Cysteine-based mannoside glycoclusters: Synthetic routes and antiadhesive properties

Article abstract of DOI:10.1002/ejoc.201000185

Clustermannosides of different valency were synthesised based on cysteine. By employing the orthogonally protected amino acid as scaffold molecule, a variety of structurally varied products can be obtained according to different synthetic routes. Testing of the prepared glycoclusters as inhibitors of type 1 fimbriae mediated bacterial adhesion is reported, giving hints about the influence of sugar valency, sugar scaffolding, and the nature of the glycosidic aglycon in this testing system.

Full text of DOI:10.1002/ejoc.201000185

FULL PAPER
DOI: 10.1002/ejoc.201000185
Cysteine-Based Mannoside Glycoclusters: Synthetic Routes and Antiadhesive
Properties
Alexander Schierholt,[a] Mirja Hartmann,[a] Kirsten Schwekendiek,[a] and
Thisbe K. Lindhorst*[a]
Keywords: Glycoclusters / Cysteine scaffold / Glycopeptides / Cell adhesion / Type 1 fimbriae
Clustermannosides of different valency were synthesised routes. Testing of the prepared glycoclusters as inhibitors of
based on cysteine. By employing the orthogonally protected type 1 fimbriae mediated bacterial adhesion is reported, giving
amino acid as scaffold molecule, a variety of structurally varied hints about the influence of sugar valency, sugar scaffolding,
products can be obtained according to different synthetic and the nature of the glycosidic aglycon in this testing system.
Introduction
Molecular recognition of carbohydrates is associated
with numerous processes in cell biology, comprising cell ad-
hesion and cell communication, signaling, inflammation,
and various disease states, such as i.a. cancer and metasta-
sis.[1] Because of the enormous structural diversity of the
carbohydrates and the manyfold aspects of their supra-
molecular assembly on eukaryotic cell surfaces it is a major
challenge to understand and investigate the molecular de-
tails of the mechanisms underlying glycobiology. In this
context multivalent glycoconjugates and glycomimetics
have been developed into a valuable class of molecular
tools. Multivalent glycomimetics, such as glycodendrimers
and glycoclusters, provide means to mimic and vary some
of the complexity of the carbohydrate regime in order to
study multivalency effects in carbohydrate–protein interac-
tions and other binding effects in biological tests.[2]
Glycoproteins and glycopeptides have been shown to be
involved in many important molecular recognition pro-
cesses.[3] Consequently, synthetic glycopeptides, glycoamino
acids as well as glycopeptide clusters have been designed
and synthesised and were studied as ligands of carbo-
hydrate-binding proteins such as the lectins.[4] Here a new
approach is reported in which the amino acid cysteine is
used as scaffold molecule (Figure 1). Both, the cysteine
amino function and its carboxylic group can be addressed
in peptide coupling reactions by employing suitably func-
tionalised carbohydrates to give rise to divalent glycosides
Figure 1. The amino acid cysteine can be used as scaffold molecule
for the synthesis of glycoclusters with different valencies. When the
carboxy group and the amino group of cysteine are addressed in
[a] Otto Diels Institute of Organic Chemistry, Christiana Albertina
peptide coupling reactions with appropriately functionalised glyco-
University of Kiel,
sides, divalent glycoamino acids of type I can be obtained. A subse-
Otto-Hahn-Platz 4, 24098 Kiel, Germany
quent nucleophilic displacement step, involving the cysteine mer-
Fax: +49-431-8807410
E-mail: tklind@oc.uni-kiel.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201000185.
capto function, affords trivalent glycoclusters of type II. In ad-
dition, glycocysteine derivatives of type I enclose the option to ac-
cess the respective tetravalent disulfide dimers of type III.
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Cysteine-Based Mannoside Glycoclusters
of type I. The sugar valency of the cysteine-based glycoclus-
ters can be increased by functionalisation of the cysteine
mercapto function, leading to type II glycoclusters, or by
disulfide formation to achieve dimerised glycoclusters of
type III. For a first evaluation of their ligand properties,
mannose was employed in this account, and the respective
cysteine-based mannose clusters were tested with type 1-
fimbriated Eschericha coli bacteria.[5]
Results and Discussion
The synthesis of trivalent cluster mannosides of type II
(Figure 1) was started by using O-protected mannosides.
According to a synthetic route, which was published ear-
lier,[6] the 2-azidoethyl mannoside 1, which is available from
mannose in three simple steps,[7] was conjugated to the or-
thogonally protected -cysteine derivative Fmoc-Cys(Trt)-
OH (2) in a Staudinger ligation. This led to the protected
glycoamino acid 3 in high yield (Scheme 1). Then, cleavage
of the Fmoc protecting group gave amine 4, which was
ready for the next peptide coupling step by using an appro-
priate carboxylic acid derivative such as the mannoside 9.
This was synthesised in three steps by starting with (4-hy-
droxyphenyl)acetic acid (5). Acid-catalysed treatment with
allyl alcohol led to the allyl ester 6,[8] which was treated
with the mannose pentaacetate 7 in a Lewis acid catalysed
glycosylation reaction to afford the fully protected manno-
side 8. The allyl ester protecting group was cleaved chemo-
selectively after isomerisation of the double bond to lead to
the desired O-acetylated carboxylic acid mannoside 9. This
was subjected without extensive purification to peptide cou-
pling with the amine 4 by using 2-(7-aza-1H-benzotriazol-
Scheme 1. Synthesis of the bis(mannosidic) -cysteine derivative 11.
Reagents and conditions: (a) DIC, HOBt, nBu3P, THF, 0 °C Ǟ
room temp., overnight, 78%; (b) morpholine, DMF, room temp.,
1.5 h, 83%; (c) allyl alcohol, H2SO4, 6 h, 100 °C, 84%; (d) BF3–
Et2O, CH2Cl2, 0 °C Ǟ room temp., overnight, 62%; (e) Pd-
(PPh3)4, morpholine, THF, room temp., 3 h, 69%; (f) HATU, DI-
PEA, DMF, 0 °C Ǟ room temp., overnight, 60%; (g) TFA/CH2Cl2
(1:1), TES, room temp., 2 h, quant.
1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
(HATU) and diisopropyl(ethyl)amine (DIPEA) to afford
the divalent glycoamino acid 10 in good yield. For subse-
quent functionalisation of the mercapto function deprotec-
tion of the S-trityl protecting group was performed by using
TFA[9] to yield the thiol 11 in a quantitative reaction.
In order to achieve a trivalent cluster glycoside, an ap-
propriate mannoside was seeked to react with thiol 11 ac- protection is dispensable. Thus, the OH-free 2-aminoethyl
cording to a Williamson-type thioether synthesis. Thus, p- mannoside 17[12] was employed in a peptide coupling reac-
cresol was mannosylated by employing the mannosyl tri- tion with the cysteine derivative 2 (Scheme 3). Standard
chloroacetimidate 12 as the glycosyl donor.[10] The Lewis conditions by using HATU and DIPEA led to the desired
acid catalysed stereospecific reaction gave the α-glycoside glycoamino acid 18 in very good yield. Removal of the
13 in high yield, which was subsequently subjected to radi- Fmoc protecting group gave the free amine 19, which was
cal bromination by using azobis(isobutyronitrile) (AIBN) further treated in two different reactions. Firstly, chemose-
and dimethyldibromohydanthoin (DBDMH) to result in lective N-acetylation was performed to achieve the model
the benzyl bromide 14 (Scheme 2). The subsequent nucleo- compound 20, which was used to elaborate thiol deprotec-
philic displacement reaction with 11 proceeded in accept- tion in order to obtain the target disulfide glycoclusters.
able yields when ultrasound was applied; no de-O-acetyl- Removal of the S-trityl protection group and concurrent
ation was observed in this step. The pure protected trivalent oxidation to the disulfide dimer 21 was possible in only
glycocluster 15 was obtained after laborious gel permeation 15 min by using iodine in methanol.[13] Purification of 21
chromatography (GPC). Deprotection under Zemplén con- by silica gel chromatography was very convenient in this
ditions[11] gave the unprotected trivalent cluster mannoside case. Enouraged by these promising results, synthesis of the
16.
tetravalent glycocluster disulfide 24 was tackled next. The
For the preparation of cluster glycosides of type III (cf. amine 19 was conjugated to the carboxy-functionalised
Figure 1), a synthetic approach was envisioned in which O- mannoside 22,[14] again by using standard peptide coupling
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A. Schierholt, M. Hartmann, K. Schwekendiek, T. K. Lindhorst
FULL PAPER
conditions, to yield the S-trityl-protected bis(mannoside) 23
in moderate yield. Application of the approved oxidising
deprotection conditions, iodine in methanol, indeed led to
the cluster mannoside 24. However, in this case, purification
was problematic and required a combination of reverse-
phase medium-pressure chromatography (RP-MPLC) and
subsequent GPC, thus lowering the yield considerably.
Testing Ligand Properties
It was shown that structurally varied glycoclusters can be
easily obtained based on cysteine as the scaffold molecule.
For a first evaluation of their properties as lectin ligands, a
collection of the herein described cluster mannosides were
tested as inhibitors of type 1-fimbriated bacterial adhesion
by employing uropathogenic Escherichia coli cells.[5] This
important adhesion system is well described, and the X-
ray structure of the participating lectin, FimH, has been
solved.[15] It has been shown that sugar valency of a carbo-
hydrate ligand and the nature of the glycosidic aglycon moi-
eties have an influence on type 1 fimbriae mediated bacte-
rial binding.[16] Therefore, the mono- to tetravalent glyco-
clusters and mannosides 22, 21, 16, and 24 were checked
for their inhibitory potencies in an inhibition adhesion as-
say. Serial dilutions of the respective inhibitor were incu-
bated with fluorescent E. coli cells in mannan-coated 96-
well microtiter plates.[17] Inhibition curves were determined
for each tested glycocluster, from which IC50 values were
deduced. IC50 values in this case reflect the inhibitor con-
centration, which causes 50% inhibition of bacterial bind-
ing to mannan. On each individual test plate methyl α--
mannoside (MeMan), which is typically used as standard
inhibitor in this assay, was tested in parallel. Hence, the
IC50 values obtained for the tested glycoclusters could be
Scheme 2. Synthesis of the trivalent cluster mannoside 16. Reagents
and conditions: (a) TMSOTf, CH2Cl2, room temp., overnight,
81%; (b) AIBN, DBDMH, benzene, hν, 1.5 h, reflux, 80%; (c)
NaH, NaI, DMF, ultrasound, room temp., 3 h, 34%; (d) NaOMe,
MeOH, room temp., 3 h, 98%.
Scheme 3. Synthesis of disulfide glycoclusters 21 and 24. Reagents and conditions: (a) 2, HATU, DIPEA, DMF, 0 °C Ǟ room temp.,
overnight, 85%; (b) morpholine, DMF, room temp., quant.; (c) Ac2O, DIPEA, room temp., 4 h, quant.; (d) I2, MeOH, room temp.,
15 min, 60% for 21, 39% for 24; (e) HATU, DIPEA, DMF, 0 °C Ǟ room temp., overnight, 45%.
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Cysteine-Based Mannoside Glycoclusters
referenced to the IC50 value that was determined for Me- of that of 16 [RIP(24) = 63]. It can be assumed that, in
Man on the same plate leading to a relative inhibitory po- addition to the effect of scaffolding, also the carbohydrate
tency (RIP) for every tested compound. Thus, RIP values environment of a glycosidic moiety effects its ligand proper-
allow to compare the inhibitory potencies of all tested in- ties. This observation might be of considerable importance
hibitors, even when they were not examined in the same in a natural adhesion system and needs further investiga-
experiment, because, they are consistently referenced.
Testing results are collected in Figure 2. In addition to
tion.
The divalent cluster mannoside 21, lacking any aromatic
the measured IC50 values and obtained RIP values, valency- aglycon moiety, showed only a slightly improved inhibitory
corrected RIPvc values are depicted, in order to assess the potency when compared to MeMan. This finding can be
inhibitory potency of a compound irrespective of its man- assigned to a statistical multivalency effect, which is, how-
noside valency. This approach translates the RIP value of 6 ever, not very pronounced in this case. Overall, the aromatic
measured for the divalent cluster mannoside 21 into a RIPvc nature of the linker moieties in the tested mannosides seem
value of 3, for example.
to exert a dominant effect on receptor binding, much more
than mannoside valency. Thus, for judgment of the valency-
corrected RIP values (RIPvc) the ratio of aromatic to ali-
phatic aglycon residues in the respective cluster mannoside
should be taken into account. The trivalent cluster mannos-
ide 16, which performed best in the testing system, can
profit from a combination of ligand multivalency and a
high contribution of two phenyl moieties to π–π stacking
stabilisation of the lectin–ligand complex. In the higher-va-
lent cluster 24, on the other hand, only 50% of all aglycon
linkers are aromatic, thus its RIPvc is significantly lowered.
Conclusions
It has been especially difficult to design high-affinity
carbohydrate ligands for lectin proteins.[18] This research
field is complicated by a number of effects, which are affili-
ated with weak binding and multivalency. In addition to the
configuration of the glycone part of a glycosidic ligand, the
aglycon moiety can contribute to binding and, moreover,
the aglycon can also affect the conformational properties of
a given ligand such as its overall flexibility and its mini-
mum-energy conformation. A multitude of published stud-
ies[16,19] as well as the herein described work on carbo-
hydrate binding of type 1-fimbriated E. coli have shown,
that also in this testing system, which seems to be ruled
by the monovalent α--mannoside-specific fimbrial lectin
FimH, multivalency makes a difference, and even more the
Figure 2. Inhibitory potencies of mannoside 22 and the di- to tetra-
valent clusters 21, 16, and 24 obtained from triplicate testing. The
relative inhibitory potencies (RIP) are referenced to the IC50 value
obtained for an individual MeMan standard on the same test plate,
with RIP(MeMan) ϵ 1 (see Exp. Sect.). RIPvc = valence corrected
RIP; SD = standard deviation.
To interpret the obtained testing results, two important nature of the mannosidic aglycon moieties and mannoside
features of the inhibitor molecules must be considered, scaffolding are important. With respect to the investigation
namely their mannose valency and the properties of the ag- of these effects and their meaning in type 1 fimbriae medi-
lycon moieties that link a mannosidic moiety to the cysteine ated bacterial adhesion the current study paves the way to
scaffold. The monovalent mannoside 22 gave an IC50 value a future project by highlighting some effects of structural
of 70 µ, corresponding to an inhibitory potency, which is variation of cysteine-scaffolded cluster mannosides on the
38 times higher than that of the reference compound Me- inhibitory potency of the respective glycoclusters.
Man. The elevated inhibitory potency can be attributed to
Moreover, a synthetic concept was introduced, which is
π–π stacking interactions of the phenyl aglycon with two suited to easily vary carbohydrate valency and aglycon
tyrosine residues flanking the entrance of the bacterial ad- properties of cluster glycosides, which are of interest for
hesin FimH, an effect that is known and expected.[15,16] biological testing. Carbohydrate clustering on a cysteine
When two phenyl mannoside moieties were scaffolded in scaffold was shown to even work with unprotected mannos-
one molecule such as in the case of 16 and 24, different ides, and in addition, this project can be easily extended
effects were seen. Whereas in the case of 16 the RIP value towards the synthesis of glycoclusters of a “mixed” type[20]
went up to ca. 180, the potency of the tetravalent cluster and to solid-phase synthesis. It is obvious that the exem-
mannoside 24 as inhbitor of type 1 fimbriae mediated bac- plified approach can be used as versatile tool box for a rela-
terial adhesion was weaker, only reaching about one third tively rapid access to variable synthetic lectin ligands.
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A. Schierholt, M. Hartmann, K. Schwekendiek, T. K. Lindhorst
FULL PAPER
General Procedure for Disulfide Formation: The protected thiol
(1 equiv.) was dissolved in dry MeOH (2 mL), and a solution of
iodine (1.5 equiv. in 5 mL dry MeOH) was added. The reaction
mixture was stirred at room temperature for 15 min and then
quenched by addition of sodium thiosulfate solution (3 mL, 0.01 
in MeOH), filtered and concentrated under reduced pressure. The
crude product was purified by silica gel chromatography.
Experimental Section
General: All solvents were distilled prior to use. Commercially
available starting materials, reagents and anhydrous DMF were
used without further purification unless otherwise noted. Air- and/
or moisture-sensitive reactions were carried out under nitrogen.
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 treatment. Flash chromatography was performed on silica gel
60 (230–400 mesh, particle size 0.040–0.063 mm, Merck). Prepara-
tive MPLC was performed with an apparatus of BÜCHI Labor-
technik GmbH by using a LiChroprep RP18 (40–60 µm, Merck)
column for reversed-phase silica gel chromatography. 1H and 13C
NMR spectra were recorded with Bruker DRX-500 and AV-600
General Procedure for Deacetylation: The acetylated compound was
dissolved in dry methanol (10 mL/g), and sodium methoxide
(100 µL, 1  in MeOH) was added under nitrogen. The reaction
mixture was stirred at room temperature until the reaction was
complete (minimum 3 h, maximum overnight), then it was neutral-
ised by the addition of Amberlite IR 120 (H+), filtered, and the
solution was concentrated under reduced pressure. The crude prod-
uct was purified by chromatography on normal-phase or RP silica
gel.
spectrometers. 2D NMR techniques (1H-1H COSY, H-13C HSQC
1
1
and H-13C HMBC) were used for full assignment of the spectra.
Chemical shifts were reported relative to the following shifts:
CHCl3 (internal tetramethylsilane: 1H: δ = 0.00 ppm; 13C: δ =
77.00 ppm), MeOH (1H: δ = 3.31 ppm; 13C: δ = 49.05 ppm) or H2O
(δ = 4.65 ppm). For signal assignment, nuclei of the sugar ring,
which are coupled to the cysteine amino group are primed, those
of the glycoside ligated to the thiol function are double-primed.
ESI-MS measurements were recorded with a Mariner ESI-ToF
5280 (Applied Biosystems) instrument and MALDI-MS measure-
ments with a MALDI-ToF-MS Biflex III (Bruker) instrument. Op-
tical rotations were measured with a Perkin–Elmer 241 polarimeter
(NaD line: 589 nm; length of cell: 1 dm).
General Procedure for Fmoc Deprotection: The Fmoc-protected
amine (1 equiv.) and morpholine (6 equiv.) were dissolved in DMF
(10 mL) and stirred at room temperature for 4 h. Then, the solvent
was removed under reduced pressure and the crude product dis-
solved in MeOH. The white precipitate formed was filtered off and
the solvent removed under reduced pressure to give the desired de-
protected amine.
N-[2-(2,3,4,6-Tetra-O-acetyl-α-
D-mannopyranosyloxy)ethyl]-S-(tri-
phenylmethyl)- -cysteinamide (4): According to the general pro-
L
cedure for Fmoc deprotection, mannoside 3 (3.93 g, 4.10 mmol)
and morpholine (2.63 mL, 30.1 mmol) were allowed to react in dry
DMF (10 mL) for 1.5 h. The product 4 (2.40 g, 83%) was obtained
after silica gel chromatography (EtOAc/toluene, 1:1) as a pale yel-
Bacteria Culture: E. coli bacteria of strain PKL1162 were grown in
LB media + AMP + CAM (100 mg of ampicillin, 50 mg of chlor-
amphenicol/L) at 37 °C under slight agitation.
1
low syrup. H NMR (600 MHz, [D4]MeOH): δ = 7.47–7.44 (m, 6
3
3
GFP-Based Bacterial Adhesion Assay:[17] Black (Thermo Fisher
Scientific, Nunc Maxisorp) 96-well plates were filled with a solu-
tion of mannan from Saccharomyces cerevisiae (1.2 mg/mL in car-
bonate buffer, pH = 9.5; 100 µL solution per well) and allowed
to dry in at 37 °C overnight. The plates were washed with PBST
(3ϫ150 µL/well) and stored at 4 °C. Before use, the wells were
blocked with BSA (5% in PBS, 120 µL/well) at 37 °C for 2 h and
then washed with PBST (3ϫ150 µL/well). Serial dilutions of the
examined inhibitor (14, 21, 22, or 24) were prepared in the man-
nan-coated, BSA-blocked 96-well plates. The bacteria suspension
(2 mg/mL in PBS buffer, pH = 7.2; 50 µL solution per well) was
added, and the plates were agitated (80 rpm) and incubated at
37 °C for 45 min. After washing with PBS (3ϫ150 µL/well), the
wells were filled with PBS (100 µL/well), and the fluorescence in-
tensity (485 nm/535 nm) was determined. The percentage inhibition
was calculated as {[F(nI) – F(I)]ϫ100ϫ[F(nI)]–1} (F: fluorescence;
nI: no inhibitor, I: with inhibitor). All assays were performed by
using triplicate samples of each concentration. Relative inhibitory
potencies (RIPs) are based on the average IC50 value of duplicate
MeMan samples on the same plate [RIP(MeMan) ϵ 1], and the
RIP values reported in Figure 2 are average values of three RIPs.
LB: lysogeny broth; rpm: revolutions per minute; PBS: phosphate-
buffered saline; PBST: PBS + 0.05% Tween 20.
H, Ar-H), 7.34 (t, J = 7.4 Hz, 6 H, Ar-H), 7.27 (t, J = 7.3 Hz, 3
3
3
3
H, Ar-H), 5.33 (dd, J = 3.4, J = 10.0 Hz, 1 H, 3-H), 5.30 (dd, J
3
3
= 3.4, J = 1.7 Hz, 1 H, 2-H), 5.29 (dd ≈ t, J = 10.0 Hz, 1 H, 4-
H), 4.92 (d, 3J = 1.7 Hz, 1 H, 1-H), 4.27 (dd, 3J = 4.9, 2J = 12.3 Hz,
1 H, 6a-H), 4.11 (dd, 3J = 2.3, 2J = 12.3 Hz, 1 H, 6b-H), 4.07 (ddd,
3J = 2.3, 3J = 4.9, 3J = 10.0 Hz, 1 H, 5-H), 3.81 (mc, 1 H,
OCHHCH2), 3.73–3.54 (m, 3 H, OCHHCH2, CH2CH2N), 3.20–
3
2
3.15 (m, 1 H, α-H), 2.61 (dd, J = 6.5, J = 12.2 Hz, 1 H, βa-H),
3
2
2.45 (dd, J = 6.5, J = 12.2 Hz, 1 H, βb-H), 2.13, 2.06, 2.04, 2.00
(each s, each 3 H, 4 OAc) ppm. 13C NMR (150 MHz, [D4]MeOH):
δ = 171.3 (COC-α), 170.1, 169.9, 169.4, 169.2 (4 COCH3), 96.7 (C-
1), 68.6 (C-3), 68.3 (C-2), 67.9 (C-5), 67.2 (OCH2CH2), 65.6 (C-4),
62.2 (C-6), 53.6 (C-α), 39.8 (CH2CH2N), 32.9 (C-β), 20.6, 20.5,
20.5, 20.4 (4 COCH3) ppm. HRESI-MS: calcd. for [C38H44N2O11S
+ H]+ 737.2744; found 737.2739.
Allyl [4-(2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyloxy)phenyl]-
acetate (8): The allyl ester 6[8] (4.06 g, 21.1 mmol) and mannose
pentaacetate 7 (4.13 g, 10.8 mmol) were combined in a Schlenk
flask and dried in vacuo for more than 70 min. This mixture was
dissolved in dry CH2Cl2 (7 mL), and BF3–Et2O (159 µL,
23.5 mmol) was added at 0 °C. The mixture was stirred at 0 °C for
30 min and then stirred at room temperature overnight. Ice-cold
water (100 mL) was added and the mixture extracted with CH2Cl2
(3 ϫ 30 mL). The combined organic phases were subsequently
washed with aq. NaHCO3 and water (40 mL each), dried with
MgSO4, filtered, and the filtrate was concentrated under reduced
pressure. The crude product was purified by silica gel chromatog-
raphy (cyclohexane/EtOAc, 3:2) to yield 8 (3.50 g, 62 %) as a
General Procedure for Peptide Coupling: The carboxy-function-
alised mannoside (1–1.2 equiv.) and HATU (1.1–1.2 equiv.) were
dissolved in dry DMF under nitrogen and cooled to 0 °C. To this
solution DIPEA (1.1–1.2 equiv.) was added, and the mixture was
shaken for 2 min. Then the amine (1.4–2 equiv.), dissolved in dry
DMF (4–10 mL), was added dropwise. After 4 h, the cooling was colourless syrup. 1H NMR (600 MHz, CDCl3): δ = 7.22 (d, 3J =
removed and the reaction mixture stirred at room temperature
overnight. The solvent was removed under reduced pressure and
the crude product purified by silica gel chromatography.
6.6 Hz, 2 H, Ar-H), 7.05 (3J = 6.6 Hz, 2 H, Ar-H), 5.90 (dddd ≈
3
ddt, 3J = 5.7, 3J = 10.4, J = 17.2 Hz, 1 H, CH2CHCH2), 5.56 (dd,
3
3
3J = 3.5, J = 10.0 Hz, 1 H, 3-H), 5.50 (d, J = 1.9 Hz, 1 H, 1-H),
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Cysteine-Based Mannoside Glycoclusters
5.44 (dd, 3J = 1.9, 3J = 3.5 Hz, 1 H 2-H), 5.35 (dd ≈ t, 3J = 10.0 Hz, 3J = 1.7 Hz, 1 H, 1-H), 5.44 (dd, J = 1.9, J = 3.5 Hz, 1 H, 2-H),
3
3
1 H, 4-H), 5.28 (dddd ≈ dq, 4J = 1.4, 3J = 10.4, 3J = 17.2 Hz, 1
H, CH2CHCHH), 5.22 (dddd ≈ dq, 4J = 1.4, 3J = 10.4 Hz, 1 H,
5.36 (dd ≈ t, 3J = 10.1 Hz, 1 H, 4-H), 4.28 (dd, 3J = 5.2, 2J =
3 3 3
12.1 Hz, 1 H, 6a-H), 4.11 (ddd, J = 2.3, J = 5.2, J = 10.1 Hz, 1
3
2
CH2CHCHH), 4.65–4.53 (m, 2 H, CH2CHCH2), 4.28 (dd, 3J = H, 5-H), 4.07 (dd, J = 2.3, J = 12.1 Hz, 1 H, 6b-H), 2.30, 2.19,
2
5.2, J = 12.2 Hz, 1 H, 6a-H), 4.15–4.10 (m, 1 H, 5-H), 4.07 (dd,
3J = 2.4, 2J = 12.2 Hz, 1 H, 6b-H), 3.60 (s, 2 H, OCH2C-Ar), 2.05,
2.04, 2.03, 2.03 (each s, each 3 H, 4 OAc) ppm. 13C NMR
(150 MHz, CDCl3): δ = 171.1, 170.5, 169.9, 169.9, 169.7 (4
2.05, 2.04, 2.03 (each s, each 3 H, 4 OAc, CH3-Ar) ppm. 13C NMR
(150 MHz, CDCl3): δ = 170.6, 170.0, 170.0, 169.8 (4 COCH3),
153.5, 132.5, 130.0, 116.4 (C-Ar), 96.0 (C-1), 65.3, 69.0, 68.9, 60.0
(C-2, C-3, C-4, C-5), 62.2 (C-6), 20.9, 20.7, 20.7, 20.6 (4 COCH3,
COCH3, COOallyl), 154.8 (C-Ar), 132.0 (CH2CHCH2), 130.5, Ar-CH3) ppm. HRESI-MS: calcd. for [C21H26O10 + Na]+ 461.1424;
128.6 (C-Ar), 118.3 (CH2CHCH2), 116.6 (C-Ar), 95.9 (C-1), 69.4 found 461.1439.
(C-2), 69.2 (C-5), 68.9 (C-3), 66.0 (C-4), 65.4 (CH2CHCH2), 62.1
4-(Bromomethyl)phenyl 2,3,4,6-Tetra-O-acetyl-α-D-mannopyran-
(C-6), 40.4 (CH2COO), 21.0, 20.8, 20.6, 20.6 (4 COCH3) ppm.
HRESI-MS: calcd. for [C25H30O12 + Na]+ 545.1635; found
545.1629.
oside (14): Mannoside 13 (206 mg, 470 µmol), 1,3-dibromo-5,5-di-
methylhydantoine (DBDMH) (60 mg, 210 µmol) and AIBN (3 mg,
20 µmol) were dissolved in dry benzene (10 mL) under nitrogen.
The reaction mixture was refluxed for 90 min and simultaneously
irradiated with a 150 W tungsten lamp. The solvent was evapo-
rated, and the crude product was purified by flash chromatography
on silica gel (cyclohexane/EtOAc, 3:1). The title compound was
obtained as a colourless solid (194 mg, 80%). [α]2D0 = +56.8 (c =
N-[4-(2,3,4,6-Tetra-O-acetyl-α-
D-mannopyranosyloxy)phenyl]acetyl-
S-(triphenylmethyl)- -cysteine [2-(2,3,4,6-Tetra-O-acetyl-α-
L
D-man-
nopyranosyloxy)ethyl]amide (10): The allyl ester 8 (3.20 g,
6.12 mmol) and Pd(PPh3)4 (50 mg, 43 µmol) were dissolved in dry
THF (20 mL) under nitrogen. Morpholine (2.2 mL, 25.1 mmol)
was added, and the mixture was stirred at room temperature for
3 h. The solvent was evaporated, and the crude product was filtered
through a short silica gel column and washed (cyclohexane/EtOAc,
1:1) to give the carboxylic acid 9 (2.03 g, 69%). A portion of this
product (680 mg, 1.41 mmol) was treated with the amine 4 (865 mg,
1.17 mmol) by addition of HATU (535 mg, 1.40 mmol) and DI-
PEA (244 µL, 1.40 mmol) in DMF (4 mL) according to the general
coupling procedure. Silica gel column chromatography (CH2Cl2/
EtOAc, 1:1) gave the title compound 10 (857 mg, 60%) as a colour-
1
3
1.25 in CH2Cl2). H NMR (500 MHz, CDCl3): δ = 7.34 (d, J =
3
3
8.7 Hz, 2 H, Ar-H), 7.06 (d, J = 8.7 Hz, 2 H, Ar-H), 5.55 (dd, J
= 3.6, 3J = 10.0 Hz, 1 H, 3-H), 5.53 (d, 3J = 1.8 Hz, 1 H, 1-H),
5.43 (dd, 3J = 1.8, 3J = 3.6 Hz, 1 H, 2-H), 5.36 (dd ≈ t, 3J = 10.0 Hz,
3
2
1 H, 4-H), 4.48 (s, 2 H, CH2Br), 4.27 (dd, J = 5.8, J = 12.7 Hz,
1 H, 6a-H), 4.04–4.09 (m, 2 H, 5-H, 6b-H), 2.20, 2.05, 2.04, 2.03
(each s, each 3 H, 4 OAc) ppm. 13C NMR (500 MHz, CDCl3): δ =
170.6, 170.0, 170.0, 169.7 (4 COCH3), 155.5, 132.5, 130.5, 116.7
(C-Ar), 95.6 (C-1), 69.3, 69.2, 68.8, 65.9 (C-2, C-3, C-4, C-5), 62.1
(C-6), 33.1 (CH2Br), 20.9, 20.7, 20.7, 20.7 (4 COCH3) ppm.
HRESI-MS: calcd. for [C21H25BrO10 + Na]+ 539.0523, 541.0506;
found 539.0468, 541.0482.
1
less foam. [α]2D0 = +44.2 (c = 1.1 in MeOH). H NMR (600 MHz,
[D4]MeOH): δ = 7.38–7.35 (m, 6 H, Ar-H), 7.34–7.28 (m, 9 H, Ar-
H), 7.26 (d, 3J = 7.3 Hz, 2 H, Ar-H), 7.08 (d, 3J = 8.5 Hz, 2 H,
Ar-H), 5.57 (d, 3J = 1.8 Hz, 1 H, 1Ј-H), 5.50 (dd, 3J = 3.4, 3J =
10.0 Hz, 1 H, 3-H), 5.47 (dd, 3J = 1.8, 3J = 3.4 Hz, 1 H, 2Ј-H),
5.35 (dd ≈ t, 3J = 10.3 Hz, 1 H, 4Ј-H), 5.33 (dd, 3J = 3.4, 3J =
10.3 Hz, 1 H, 3Ј-H), 5.29 (dd, 3J = 1.6, 3J = 3.2 Hz, 1 H, 2-H),
N-[4-(2,3,4,6-Tetra-O-acetyl-α-
[4-(2,3,4,6-tetra-O-acetyl-α- -mannopyranosyl)benzyl]-
(2,3,4,6-Tetra-O-acetyl-α- -mannopyranosyloxy)ethyl]amide (15):
D
-mannopyranosyl)phenyl]acetyl-S-
D
L-cysteine [2-
D
The trityl-protected sulfide 10 (650 mg, 541 µmol) was dissolved in
dry CH2Cl2 (5 mL) under nitrogen. Then triethylsilane (0.41 mL,
2.5 mmol) and trifluoroacetic acid (190 µL, 2.5 mmol) were added,
and the reaction mixture was stirred at room temperature for 2 h.
The solvent was evaporated, and the crude product was purified
on a short silica gel column (CH2Cl2/EtOAc, 1:1) to give the de-
sired product 11 (512 mg, 99%). A portion of this thiol 11 (100 mg,
104 µmol) and sodium hydride (2.5 mg, 0.1 mmol) were suspended
in dry DMF (5 mL) and treated with ultrasound for 10 min. Then,
the benzyl bromide 14 (49 mg, 95 µmol) was added, and the reac-
tion mixture was stirred with ultrasound for further 3 h. The mix-
ture was concentrated under reduced pressure, and the crude prod-
uct was purified by GPC on Sephadex LH-20 (eluent: MeOH) to
yield the glycocluster 15 (45 mg, 34%) as a colourless solid. 1H
NMR (500 MHz, [D4]MeOH): δ = 7.31–7.28 (m, 4 H, ArЈЈ-H, ArЈ-
H), 7.12–7.08 (m, 4 H, ArЈЈ-H, ArЈ-H), 5.64 (br. s, 1 H, 1Ј-H), 5.54
3
5.25 (dd ≈ t, J = 10.0 Hz, 1 H, 4-H), 4.86 (br. s, 1 H, 1-H), 4.26–
4.22 (m, 2 H, 6a-H, α-H), 4.18 (dd, 3J = 4.2, 2J = 12.3 Hz, 1 H,
2
6Јa-H), 4.09–4.02 (m, 3 H, 6b-H, 5-H, 5Ј-H), 3.97 (dd, 3J = 2.5, J
= 12.3 Hz, 1 H, 6Јb-H), 3.78–3.73 (m, 2 H, OCH2CH2) 3.56–3.51
3
(m, 2 H, CH2C-Ar), 3.42–3.30 (m, 2 H, OCH2CH2), 2.59 (dd, J
2
= 6.0, J = 12.5 Hz, 1 H, βa-H), 2.52–2.46 (m, 1 H, βb-H), 2.13,
2.12, 2.05, 2.04, 1.99, 1.97 (each s, 24 H, 8 OAc) ppm. 13C NMR
(150 MHz, [D4]MeOH): δ = 173.6 (CH2CONH), 173.0 (α-CONH),
172.2, 171.6, 171.5, 171.0 (8 COCH3), 156.0, 145.9, 131.5, 131.4,
130.7, 129.1, 127.9, 117.9 (C-Ar), 98.9 (C-1), 97.1 (C-1Ј), 70.8 (C-
2), 70.6 (C-2Ј), 70.6 (C-5Ј), 70.5 (C-3), 70.5 (C-5), 70.4 (C-3Ј), 69.9
(C-4Ј), 69.8 (C-4), 67.9 (CPh3), 67.7 (OCH2CH2), 63.5 (C-6Ј), 63.2
(C-6), 53.8 (C-α), 42.8 (CH2CH2N), 40.3 (CH2C-Ar), 35.2 (C-β),
20.8, 20.7, 20.6, 20.5 (8 COCH3) ppm. HRESI-MS: calcd. for
[C38H44N2O11S + Na]+ 1223.3882; found 1223.3877.
4-Methylphenyl 2,3,4,6-Tetra-O-acetyl-α-D-mannopyranoside (13): (br. s, 1 H, 1ЈЈ-H), 5.53–5.45 (m, 4 H, 3Ј-H, 3ЈЈ-H, 3-H, 4-H), 5.42
3
The trichloroacetimidate 12 (1.00 g, 2.03 mmol) and p-cresol
(264 mg, 2.44 mmol) were coevaporated with toluene (5 mL). This
mixture was dissolved in dry CH2Cl2 (20 mL) and cooled to 0 °C.
TMSOTf (10 mL, 0.01 mmol in dry CH2Cl2) was added dropwise
over 30 min. The solution was stirred at room temperature over-
night and then washed with satd. NaHCO3 solution (3ϫ 5 mL).
The organic phase was dried with MgSO4 and filtered. The solvent
was evaporated, and the crude product was purified by column
chromatography (cyclohexane/EtOAc, 1:1) to obtain the title com-
pound 13 (750 mg, 84%) as a colourless solid. 1H NMR (300 MHz,
CDCl3): δ = 7.09 (d, 3J = 8.6 Hz, 2 H, Ar-H), 6.97 (d, 3J = 8.6 Hz,
(dd, 3J = 1.8, J = 3.5 Hz, 1 H, 2Ј-H), 5.38–5.30 (m, 3 H, 2-H, 2ЈЈ-
3
H, 4ЈЈ-H), 5.26 (dd ≈ t, J = 10.2 Hz, 1 H, 4Ј-H), 4.88 (br. s, 1 H,
1-H), 4.56–4.50 (m, 1 H, α-H), 4.31–4.21 (m, 4 H, 5ЈЈ-H, 6a-H,
6aЈ-H, 6aЈЈ-H), 4.16–4.05 (m, 5 H, 5-H, 5Ј-H, 6b-H, 6bЈ-H, 6bЈЈ-
H), 3.84–3.80 (m, 1 H, OCHHCH2), 3.74–3.71 (m, 2 H, SCH2C-
Ar), 3.62–3.50 (m, 4 H, OCHHCH2, CH2N, CArCHHCO), 3.46–
3.42 (m, 1 H, Ar-CHHCO), 2.90–2.85 (m, 1 H, βa-H), 2.72–2.66
(m, 1 H, βb-H), 2.22, 2.17, 2.10, 2.09, 2.05, 2.05, 2.04, 2.01, 2.00,
2.00, 1.99, 1.99 (each s, each 3 H, 12 OAc) ppm. 13C NMR
(150 MHz, [D4]MeOH): δ = 173.7 (CH2CONH), 173.1 (α-CO),
172.9, 172.8, 172.5, 172.4, 172.2, 171.6, 171.5, 171.5 (12 COCH3),
156.0, 155.0, 134.1, 131.6, 131.5, 131.4, 118.0, 117.9 (C-Ar), 98.9
3
3
2 H, Ar-H), 5.56 (dd, J = 3.5, J = 10.0 Hz, 1 H, 3-H), 5.47 (d,
Eur. J. Org. Chem. 2010, 3120–3128
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3125

A. Schierholt, M. Hartmann, K. Schwekendiek, T. K. Lindhorst
FULL PAPER
(C-1), 97.2 (C-1Ј), 97.2 (C-1ЈЈ), 70.8 (C-2), 70.7 (C-2Ј), 70.6 (C-5Ј), (1.17 mL, 13.4 mmol) were allowed to react in DMF (10 mL). The
70.5 (C-2ЈЈ), 70.4 (C-3Ј), 70.0 (C-5), 69.9 (C-3), 69.9 (C-5ЈЈ), 69.8 title compound (1.27 g, quant.) was used in the next step without
(C-3ЈЈ), 67.5 (OCH2CH2), 67.2 (C-4ЈЈ), 67.2 (C-4Ј), 67.1 (C-4), 63.5 further purification. 1H NMR (500 MHz, [D4]MeOH): δ = 7.45 (d,
3
3
(C-6Ј), 63.5 (C-6ЈЈ), 63.3 (C-6), 54.0 (C-α), 42.7 (CH2CH2N), 40.3 3J = 8.0 Hz, 6 H, Ar-H), 7.34 (dd, J = 7.6, J = 7.6 Hz, 6 H, Ar-
3
(CH2C-Ar), 36.3 (Ar-CH2S), 34.2 (C-β), 20.8, 20.7, 20.6, 20.5 (12 H), 7.26 (t, J = 7.3 Hz, 3 H, Ar-H), 4.79 (d, J = 1.5 Hz, 1 H, 1-
3
3
COCH3) ppm. HRESI-MS: calcd. for [C62H78N2O32S + Na]+
1417.4156; found 1417.4151.
H), 3.87–3.82 (m, 2 H, 6a-H, 2-H), 3.79 (ddd, J = 4.5, J = 6.4,
2J = 10.1 Hz, 1 H, OCHHCH2), 3.74–3.68 (m, 2 H, 3-H, 6b-H),
3.63 (dd ≈ t, J = 9.6 Hz, 1 H, 4-H), 3.59–3.43 (m, 2 H, OCHHCH2,
N-[4-(α-
D
-Mannopyranosyloxy)phenyl]acetyl-S-[4-(α-
D-manno-
3
5-H), 3.46–3.39 (m, 2 H, OCH2CH2), 3.17 (dd ≈ t, J = 6.5 Hz, 1
pyranosyl)benzyl]-
L
-cysteine [2-(α- -Mannopyranosyloxy)ethyl]-
D
3
2
3
H, α-H), 2.59 (dd, J = 6.0, J = 12.1 Hz, 1 H, βa-H), 2.44 (dd, J
amide (16): The protected glycocluster 15 (45 mg, 32 µmol) was
treated according to the general procedure for deacetylation. The
crude product was purified by HPLC (RP-8) to yield 16 (28 mg,
98%) as a colourless lyophilisate. HPLC: tR = 11.3 min (A = water,
B = acetonitrile + 1% TFA, 20% B Ǟ 80% B, 100 min, 10 mL/
min]. [α]2D0 = +41.9 (c = 1.1 in MeOH). 1H NMR (500 MHz, D2O):
= 7.0, J = 12.1 Hz, 1 H, βb-H) ppm. 13C NMR (150 MHz, [D4]-
2
MeOH): δ = 164.8 (COC-α), 146.1, 130.7, 128.9, 127.8 (C-Ar),
101.6 (C-1), 74.8 (C-5), 72.6 (C-3), 72.0 (C-2), 68.7 (C-4), 67.1
(OCH2CH2), 62.9 (C-6), 55.3 (C-α), 40.2 (OCH2CH2), 38.2 (C-β)
ppm. HRESI-MS: calcd. for [C30H36N2O7S + H]+ 569.2321; found
569.2402.
3
3
δ = 7.29 (d, J = 8.8 Hz, 2 H, ArЈЈ-H), 7.23 (d, J = 8.3 Hz, 2 H,
N-Acetyl-S-(triphenylmethyl)-L-cysteine [2-(α-D-Mannopyranosyl-
3
3
ArЈ-H), 7.13 (d, J = 8.8 Hz, 2 H, ArЈЈ-H), 7.06 (d, J = 8.3 Hz, 2
H, ArЈ-H), 5.59 (d, J = 1.5 Hz, 1 H, 1ЈЈ-H), 5.58 (br. s, 1 H, 1Ј-
oxy)ethyl]amide (20): Glycoamino acid 19 (420 mg, 739 µmol) was
dissolved in DMF (5 mL), DIPEA (532 µL, 2.96 mmol) and acetic
anhydride (280 µL, 2.96 mmol) were added, and the solution was
stirred at room temperature for 4 h. The crude product was purified
by silica gel chromatography (cyclohexane/EtOAc/MeOH, 1:4:2) to
yield the title compound (452 mg, quant.) as a colourless foam. 1H
NMR (500 MHz, [D6]DMSO): δ = 8.09 (d, 3J = 8.4 Hz, 1 H,
NHAc), 7.92 (t, J = 5.6 Hz, 1 H, CH2NHCO), 7.36–7.23 (m, 15
H, Ar-H), 4.59 (d, J = 1.5 Hz, 1 H, 1-H), 4.31 (dd, J = 6.2, J =
8.0 Hz, 1 H, α-H), 3.66–3.59 (m, 2 H, 6a-H, 2-H), 3.54 (dd, J =
3
H), 4.83 (d, 3J = 1.4 Hz, 1 H, 1-H), 4.32 (dd, 3J = 1.5, 3J = 8.3 Hz,
1 H, 2-H), 4.18–4.15 (m, 2 H, 2Ј-H, 2ЈЈ-H), 4.08–4.05 (m, 2 H, 3Ј-
H, 3ЈЈ-H), 4.05–4.00 (m, 1 H, OCHHCH2), 3.93–3.88 (m, 2 H, α-
H, OCHHCH2), 3.86–3.83 (m, 2 H, 6a-H, 6b-H), 3.79–3.70 (m, 7
H, 6Јa-H, 6ЈЈa-H, 6Јb-H, 6ЈЈb-H, 3-H, 4Ј-H, 4ЈЈ-H), 3.69–3.66 (m,
3
4 H, CArCH2S, 5Ј-H, 5ЈЈ-H), 3.63 (dd ≈ t, J = 9.2 Hz, 1 H, 4-H),
3
3.59–3.56 (m, 3 H, Ar-CH2CO, 5-H), 3.45–3.42 (m, 1 H, CHHN),
3
3
3
3
2
3.39–3.36 (m, 1 H, CHHN), 2.92 (dd, J = 5.5, J = 14.1 Hz, 1 H,
βa-H), 2.74 (dd, 3J = 8.3, 2J = 14.1 Hz, 1 H, βb-H) ppm. 13C NMR
(125 MHz, D2O): δ = 176.4 (CH2CON), 174.2 (α-CON), 156.6,
156.5, 134.3, 132.4, 132.1, 131.1, 119.2 (C-Ar), 101.9 (C-1), 100.1
(C-1Ј, C-1ЈЈ), 75.3 (C-5Ј, C-5ЈЈ), 74.7 (C-5), 72.5 (C-3Ј, C-3ЈЈ), 72.4
(C-3), 72.0 (C-2), 71.9 (C-2Ј, C-2ЈЈ), 68.7 (C-4), 68.5 (C-4ЈЈ), 68.4
(C-4Ј), 68.1 (OCH2CH2), 62.9 (C-6Ј, C-6ЈЈ), 62.6 (C-6), 55.1 (C-α),
42.9 (CH2C-Ar), 40.8 (CH2N), 36.9 (Ar-CH2S), 34.5 (C-β) ppm.
MALDI-ToF-MS: calcd. for [C38H54N2O20S + Na]+ 914.03; found
913.30.
3
2
5.9, J = 10.1 Hz, 1 H, OCHHCH2), 3.48–3.33 (m, 5 H, 3-H, 4-H,
3
2
6b-H, OCHHCH2-H, 5-H), 3.22 (dd, J = 6.9, J = 12.6 Hz, 1 H,
OCH2CHH), 3.18 (dd, 3J = 7.0, J = 13.2 Hz, 1 H, OCH2CHH)
2
3
2
3
2
2.35 (dd, J = 6.1, J = 11.7 Hz, 1 H, βa-H), 2.29 (dd, J = 7.9, J
= 11.7 Hz, 1 H, βb-H), 1.83 (s, 3 H, NHAc) ppm. 13C NMR
(150 MHz, [D6]DMSO): δ = 169.7 (COCH3), 169.1 (COC-α),
144.2, 129.0, 127.9, 126.6 (C-Ar), 99.9 (C-1), 73.8 (C-5), 70.8 (C-
3), 70.1 (C-2), 66.9 (C-4), 65.8 (CPh3), 65.0 (OCH2CH2), 61.1 (C-
6), 51.5 (C-α), 38.4 (OCH2CH2), 33.8 (C-β), 22.3 (COCH3) ppm.
HRESI-MS: calcd. for [C32H38N2O8S + Na]+ 633.2247; found
633.2257.
N-(Fluoren-9-ylmethoxycarbonyl)-S-(triphenylmethyl)-
L-cysteine [2-
(α- -Mannopyranosyloxy)ethyl]amide (18): According to the gene-
D
ral coupling procedure, Fmoc-Cys(Trt)-OH (2) (1.28 g, 2.18 mmol),
the mannoside 17 (970 mg, 4.34 mmol), HATU (913 mg,
2.40 mmol) and DIPEA (418 µL, 2.40 mmol) were allowed to react
in DMF (4 mL). Silica gel column chromatography (cyclohexane/
EtOAc/MeOH, 4:7:1) gave the title compound (326 mg, 81%) as a
N,NЈ-Diacetyl-L-cystine {Bis[2-(α-D-mannopyranosyloxy)ethyl]}-
diamide (21): According to the general procedure for disulfide for-
mation, the tritylated thiol 20 (100 mg, 164 µmol) and iodine
(63 mg, 250 µmol) were allowed to react in methanol (3 mL). The
crude product was purified by silica gel chromatography (EtOAc/
MeOH, 3:1 Ǟ EtOAc/MeOH, 1:1) to yield the disulfide 21 (36 mg,
60%) as a colourless foam. [α]2D0 = –10.5 (c = 1.1 in MeOH). 1H
NMR (600 MHz, [D4]MeOH): δ = 4.81 (d, 3J = 1.8 Hz, 2 H, 1-H),
1
colourless foam. H NMR (500 MHz, [D4]MeOH): δ = 7.84–7.79
3
(m, 2 H, Ar-H), 7.70 (t, J = 6.5 Hz, 2 H, Ar-H), 7.44–7.41 (m, 8
3
H, Ar-H), 7.33–7.23 (m, 11 H, Ar-H), 4.77 (d, J = 1.6 Hz, 1 H,
1-H), 4.48 (dd, 3J = 7.2, 3J = 10.1 Hz, 1 H, CHH-Fmoc), 4.33 (dd,
3
3
3
3
4.78 (dd, J = 5.9, J = 8.3 Hz, 1 H, α-H), 4.73 (dd, J = 5.6, J =
3
3
3
3J = 7.0, J = 10.1 Hz, 1 H, CHH-Fmoc), 4.27 (dd, J = 7.1, J =
3
2
8.7 Hz, 1 H, αЈ-H), 3.88 (dd, J = 2.4, J = 11.9 Hz, 2 H, 6a-H),
3
3
6.7 Hz, 1 H, CH-Fmoc), 3.96 (dd, J = 5.7, J = 8.4 Hz, 1 H, α-
H), 3.86–3.81 (m, 2 H, 6a-H, 2-H), 3.76–3.69 (m, 3 H, OCHHCH2,
3-H, 6b-H), 3.63 (dd ≈ t, J = 9.6 Hz, 1 H, 4-H), 3.55–3.48 (m, 2
3
3
3
3
3.86 (dd, J = 1.7, J = 3.3 Hz, 2 H, 2-H), 3.82 (ddd, J = 4.4, J
= 6.7, 2J = 10.9 Hz, 2 H, OCHHCH2), 3.78–3.72 (m, 4 H, 3-H, 6b-
H), 3.64 (dd ≈ t, J = 9.4 Hz, 2 H, 4-H), 3.60–3.55 (m, 4 H,
OCHHCH2, 5-H), 3.53–3.47 (m, 2 H, OCH2CHH), 3.46–3.41 (m,
3
H, 5-H, OCHHCH2), 3.44–3.36 (m, 2 H, OCH2CH2), 2.63 (dd, J
2
3
2
= 5.2, J = 12.5 Hz, 1 H, βa-H), 2.52 (dd, J = 8.6, J = 12.5 Hz,
1 H, βb-H) ppm. 13C NMR (150 MHz, [D4]MeOH): δ = 172.8
(COC-α), 158.1 (COO-Fmoc), 145.9, 145.1, 142.6, 130.7, 128.9,
128.7, 128.2, 127.9, 126.2, 120.9 (C-Ar), 101.6 (C-1), 74.7 (C-5),
72.6 (C-3), 72.0 (C-2), 68.7 (C-4), 68.1 (CH2-Fmoc), 67.0
(OCH2CH2), 62.9 (C-6), 55.6 (C-α), 48.4 (CH-Fmoc), 40.3
(OCH2 CH2 ), 3 5. 1 ( C-β) ppm. HRESI-MS: calcd. for
[C45H46N2O9S + Na]+ 813.2822; found 813.2382.
3
2
2 H, OCH2CHH), 3.40–3.36 (m, 1 H, βa-H), 3.23 (dd, J = 5.6, J
3
2
= 13.8 Hz, 1 H, βЈa-H), 3.19 (dd, J = 5.6, J = 13.8 Hz, 1 H, βb-
H), 2.98 (dd, 3J = 8.7, 2J = 13.8 Hz, 1 H, βЈb-H), 2.06 (s, 6 H,
NHAc) ppm. 13C NMR (150 MHz, [D4]MeOH): δ = 173.5, 173.4
(COCH3), 172.6, 172.3 (COC-α), 101.6 (C-1), 74.7 (C-5), 72.5 (C-
3), 72.0 (C-2), 68.8 (C-4), 66.9 (OCH2CH2), 63.0 (C-6), 54.1, 54.0
(C-α), 41.4, 41.0 (C-β), 40.5 (OCH2CH2), 22.6 (COCH3) ppm.
HRESI-MS: calcd. for [C26H46N4O16S2 + Na]+ 757.2248; found
757.2207.
N-[2-(α-D-Mannopyranosyloxy)ethyl]-S-(triphenylmethyl)-L-cysteine
Amide (19): According to the general procedure for Fmoc deprotec-
tion, glycoamino acid 18 (1.77 g, 2.24 mmol) and morpholine
N-[4-(α-
D
-Mannopyranosyloxy)phenyl]acetyl-S-(triphenylmethyl)-
-Mannopyranosyloxy)ethyl]amide (23): According
L-cysteine [2-(α-
D
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Eur. J. Org. Chem. 2010, 3120–3128

Cysteine-Based Mannoside Glycoclusters
to the general coupling procedure, the amine 19 (1.27 g,
2.24 mmol), the carboxylic acid 22 (503 mg, 1.60 mmol), HATU
(669 mg, 1.76 mmol) and DIPEA (306 µL, 1.76 mmol) were al-
lowed to react in DMF (4 mL). Silica gel column chromatography
(cyclohexane/EtOAc/MeOH, 1:4:2) yielded the title compound
(632 mg, 46%) as a colourless foam. [α]2D0 = +51.4 (c = 1.0 in
MeOH). 1H NMR (600 MHz, [D4]MeOH): δ = 7.39 (d, 3J =
8.5 Hz, 6 H, Ar-H), 7.31 (dd, 3J = 7.5, 3J = 7.5 Hz, 6 H, Ar-H),
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3
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7.28–7.23 (m, 5 H, Ar-H), 7.07 (d, J = 8.7 Hz, 2 H, Ar-H), 5.48
3
3
(d, J = 1.9 Hz, 1 H, 1Ј-H), 4.77 (d, J = 1.6 Hz, 1 H, 1-H), 4.20
(dd, 3J = 6.1, 3J = 8.1 Hz, 1 H, H-α), 4.03 (dd, 3J = 1.9, 3J =
3.4 Hz, 1 H, 2Ј-H), 3.94 (dd, 3J = 3.4, 3J = 9.5 Hz, 1 H, 3Ј-H),
3.85–3.82 (m, 2 H, 2-H, 6a-H), 3.79 (dd ≈ t, J = 9.7 Hz, 1 H, 4Ј-
H), 3.75–3.70 (m, 5 H, 6aЈ-H, 6bЈ-H, 6b-H, 3-H, OCHHCH2), 3.63
(dd ≈ t, J = 9.5 Hz, 1 H, 4-H), 3.63 (mc, 1 H, 5Ј-H), 3.56–3.51 (m,
4 H, 5-H, COCH2C-Ar, OCHHCH2), 3.38–3.37 (m, 2 H,
OCH2CH2), 2.60 (dd, 3J = 6.0, 2J = 12.4 Hz, 1 H, βa-H), 2.50 (dd,
3J = 8.1 H, 12.4 Hz, 1 H, βb-H) ppm. 13C NMR (150 MHz, [D4]-
MeOH): δ = 174.0 (NHCOCH2), 172.4 (COC-α), 157.0, 145.9,
131.3, 130.6, 130.4, 129.1, 127.9 (C-Ar), 117.8 (C-Ar), 101.6 (C-1),
100.2 (C-1Ј), 75.2 (C-5Ј), 74.6 (C-5), 72.5 (C-3), 72.4 (C-3Ј), 72.0
(C-2), 72.0 (C-2Ј), 68.7 (C-4), 68.2 (C-4Ј), 67.9 (CPh3), 66.8
(OCH2CH2), 62.9 (C-6), 62.5 (C-6Ј), 53.9 (C-α), 42.7 (COCH2C-
Ar), 40.3 (OCH2CH2), 34.8 (C-β) ppm. MALDI-ToF-MS: calcd.
for [C44H52N2O14S + Na]+ 887.30; found 888.26; calcd. for
[C44H52N2O14S + K]+ 903.27; found 904.20. HRESI-MS: calcd. for
[C44H52N2O14S + Na]+ 887.3037; found 887.3081.
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N,NЈ-Bis{[4-(α-
D-mannopyranosyloxy)phenyl]acetyl}-L-cystine
{Bis[2-(α- -mannopyranosyloxy)ethyl]}diamide (24): According to
D
the general procedure for disulfide formation, the tritylated thiol
21 (112 mg, 129 µmol) and iodine (49 mg, 190 µmol) were allowed
to react in methanol (3 mL). The crude product was purified by
RP-MPLC (MeCN/H2O, 1:1) followed by GPC on Sephadex LH-
20 (MeOH) to yield pure 24 (31 mg, 39%) as a white lyophilisate.
[α]2D0 = +36.1 (c = 0.6 in MeOH). 1H NMR (600 MHz, [D4]MeOH):
δ = 7.28 (d, 3J = 8.8 Hz, 4 H, Ar-H), 7.10 (d, 3J = 8.7 Hz, 4 H,
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3
3
Ar-H), 5.50 (d, J = 1.8 Hz, 2 H, 1Ј-H), 4.80 (d, J = 1.8 Hz, 2 H,
3
3
1-H), 4.76 (dd, 3J = 5.2, J = 9.1 Hz, 2 H, α-H), 4.04 (dd, J = 1.8,
3J = 3.4 Hz, 2 H, 2Ј-H), 3.94 (dd, J = 3.4, J = 9.5 Hz, 2 H, 3Ј-
H), 3.90–3.74 (m, 8 H, 2-H, 4Ј-H, 6a-H, 6Јa-H), 3.66–3.62 (m, 12
H, 3-H, 4-H, 5Ј-H, 6b-H, 6Јb-H, OCHHCH2), 3.58–3.51 (m, 8 H,
5-H, OCH2C-Ar, OCHHCH2), 3.45–3.40 (m, 4 H, OCH2CH2),
3
3
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3
2
3
2
3.14 (dd, J = 5.3, J = 13.9 Hz, 2 H, βa-H), 2.96 (dd, J = 9.1, J
= 13.9 Hz, 2 H, βb-H) ppm. 13C NMR (150 MHz, [D4]MeOH): δ
= 174.4 (NHCOCH2), 172.4 (NHCOC-α), 156.9, 131.4, 130.4,
118.0 (C-Ar), 101.6 (C-1), 100.3 (C-1Ј), 75.2 (C-5Ј), 74.7 (C-5), 72.5
(C-3), 72.4 (C-3Ј), 72.0 (C-2), 71.9 (C-2Ј), 68.8 (C-4), 68.4 (C-4Ј),
66.9 (OCH2CH2), 62.9 (C-6), 62.6 (C-6Ј), 54.2 (C-α), 42.7
(COCH2C-Ar), 41.4 (C-β), 40.4 (OCH2CH2) ppm. HRESI-MS:
calcd. for [C50H74N4O28S2 + Na]+ 1265.3829; found 1265.3768;
calcd. for [C50H74N4O28S2 + K]+ 1281.3568; found 1281.3164.
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Supporting Information (see also the footnote on the first page of
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Acknowledgments
We are grateful to Mr. cand. chem. Alexander Thrun and in par-
ticular to Dipl.-Chem. Johannes W. Wehner for their valuable con-
tributions and their outstanding commitment as trainees of A. S.
and we acknowledge financial support by Christiana Albertina
University of Kiel (CAU).
Eur. J. Org. Chem. 2010, 3120–3128
© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3127

A. Schierholt, M. Hartmann, K. Schwekendiek, T. K. Lindhorst
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Received: February 10, 2010
Published Online: April 26, 2010
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Eur. J. Org. Chem. 2010, 3120–3128