),
30.3/1.84 (OCH2CH2CH2S), 29.7/2.63 (OCH2CH2CH2S).
3
3
1 H, H-3¢), 3.85 (dd, J5,6a = 2.2 Hz, J6a,6b = 10.7 Hz, 1 H, H-6a),
3
3
3.82 (dd, J1,2 = 1.8 Hz, J2,3 = 3.4 Hz, 1 H, H-2), 3.77–3.65 (m,
6 H, OCHHCH2, H-4¢, H-6b, H-6a¢, H-6b¢, H-3), 3.55 (s, 2 H,
CH2CONH), 3.61–3.50 (m, 4 H, H-5¢, H-4, H-5, OCHHCH2), 3.41
(mc, 2 H, OCH2CH2), 2.87 (dd, 3JH–aCys,H–baCys = 5.8 Hz, 2JH–baCys,H–bbCys
=
MS (MALDI-TOF, CCA): m/z [M
+
Na]+ calcd for
3
13.6 Hz, 1 H, H-baCys), 2.78 (dd, JH–aCys,H–bbCys = 7.2 Hz,
C74H116N6NaO42S4: 1911.58; found: 1911.53.
2JH–baCys,H–bbCys = 13.6 Hz, 1 H, H-bbCys).
13C NMR (125 MHz, CD3OD): d = 174.4 (CONH), 172.4 (COC-
a
Cys), 157.0 (Ar-C), 131.4 (Ar-CH), 130.5 (Ar-CH), 118.0 (Ar-C),
Supporting Information for this article is available online at
101.7 (C-1), 100.2 (C-1¢), 75.3 (C-5¢), 74.8 (C-5), 72.5 (C-3), 72.4
(C-3¢), 72.1 (C-2), 72.0 (C-2¢), 68.8 (C-4), 68.3 (C-4¢), 67.0
(OCH2CH2), 63.0 (C-6), 62.6 (C-6¢), 57.2 (C-aCys), 42.8
(CH2CONH), 40.4 (OCH2CH2), 26.9 (C-bCys).
http://www.thieme-connect.com/ejournals/toc/synthesis.
Acknowledgment
HRMS (ESI): m/z [M + Na]+ calcd for C25H38N2NaO14S: 645.1936;
found: 645.2093.
We thank Christiana Albertina University (CAU) and Evonik Foun-
dation (stipend for J.W.W.) for financial support and Dr. Frank
Sönnichsen for NMR experiments with the shift reagent Eu(hfc)3.
Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York

3082
J. W. Wehner, T. K. Lindhorst
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Synthesis 2010, No. 18, 3070–3082 © Thieme Stuttgart · New York