Are multivalent cluster glycosides a means of controlling ligand density of glycoarrays?

Article abstract of DOI:10.1016/j.carres.2013.01.023

Bacterial adhesion to the glycocalyx of human host cells is of biological and medicinal importance. This process is often initiated by the interaction of bacterial lectins and specific carbohydrate ligands. Thus, adhesion of bacterial cells to glycosylated surfaces is a suitable model system to study various parameters of lectin-mediated carbohydrate recognition. Glycoarrays have become important tools to study such lectin-mediated carbohydrate recognition. However, it is difficult to adjust the characteristics of a specific glycoarray regarding its carbohydrate density or the clustering of sugar ligands, respectively. Thus, we have made an attempt to use synthetic cluster glycosides of different valencies to vary carbohydrate density on a polystyrene surface. A series of mono-, di- and trivalent mannosides were synthesised for immobilisation on pre-functionalised polystyrene microtiter plates and the resulting glycoarrays were tested as adhesive surfaces in mannose-specific adhesion of Escherichia coli. Our measurements give first promising hints about the potential of this approach to alter ligand density of glycoarrays in a systematic way.

Full text of DOI:10.1016/j.carres.2013.01.023

Carbohydrate Research 371 (2013) 22–31
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Are multivalent cluster glycosides a means of controlling ligand
density of glycoarrays?
⇑
Johannes W. Wehner, Mirja Hartmann, Thisbe K. Lindhorst
Otto Diels Institute of Organic Chemistry, Christiana Albertina University of Kiel, Otto-Hahn-Platz 3/4, 24098 Kiel, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Bacterial adhesion to the glycocalyx of human host cells is of biological and medicinal importance. This
process is often initiated by the interaction of bacterial lectins and specific carbohydrate ligands. Thus,
adhesion of bacterial cells to glycosylated surfaces is a suitable model system to study various parameters
of lectin-mediated carbohydrate recognition. Glycoarrays have become important tools to study such lec-
tin-mediated carbohydrate recognition. However, it is difficult to adjust the characteristics of a specific
glycoarray regarding its carbohydrate density or the clustering of sugar ligands, respectively. Thus, we
have made an attempt to use synthetic cluster glycosides of different valencies to vary carbohydrate den-
sity on a polystyrene surface. A series of mono-, di- and trivalent mannosides were synthesised for immo-
bilisation on pre-functionalised polystyrene microtiter plates and the resulting glycoarrays were tested
as adhesive surfaces in mannose-specific adhesion of Escherichia coli. Our measurements give first prom-
ising hints about the potential of this approach to alter ligand density of glycoarrays in a systematic way.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 2 December 2012
Received in revised form 28 January 2013
Accepted 30 January 2013
Available online 8 February 2013
Keywords:
Bacterial adhesion
Glycoarrays
Cluster glycosides
Density control
Synthesis
Adhesion assays
1. Introduction
ple, carbohydrate spacing has been accomplished on DNA arrays,24
and carbohydrate density on a surface has been varied using neogly-
Every eukaryotic cell is covered with a layer of complex glyco-
conjugates, the so-called glycocalyx, which plays a crucial role in
processes like cell-cell communication, cellular adhesion, inflam-
mation and signalling.1–3 Due to the structural complexity of the
glycoconjugates embedded into the cell membrane, elucidation
of the molecular details of carbohydrate recognition is a demand-
ing task. A specific scenario which is suited to test the details of
carbohydrate recognition on a surface is bacterial adhesion to fab-
ricated glycosylated surfaces and glycoarrays, respectively.4–11
Bacteria utilise lectins, expressed as part of long protein append-
ages, called fimbriae, to attach to the glycosylated surface of their
host cells.12,13 This process facilitates bacterial colonisation of cell
surfaces and biofilm formation and is connected to, i.a., severe
inflammatory diseases of the host.
A key question in the investigation of carbohydrate recognition
processes occurring on the cell surface is the composition of the gly-
cocalyx. It is determined by the chemical nature of the carbohydrate
constituents of the various glycoconjugates. But, moreover, the nat-
ure of the glycocalyx is regulated by additional parameters. These
comprise (i) multivalency of sugar epitopes,14–16 (ii) their orienta-
tion and conformational flexibility of their presentation17–19 as well
as (iii) ligand density and spacing of interaction partners.20–23 Such
parameters are difficult to adjust and to analyse and related research
typically requires complex and demanding approaches. For exam-
coproteins.25 Indeed, glycoarrays offer an opportunity to address
many important features of glycosylated surfaces but the effort to
systematically vary and characterise their properties has remained
high.26–29
Here, we report on a study where cluster glycosides of different
valencies were used in an attempt to vary carbohydrate ligand den-
sity of a glycoarray in a facile way. It is known from surface chemis-
try, that the size of molecules used for surface decoration influences
the density of the formed molecular layer as a consequence of steric
hindrance during the immobilisation process.30,31 Thus, we have
made mono-, di- and trivalent (cluster) mannosides to functionalise
polystyrene microtiter plates. Then, mannose-specific adhesion of
bacterialcells to the respectiveglycoarrayswas assayedin three par-
allel test series employing systematic concentration variation
(Fig. 1). We were interested to investigate, which different influ-
ences concentration and valency of sugar ligands have on the ‘stick-
iness’ of a glycosylated surface. This was systematically studied with
three different types of mannose-coated surfaces which were em-
ployed in a lectin-mediated bacterial adhesion assay.
2. Results and discussion
2.1. Synthesis of mannosides
A series of mono-, di- and trivalent cluster mannosides were
synthesised in two variations. One set of mannosides was equipped
⇑
Corresponding author. Tel.: +49 431 880 2023; fax: +49 431 880 7410.
E-mail address: tklind@oc.uni-kiel.de (T.K. Lindhorst).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.01.023

J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
23
Figure 1. Bacterial adhesion of type 1 fimbriated E. coli to three different types of glycoarrays, fabricated from mono-, di- and trivalent (cluster) mannosides (from left to
right). Hypothetically, each type of glycoarray could show different concentration dependencies in bacterial adhesion assays. When higher sample concentrations are used for
immobilisation, density of exposed mannosyl ligands should be comparable in all three cases owing to steric circumstances. But in case of more ‘diluted’ glycoarrays, the
surface that is decorated with cluster mannosides could be more adhesive then the simple mannoside-coated surface, because ligand clustering is maintained on the
multivalent scaffold.
with a primary amino function to allow covalent immobilisation of
pre-functionalised microtiter plates. A second set was prepared
accordingly, but with the amino group in N-acetylated form. The
latter mannosides were used as soluble inhibitors of bacterial
adhesion to a mannan-coated surface to determine their IC50
values.
the intermediate and N-Boc removal applying TFA in dichloro-
methane in 97% overall yield.
By analogy to the synthesis of 4 and 5, the preparation of the
divalent mannosides 8 and 9 started with Staudinger ligation of
mannoside 1 with diacid 6.35 This step initially led to cluster man-
noside 7. Then, N-Boc deprotection, N,O-acetylation and deprotec-
tion of the O-acetyl groups yielded the N-acetyl-protected
mannoside 8. The free amine 9 was obtained as bis-trifluoroacetate
after Zemplén deprotection of 7 followed by acidic N-Boc removal
applying TFA in dichloromethane.
The synthetic routes started with azidoethyl mannoside 132
which could be conjugated to the appropriate mono-, di- and tri-
functional carboxylic acid derivatives 2, 6 and 12 (Schemes 1 and
2) employing Staudinger ligation.33
Mannoside 3 was obtained from reaction of 1 with commer-
cially available N-Boc-protected methionine 2. This fully protected
mannoside was then treated with TFA to remove the N-Boc pro-
tecting group, followed by N,O-acetylation and de-O-acetylation
according to Zemplén34 to yield the OH-free N-acetylated deriva-
tive 4 in very good yield after purification by reversed phase chro-
matography (Scheme 1). The free amine 5 was obtained in the form
of its TFA salt after de-O-acetylation of mannoside 3, RP-MPLC of
For the preparation of the trivalent cluster mannosides 15 and
16, the tris-carboxylic acid 12 was employed. This was prepared
from the well-known trivalent wedge-type molecule 1036,37 which
was coupled to Fmoc-Cys(Bn)-OH applying HBTU/DIPEA following
a literature-known procedure9 yielding 11 in 87% yield. Acidic
cleavage of the Bu-esters using formic acid gave 12. Then, three-
fold Staudinger ligation of 12 and mannoside 1 was successfully
accomplished to obtain 13 in fair yield (Scheme 2). For preparation
t
OR1
O
OR1
O
NHBoc
R1O
R1O
HO
R1 = Ac, R2 = Boc
R1 = H, R2 = Ac
2
3
4
O
O
NHR2
SCH3
N
H
b)
SCH3
a)
OAc
OAc
c)
O
OR1
AcO
AcO
5
R1 = H, R2 = H.TFA
OR1
O
R1O
R1O
O
1
N3
O
d)
NHR2
e)
O
O
N
O
N
H
NHBoc
7
8
R1 = Ac, R2 = Boc
HO
N
R1O
O
R1O
R1O
R1O
6
HN
R1 = H, R2 = Ac
O
HO
O
f)
9
R1 = H, R2 = H.2 TFA
Scheme 1. Synthesis of mono- and divalent NHAc- and NH2-functionalised mannosides 4, 5, 8 and 9. Reagents and conditions: (a) P(tBu)3, DIC, dry THF, 0 °C ? rt, overnight,
72%, (b) (i) TFA, CH2Cl2, rt, 4 h; (ii) acetic anhydride, pyridine, rt, overnight; (iii) Na, dry MeOH, rt, overnight, 88% over three steps; (c) (i) Na, dry MeOH, rt, 4.5 h; (ii) TFA,
CH2Cl2, rt, 1.5 h, 97% over two steps; (d) HOBt, P(tBu)3, DIC, dry THF, 0 °C ? rt, 40 h, 55%, (e) (i) TFA, CH2Cl2, rt, 9 h; (ii) acetic anhydride, pyridine, rt, overnight; (iii) Na, dry
MeOH, rt, overnight, 86% over three steps. (f) (i) Na, dry MeOH, rt, overnight; (ii) TFA, CH2Cl2, rt, overnight, 99% over two steps.

24
J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
O
O
SBn
SBn
RO
RO
O
O
HO
H
N
NHFmoc
NH2
NHFmoc
O
O
O
O
11 R = tBu
a)
RO
O
b)
O
O
12 R = H
10
OAc
OAc
O
c)
AcO
AcO
1
O
OR1
OR1
N3
OAc
OAc
O
O
R1O
R1O
AcO
AcO
O
O
O
SBn
NHR2
O
SBn
NHFmoc
N
H
R1O
N
AcO
H
N
d)
H
N
O
O
H
R1O
R1O
H
N
H
N
AcO
AcO
O
O
O
O
O
O
R1O
AcO
H
H
N
N
R1O
O
O
O
O
13
AcO
O
O
R1O
R1O
14
R
1 = Ac, R2 = H
AcO
AcO
OR1
e)
OAc
15 R1 = H, R2 = Ac
f)
16 R1 = H, R2 = H
Scheme 2. Synthesis of trivalent NHAc- and NH2-functionalised cluster mannosides 15 and 16. Reagents and conditions: (a) HBTU, DIPEA, dry DMF, rt, overnight, 87%; (b)
formic acid, rt, overnight, quant.; (c) HOBt, P(tBu)3, DIC, dry THF, 0 °C ? rt, overnight, 62%, (d) piperidine, DMF, rt, 1.5 h, 98%; (e) (i) acetic anhydride, pyridine, rt, 2 h; (ii) Na,
dry MeOH, rt, 2 h, 94% over two steps; (f) Na, dry MeOH, rt, overnight, 99%.
of the free amine 14, the Fmoc protecting group was removed with
piperidine in DMF. Next, the N-acetylated OH-unprotected cluster
mannoside 15 was furnished in a sequence of N,O-acetylation and
Zemplén deprotection of O-acetates. The unprotected free amine
16 was obtained after O-deprotection of 14 in quantitative yield.
in Table 1. These RIPvc values show that on a valency-corrected ba-
sis only the trivalent cluster glycoside has a significant advantage
over MeMan with RIPvc = 2. This small multivalency effect can
most probably be attributed to a statistical advantage of the triva-
lent cluster mannoside over simple mannosides: as soon as one
mannosyl residue departs from the FimH carbohydrate binding
site, the next ligand is already pre-positioned on the same scaffold
and immediately in place to complex with the lectin’s carbohy-
drate recognition domain.
2.2. Adhesion inhibition assay with 4, 8 and 15
Next, the synthesised N-acetylated mannosides 4, 8 und 15
were tested as inhibitors of
a
-
D
-mannoside-specific adhesion of
Based on the little differences seen in the RIPvc values and with
regard to many earlier control studies13 we can assume that the
different natures of the glycocluster scaffold moieties exert no rel-
evant influence on the inhibitory potencies of the respective glyco-
cluster. Thus, after the evaluation of mannosides 4, 8 and 15 as
inhibitors of bacterial adhesion in solution, their amino-functional-
ised analogues 5, 9 and 16 were used to fabricate the correspond-
ing glycoarrays on appropriately activated polystyrene surfaces for
biological comparison studies.
type 1-fimbriated Escherichia coli bacteria to a mannan-coated sur-
face.38 The employed E. coli strain PKL1162 contains the GFP (green
fluorescing protein) gene and thus bacterial adhesion to the man-
nan-coated surface can be correlated to measured fluorescence
intensity. Serial dilutions of the inhibitors were employed accord-
ing to a published protocol.38 This led to inhibition curves of which
the respective IC50 values could be deduced. For a better compari-
son of inhibitors that were tested in different experiments, relative
inhibitory potencies (RIP) were calculated by referencing the mea-
sured data to the IC50 value of methyl
a-D-mannoside (MeMan),
which was tested on the respective plate.
Table 1
Inhibitory potencies of mannosides 4, 8 and 15 as determined in an adhesion-
inhibition assay on mannan-coated microtiter plates
The results of the adhesion-inhibition assays show that the
methionyl mannoside 4 has a very similar inhibitory potency as
the standard inhibitor of this system, MeMan (Table 1). The diva-
lent cluster mannoside 8 inhibits bacterial adhesion to mannan
2.5 times better than MeMan, and the trivalent cluster mannoside
15 was shown to be an approximately 6-times more potent inhib-
itor in comparison to simple MeMan. When these relative inhibi-
tory potencies (RIP values) were valency-corrected, in other
a
c
Inhibitor
IC50
(lM)
IC50 MeManb
(l
M)
RIPc
RIPvc
4 (Monovalent)
8 (Divalent)
15 (Trivalent)
3616
7152
879
5181
17667
5181
1.4
2.5
5.9
1.4
1.25
2.0
a
b
c
IC50 values are average values from triplicate results.
Tested on the same microtiter plate.
RIP: relative inhibitory potency with IP (MeMan)„1; RIPvc: valency-corrected
words, when the number of
a-D-mannosyl residues per inhibitor
RIP.
molecule was taken into account, RIPvc values resulted as depicted

J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
25
2.3. Fabrication of glycoarrays on polystyrene and testing of
their adhesive properties using E. coli cells
In case of glycoarray 17 fabricated from the simple manno-
side 5, a slight but significant decrease of adhesiveness was ob-
served going from higher (15 mM) to lower concentrations
(2 mM). This finding was expected and is not surprising, as more
dilute glycoarrays present less ligands for fimbriae-mediated
bacterial adhesion, which is consequently less strong. Interest-
ingly, a reverse course of adhesiveness was observed with sur-
faces 18 and 19, made from the di- and trivalent cluster
mannosides 9 and 16, respectively. Here, adhesiveness increased
from left to right in the concentration window shown. (At higher
concentrations, no significant differences between the three gly-
coarrays were seen, cf. Supplementary data.) In addition, the ra-
tios of adhesiveness between the three surface types at one
particular concentration also undergo a notable change. Whereas
at higher concentrations, glycoarray 17 is slightly more adhesive
than surfaces 18 and 19, this situation is reversed in case of the
more dilute glycoarrays.
The simple mannoside 5 and the di- and trivalent cluster man-
nosides 9 and 16 were immobilised on pre-activated 96 well
microtiter plates to yield surfaces 17, 18 and 19 (Scheme 3). For
immobilisation, the plates were incubated with serial dilutions of
the amino-functionalised carbohydrate derivatives in carbonate
buffer (pH 9.6). Under these conditions the free amines are formed
from the TFA salts and react with the microplate surface
Immobilisation was followed by several washing steps with
buffer (PBST) to remove carbohydrates that were not covalently
bound to the surface and unreacted surface groups were blocked
with ethanolamine. Then, the so formed glycoarrays were incu-
bated with fluorescent type 1 fimbriated E. coli (PKL1162), non-ad-
hered microorganisms were washed away and adhesion was
quantified by fluorescence read out. The results are depicted in
Figure 2.
HO
HO
OH
OH
O
HO
OH
O
HO
HO
OH
OH
OH
OH
HO
HO
OH
OH
O
O
HO
HO
O
HO
HO
O
O
HO
OH
O
O
O
HO
HO
NH
HN
O
O
HN
O
NH
O
NH
O
O
O
HN
N
NH
O
O
MeS
.
.2
NH TFA
2
NH TFA
H N
2
SBn
2
5
16
9
a)
a)
a)
OH
HO
HO
OH
HO
OH
O
HO
HO
OH
OH
OH
OH
HO
OH
HO
OH
O
O
O
O
HO
HO
O
HO
HO
O
HO
OH
O
O
O
HO
HO
NH
O
HN
O
HN
O
NH
O
NH
O
O
O
NH
HN
N
O
O
MeS
HN
SBn
NH
NH
19
17
18
Scheme 3. Preparation of glycoarrays 17, 18 and 19 on pre-functionalised microtiter plates. Reagents and conditions: (a) carbonate buffer (pH 9.6), rt, incubation with
microplates overnight.

26
J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
Figure 2. Adhesion of E. coli strain PKL1162 to the three different glycoarrays, 17 (bright grey columns), 18 (dark grey columns) and 19 (black columns), as determined by
fluorescence read-out, with standard deviations (SD) indicated. For adequate interpretation, the depicted concentrations are valency-corrected, that is, given numbers refer to
the concentrations of a-D-mannosyl moieties rather than to the sample concentration that was used for glycoarray fabrication (that means a two-fold difference for 18, a
three-fold difference for 19).
drous DMF was purchased, other solvents were dried for reactions
or distilled for chromatography. Black Immobilizer Amino™ F96
MicroWell™ plates were purchased from Nunc (Thermo Fisher Sci-
entific). Air- and/or moisture-sensitive reactions were carried out
under an atmosphere of 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 chromatog-
raphy was performed on silica gel 60 (230–400 mesh, particle size
0.040–0.063 mm, Merck). Preparative MPLC was performed on a
3. Discussion
Our findings give a first hint that the hypothesis behind this study
is promising. It has been suggested that cluster glycosides might be
used to vary ligand density of glycoarrays, which can be rudimental-
ly seen in our experiments. Apparently, concentration-dependent
fabrication of glycoarrays using glycoclusters leads to different
trends in adhesiveness depending on the cluster valency. Based on
earlier experiments, we have no reason to assume, that the effective-
ness of the immobilisation reaction on the pre-functionalised poly-
styrene plates is a limiting or otherwise critical factor in glycoarray
fabrication. Thus, steric features of the employed molecules should
be responsible for the stickiness of the resulting glycoarray. Steric
bulk on one hand and clustering of mannosyl ligands on the other
hand have to be weighed for an interpretation of the obtained data.
Presumably, at higher concentrations, immobilisation of the simple
mannoside 5 leads to a more adhesive surface than when the more
bulky glycoclusters 9 and 16 are employed. But upon dilution of
the solutions used for glycoarray fabrication, the glycoarrays 18
and 19 made from 9 and 16 show better adhesive properties than
the more simple glycoarray 17 made from 5 (with valency correction
taken into account). Thus, in the case of the more dilute glycoarrays,
BÜCHI apparatus using a LiChroprep RP-18 (40–60
lm, Merck) col-
umn for reverse phase and a LiChroprep Si 60 (40–60
l
m, Merck)
column for normal phase silica gel chromatography. 1H and 13C
were recorded on Bruker DRX-500 and AV-600 machines. 2D
NMR experiments (1H–1H COSY, 1H–13C HSQC and 1H–13C HMBC)
were performed for full assignment of the spectra. Chemical shifts
were 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 49.00 ppm) or H2O (d 4.65 ppm). ESI MS measurements were per-
formed on a Mariner ESI-ToF 5280 instrument (Applied Biosys-
tems). MALDI-TOF mass spectra were recorded on a Bruker
Biflex-III 19 kV instrument with Cl-CCA (4-chloro-
ic acid) or DHB (2,5-dihydroxybenzoic acid) as matrix. Optical rota-
tion was measured on a Perkin–Elmer polarimeter 341 (Na- -line:
a-cyanocinnam-
type 1 fimbriated E. coli cells find local high supply of a-D-mannosyl
D
ligands only in case of surfaces 18 and 19, which are glycocluster-
functionalised. This turns out favourably for cellular adhesion. The
simple glycoarray 17, on the other hand, can apparently not provide
an analogously advantageous situation in case of the sparsely cov-
ered microtiter plate.
In conclusion, it appears likely that the significant differences in
relative trends of stickiness of the three tested surfaces can be
attributed to varied ligand density arising from the specific archi-
tecture of the employed glycocluster. This is an interesting obser-
vation, which requires further biophysical studies to gain better
understanding of how adhesive processes are governed by ligand
density. We have commenced such work on other surfaces than
polystyrene to allow spectroscopic characterisation of the glycosyl-
ated surface.
589 nm, length of cell 1 dm). IR spectra were recorded on a Perkin–
Elmer Paragon 1000 FT-IR instrument. For sample preparation a
Golden Gate diamond ATR unit with a saphire stamp was used. Ele-
mental analyses were measured on a Euro-EA elemental analyser
(EuroVector) at the Institute of Inorganic Chemistry (Christiana
Albertina University of Kiel). For bacterial adhesion studies, a TE-
CAN infinite 200 multifunction microplate reader was employed.
The wavelengths of the band pass filters for excitation and emis-
sion were 485 and 535 nm, respectively. Compounds 132 and 635
were prepared according to literature-known procedures. Amber-
lyst A-21 was treated before usage as described by Srinivasan
and co-workers.39
4.2. General procedure for Staudinger ligation
4. Experimental
The corresponding azide-functionalised carbohydrate (1 equiv)
and the carboxylic acid (1.8 equiv) were combined with HOBt
(1.8 equiv) in a Schlenk flask and dried for more than 1 h in vacuo.
This mixture was dissolved in dry THF under a nitrogen atmo-
sphere and cooled to 0 °C. Then DIC (1.8 equiv) was added and
4.1. General experimental methods
Commercially available starting materials and reagents were
used without further purification unless otherwise noted. Anhy-

J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
27
the solution was stirred for 10 min, followed by the addition of tri-
n-butylphosphane (1–1.8 equiv) and stirring for 1 h at 0 °C. Then
the reaction mixture was stirred at ambient temperature, diluted
with water (50 mL) and extracted four times with dichlorometh-
ane (30 mL each). The combined organic phases were washed with
brine, dried over MgSO4, it was filtered and the filtrate concen-
trated under reduced pressure. The crude product was purified
by MPLC.
63.6 (C-6Man), 55.2 (CHMet, ), 40.1 (OCH2CH2NH), 33.2
a
(CH2CH2SCH3), 31.3 (CH2CH2SCH3), 28.7 (C(CH3)3), 20.7, 20.7,
20.6, 20.6 (4 OC(O)CH3), 15.3 (CH2CH2SCH3) ppm; MALDI-ToF MS
(DHB): calcd for C26H42N2NaO13S: m/z 645.23 [M+Na]+; found: m/
z 645.29 [M+Na]+, calcd for C26H42N2KO13S: m/z 661.20 [M+K]+;
~
m = 3345, 2978, 2937, 2493,
found: m/z 661.27 [M+K]+; IR (ATR):
1746, 1712, 1660, 1521, 1367, 1218, 1165, 1137, 1081, 1044,
978, 784 cmꢂ1
.
4.3. General procedure for N,O-acetylation
4.7. N-(Acetyl)-L-methionine-[2-(a-D-mannopyranosyloxy)
ethyl]amide (4)
The compound was dissolved in pyridine and acetic anhydride
was added. The reaction mixture was stirred at ambient tempera-
ture (minimum 2 h, maximum overnight). Then the solution was
concentrated under reduced pressure and the residue codistilled
three times with toluene (5 mL each). The crude product was puri-
fied by MPLC.
Mannoside 3 (135 mg, 217
lmol) was dissolved in dichloro-
methane (4 mL), TFA (200 L) was added and the reaction mixture
l
was refluxed for 4 h. Then, it was diluted with dichloromethane
(5 mL), Amberlyst A-21 ion exchange resin (2.00 g) was added
and the mixture was stirred for 30 min. The resin was filtered off
and it was washed with dichloromethane/methanol (1:1, 5 mL).
The solvents were removed to yield an intermediate with the ami-
4.4. General procedure for de-O-acetylation
no group deprotected (110 mg, 210
up; Rf 0.27 (methanol/dichloromethane, 1:9). Part of this material
(90.0 mg, 172 mol) was acetylated according to the general pro-
cedure for N,O-acetylation using pyridine (3 mL) and acetic anhy-
dride (500 L) overnight. Then volatile components were
removed in vacuo and the residue was codistilled with toluene.
The crude product was subjected to purification by repeated MPLC
(first: 100 g, silica column, A: methanol, B: dichloromethane, A:
1% ? 10%, 60 min; second: 50 g, silica column, A: ethyl acetate,
B: methanol, A: 100% ? 90%) to yield a fully N,O-acetylated inter-
lmol, 97%) as a colourless syr-
The acetylated compound was dissolved in dry methanol and
sodium (30–50 mg dissolved in 1 mL dry MeOH) was added under
nitrogen. The reaction mixture was stirred at ambient temperature
until the reaction was complete (minimum 1.5 h, maximum over-
night), then it was neutralised by the addition of Amberlite IR-120
ion exchange resin, filtered and the filtrate was concentrated under
reduced pressure. The crude product was purified by MPLC where
necessary.
l
l
4.5. General procedure for N-Boc-deprotection
mediate (93.0 mg, 165
(methanol/dichloromethane, 1:8). Part of this material (48 mg,
88.6 mol) was de-O-acetylated overnight according to the general
lmol, 96%) as a colourless syrup; Rf: 0.60
The Boc-protected amine was dissolved in dichloromethane and
l
TFA (118
lL-3 mL) was added. The solution was stirred at ambient
procedure. The crude product was subjected to purification by
temperature (minimum 1 h, maximum overnight). The solvent was
removed under reduced pressure and the residue codistilled three
times with toluene (5 mL each). Products used for functionalisation
of the microtiter plates were used without further purification.
MPLC (60 g, RP-18 column, A: water, B: methanol, A: 99% ? 60%,
120 min) yielding title compound (32 mg, 80.7 lmol, 95%; 88%
over three steps) as a colourless lyophylisate; Rf 0.14 (methanol/
ethyl acetate, 1:3); ½a D23
ꢀ
+27.2 (c 0.25, methanol); 1H NMR
(500 MHz, CD3OD, 300 K): d = 4.76 (d, 3J = 1.7 Hz, 1H, H-1Man),
4.6. N-(tert-Butyloxycarbonyl)-L-methionine-[2-(2,3,4,6-tetra-
4.42 (dd, 3J = 5.4 Hz, 3J = 8.9 Hz, 1H, HMet, ), 3.84 (dd, 2J = 11.8 Hz,
a
O-acetyl- -mannopyranosyloxy)ethyl]amide (3)
a
-
D
3J = 2.3 Hz, 1H, H-6aMan), 3.80 (dd, 3J = 1.7 Hz, 3J = 3.4 Hz. 1H, H-
2Man), 3.76 (mc, 1H, OCHHCH2NH), 3.71 (dd, 3J = 3.4 Hz,
3J = 9.8 Hz, 1H, H-3Man), 3.71–3.67 (m, 1H, H-6bMan), 3.58 (ddꢁt,
3J = 9.8 Hz, 1H, H-4Man), 3.55–3.49 (m, 2H, H-5Man, OCH2CHHNH),
3.48–3.42 (m, 1H, OCH2CHHNH), 3.40–3.34 (m, 1H, OCH2CHHNH),
2.59–2.46 (m, 2H, CH2CH2SCH3), 2.09 (s, 3H, CH2CH2SCH3), 2.08–
2.01 (m, 1H, CHHCH2SCH3), 2.00 (s, 3H, NHC(O)CH3), 1.94–1.85
(m, 1H, CHHCH2SCH3) ppm; 13C NMR (125 MHz, CD3OD, 300 K):
d = 174.2 (NHC(O)Met), 173.5 (NHC(O)CH3), 101.7 (C-1Man), 74.8
(C-5Man), 72.6 (C-3Man), 72.1 (C-2Man), 68.8 (C-4Man), 67.0
According to general procedure for Staudinger ligation azido-
functionalised mannoside 1 (1.00 g, 2.40 mmol), Boc-Met-OH (2,
1.08 g, 4.32 mmol) and HOBt (584 mg, 4.32 mmol) were dried for
1 h in vacuo and dissolved in anhydrous THF (20 mL). DIC
(670 lL, 4.32 mmol) and n-tributylphosphane (592 lL, 2.40 mmol)
were added and it was stirred overnight. After a standard work-up,
the crude product was subjected to MPLC (100 g, silica column, A:
ethyl acetate, B: cyclohexane, A: 20% ? 100%, 180 min) yielding
the title compound (1.08 g, 1.73 mmol, 72%) as a colourless foam;
(OCH2CH2NH), 63.0 (C-6Man), 54.1 (CHMet, ), 40.3 (OCH2CH2NH),
a
Rf 0.63 (ethyl acetate); ½a D23
ꢀ
+29.6 (c 0.5, methanol); 1H NMR
(500 MHz, CD3OD, 300 K): d = 5.30 (mc, 1H, H-3Man), 5.25 (dd,
3J = 1.9 Hz, 3J = 3.2 Hz, 1H, H-2Man), 5.24 (ddꢁt, 3J = 10.0 Hz, 1H,
H-4Man), 4.86 (br s, 1H, H-1Man), 4.27 (dd, 2J = 12.2 Hz, 3J = 5.1 Hz,
32.6 (CH2CH2SCH3), 31.2 (CH2CH2SCH3), 22.5 (NHC(O)CH3), 15.3
(CH2CH2SCH3) ppm; MALDI-ToF MS (Cl-CCA): calcd for
C
15H28N2NaO8S: m/z 419.15 [M+Na]+; found: m/z 419.19
[M+Na]+, calcd for C15H28KN2O8S: m/z 435.12 [M+K]+; found: m/z
435.19 [M+K]+; HR-ESI MS: calcd for C15H28NaN2O8S: m/z
419.1459 [M+Na]+; found: m/z 419.1439 [M+Na]+; IR (ATR):
~
1H, H-6aMan), 4.17 (dd, 3J = 5.0 Hz, 3J = 8.4 Hz, 1H, HMet, ), 4.09
a
(dd, 2J = 12.2 Hz, 3J = 1.9 Hz, 1H, H-6bMan), 4.06–4.01 (m, 1H, H-
5Man), 3.79 (mc, 1H, OCHHCH2NH), 3.60–3.54 (m, 1H,
m = 3272, 2919, 1644, 1538, 1429, 1373, 1292, 1132, 1054, 1031,
OCHHCH2NH), 3.53–3.46 (m, 1H, OCH2CHHNH), 3.44–3.38 (m,
1H, OCH2CHHNH), 2.60–2.47 (m, 2H, CH2CH2SCH3), 2.14 (s, 3H,
OC(O)CH3), 2.09 (s, 3H, CH2CH2SCH3), 2.07 (s, 3H, OC(O)CH3),
2.07–1.98 (m, 1H, CHHCH2SCH3), 2.04 (s, 3H, OC(O)CH3), 1.96 (s,
3H, OC(O)CH3), 1.91–1.82 (m, 2H, CHHCH2SCH3), 1.45 (s, 9H,
C(CH3)3) ppm; 13C NMR (125 MHz, CD3OD, 300 K): d = 175.0
(NHC(O)Met), 172.4 (OC(O)CH3), 170.6, 170.5, 170.5 (3 OC(O)CH3),
157.8 (NHC(O)O), 98.9 (C-1Man), 80.7 (C(CH3)3), 70.8 (C-2Man),
70.6 (C-3Man), 69.9 (C-5Man), 67.7 (OCH2CH2NH), 67.2 (C-4Man),
974, 915, 880, 807 cm-1; EA: calcd for C15H32N2O10 ꢃ 2 H2O: C
41.66; H 7.46; N 6.48; S 7.41; found: C 41.95; H 6.88; N 6.35; S
7.09.
4.8.
L-Methionine-[2-(a-D-mannopyranosyloxy)ethyl]amide
trifluoroacetate (5)
According to the general procedure mannoside 3 (700 mg,
1.12 mmol) was de-O-acetylated over 4.5 h and the resulting prod-

28
J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
~
uct purified by MPLC (60 g, RP-18 column, A: water, B: methanol,
A: 50% ? 0%, 120 min) to yield the OH-unprotected intermediate
(499 mg, 1.10 mmol, 98%) as a colourless syrup; Rf 0.24 (RP-18,
water/methanol, 3:1); Rf 0.44 (methanol/ethyl acetate, 1:3). Part
m
= 3323, 2976, 2940, 1743, 1659, 1528, 1432, 1367, 1275, 1216,
1169, 1135, 1081, 1043, 977, 764, 750 cmꢂ1
.
4.10. N-[2-(Acetamido)ethyl]imino dipropionic acid N0,N00-di-[2-
-mannopyranosyloxy)ethyl] diamide (8)
of this N-Boc-protected intermediate (240 mg, 528
lmol) was trea-
(a-D
ted with TFA (3 mL) for 1.5 h according to the general procedure.
After co-distillation, the residue was lyophilised to yield the title
Glycocluster 7 (161 mg, 153
lmol) was treated according to the
compound (247 mg, 527
l
ꢀ
mol, 99%, 97% over two steps) as a col-
general procedure for N-Boc deprotection with TFA (118 l
dichloromethane (2 mL) for 9 h. After co-distillation, the residue
was lyophilised yielding the O-acetylated free amine intermediate
L) in
ourless lyophilisate;
½
a 2D3
+41.1 (c 0.5, methanol); 1H NMR
(500 MHz, CD3OD, 300 K): d = 4.77 (d, 3J = 1.6 Hz, 1H, H-1Man),
3.96 (ddꢁt, 3J = 6.7 Hz, 1H, HMet, ), 3.85 (dd, 2J = 11.8 Hz,
(163 mg, 153
(methanol/ethyl acetate, 1:3). Part of this intermediate (110 mg,
103 mol) was treated according to the general procedure for
N,O-acetylation with pyridine (5 mL) and acetic anhydride
(157 L, 155 mol) overnight. Then volatile compounds were re-
lmol, quant.) as a colourless lyophilisate; Rf 0.21
a
3J = 2.1 Hz, 1H, H-6aMan), 3.83–3.77 (m, 2H, H-2Man, OCHHCH2NH),
3.71–3.64 (m, 2H, H-3Man
,
H-6bMan), 3.62–3.55 (m, 1H,
l
OCH2CHHNH), 3.57 (ddꢁt, 3J = 9.2 Hz, 1H, H-4Man), 3.55–3.52 (m,
1H, H-5Man,), 3.52–3.48 (m, 1H, OCH2CHHNH), 3.45–3.38 (m, 1H,
OCH2CHHNH), 2.58 (t, 3J = 7.6 Hz, 2H, CH2CH2SCH3), 2.18–2.06
(m, 2H, CH2CH2SCH3), 2.13 (s, 3H, CH2CH2SCH3) ppm; 13C NMR
(125 MHz, CD3OD, 300 K): d = 169.8 (NHC(O)Met), 101.6 (C-1Man),
75.0 (C-5Man), 72.6 (C-3Man), 72.0 (C-2Man), 68.8 (C-4Man), 67.0
l
l
moved and the residue was codistilled. The crude product was dis-
solved in dichloromethane (5 mL) and washed three times with
water (5 mL each). The organic layer was evaporated yielding the
crude fully N,O-protected intermediate as slightly yellow foam.
Then, according to the general procedure for de-O-acetylation, this
intermediate was treated overnight with sodium in dry methanol
(3 mL). After neutralisation, the crude product was subjected to
MPLC purification (120 g, RP-18 column, A: water, B: methanol,
A: 90% ? 50%, 120 min) yielding the title compound (58 mg,
(OCH2CH2NH), 63.1 (C-6Man), 53.7 (CHMet, ), 40.6 (OCH2CH2NH),
a
32.1 (CH2CH2SCH3), 29.9 (CH2CH2SCH3), 15.1 (CH2CH2SCH3) ppm;
MALDI-ToF MS (DHB): calcd for C13H26N2NaO7S: m/z 377.14
[M+Na]+; found: m/z 377.21 [M+Na]+; calcd for C13H26KN2O7S:
m/z 393.11 [M+K]+; found: m/z 393.21 [M+K]+; HR-ESI MS: calcd
for C13H26N2NaO7S: m/z 377.1353 [M+Na]+; found: m/z 377.1359
88.3
l
mol, 86% over three steps) as a colourless foam; ½a D23
ꢀ
+31.1
+
[M+Na] ; IR (ATR):
= 3288, 3091, 2924, 1667, 1542, 1430, 1292,
(c 0.25, methanol); 1H NMR (500 MHz, CD3OD, 300 K): d = 4.77
(d, 3J = 1.7 Hz, 2H, H-1Man), 3.84 (dd, 2J = 11.8 Hz, 3J = 2.3 Hz, 2H,
H-6aMan), 3.82 (dd, 3J = 3.4 Hz, 3J = 1.7 Hz, 2H, H-2Man), 3.77 (mc,
2H, OCHHCH2NH), 3.72–3.67 (m, 4H, H-3Man, H-6bMan), 3.60 (ddꢁt,
3J = 9.7 Hz, 3J = 9.3 Hz, 2H, H-4Man), 3.58–3.51 (m, 4H, OCHHCH2NH,
H-5Man), 3.48–3.42 (m, 2H, OCH2CHHNH), 3.39–3.33 (m, 2H,
OCH2CHHNH), 3.26 (t, 3J = 6.7 Hz, 2H, NHCH2CH2N), 2.80 (t,
3J = 6.8 Hz, 4H, NCH2CH2C(O)NH), 2.59 (t, 3J = 6.7 Hz, 2H,
NHCH2CH2N), 2.37 (t, 3J = 6.8 Hz, 4H, NCH2CH2C(O)NH), 1.96 (s,
3H, NHC(O)CH3) ppm; 13C NMR (125 MHz, CD3OD, 300 K):
d = 175.2 (C(O) NH), 173.2 (NHC(O)CH3), 101.7 (C-1Man), 74.7 (C-
~
m
1184, 1130, 1092, 1054, 1032, 974, 916, 879, 837, 800 cmꢂ1
.
4.9. N-[2-(tert-Butyloxycarbonylamino)ethyl]imino dipropionic
acid N0,N00-di-[2-(2,3,4,6-tetra-O-acteyl-
a-D-mannopyranosyloxy)
ethyl] diamide (7)
According to the general procedure for Staudinger ligation,
mannoside 1 (1.44 g, 3.45 mmol) was combined with the divalent
acid 6 (520 mg, 1.71 mmol), HOBt (462 mg, 3.42 mmol) was added
and the mixture was dried for 1 h in vacuo. Anhydrous THF
5Man), 72.6 (C-3Man), 72.1 (C-2Man), 68.7 (C-4Man), 67.3
(40 mL), DIC (533 lL, 3.42 mmol) and n-tributyl phosphane
(OCH2CH2NH), 63.0 (C-6Man), 53.3 (NHCH2CH2N), 51.0
(NCH2CH2C(O)NH), 40.3 (OCH2CH2NH), 39.9 (NHCH2CH2N), 34.6
(NCH2CH2C(O)NH), 22.7 (NHC(O)CH3) ppm; HR-ESI MS: calcd for
(1.26 mL, 5.04 mmol) were added and it was stirred for 40 h. Then,
standard work-up gave the crude product which was subjected to
MPLC purification (200 g, silica column, A: methanol, B: ethyl ace-
tate, A: 0% ? 20%, 240 min) to yield title compound (985 mg,
C26H49N4O15
:
m/z 657.3189 [M+H]+; found: m/z 657.3199
[M+H]+; IR (ATR):
= 3289, 2939, 1630, 1555, 1430, 1376, 1202,
~
m
1130, 1095, 1056, 1032, 975, 838, 801, 722 cmꢂ1
.
937 lmol, 55%) as a colourless foam; Rf 0.31 (methanol/ethyl ace-
tate, 1:9); ½a 2D3
ꢀ
+40.2 (c 0.5, methanol); 1H NMR (600 MHz, CD3OD,
298 K): d = 5.29 (dd, 3J = 10.1 Hz, 3J = 3.4 Hz, 2H, H-3Man), 5.26 (dd,
3J = 3.4 Hz, 3J = 1.6 Hz, 2H, H-2Man), 5.24 (ddꢁt, 3J = 10.1 Hz,
3J = 9.9 Hz, 2H, H-4Man), 4.88 (d, 3J = 1.6 Hz, 2H, H-1Man), 4.24 (dd,
2J = 12.2 Hz, 3J = 5.3 Hz, 2H, H-6aMan), 4.13 (dd, 2J = 12.2 Hz,
3J = 2.4 Hz, 2H, H-6bMan), 4.04 (mc, 2H, H-5Man), 3.81 (mc, 2H,
OCHHCH2NH), 3.59 (mc, 2H, OCHHCH2NH), 3.51–3.40 (m, 4H,
OCH2CH2NH), 3.16 (t, 3J = 6.1 Hz, 2H, NHCH2CH2N), 2.85–2.77 (m,
4H, NCH2CH2C(O)NH), 2.62–2.56 (m, 2H, NHCH2CH2N), 2.44–2.35
(m, 4H, NCH2CH2C(O)NH), 2.14, 2.07, 2.04, 1.96 (each s, each 6H,
OC(O)CH3), 1.44 (s, 9H, C(CH3)3) ppm; 13C NMR (150 MHz, CD3OD,
298 K): d = 175.2 (NHC(O)), 172.4, 171.6, 171.6, 171.5 (8
OC(O)CH3), 158.2 (NHC(O)O), 98.9 (C-1Man), 80.2 (C(CH3)3), 70.8
(C-2Man), 70.6 (C-3Man), 70.0 (C-5Man), 67.8 (OCH2CH2NH), 67.3
4.11. N-(2-Aminoethyl)imino dipropionic acid N0,N00-di-[2-(
mannopyranosyloxy)ethyl] diamide bis-trifluoroacetate (9)
a-D-
According to the general procedure for de-O-acetylation, the
divalent cluster mannoside 7 (71 mg, 67.7 mol) was treated over-
l
night with sodium in dry methanol (3 mL). After neutralisation, the
N-Boc-protected intermediate amine was obtained (48 mg,
67.2
water/methanol, 1:1). Part of this intermediate (35 mg, 49.0
was treated overnight according to the general procedure for N-Boc
deprotection with TFA (300 L) in dichloromethane (3 mL). After
co-distillation the residue was lyophilised yielding the title com-
l
mol, 99%) as a colourless lyophilisate; Rf 0.32 (RP-18,
l
mol)
l
pound (41 mg, 49.0
l
ꢀ
mol, quant., 99% over two steps) as a colour-
less lyophylisate; ½a D23
+33.6 (c 0.35, methanol); 1H NMR (500 MHz,
(C-4Man),
63.6
(C-6Man),
53.6
(NHCH2CH2N),
51.0
CD3OD, 300 K): d = 4.77 (d, 3J = 1.7 Hz, 2H, H-1Man), 3.85 (dd,
2J = 11.8 Hz, 3J = 2.3 Hz, 2H, H-6aMan), 3.81 (dd, 3J = 3.3 Hz,
3J = 1.7 Hz, 2H, H-2Man), 3.77 (mc, 2H, OCHHCH2NH), 3.72–3.65
(m, 4H, H-3Man, H-6bMan), 3.56 (ddꢁt, 3J = 9.7 Hz, 3J = 9.2 Hz, 2H,
H-4Man), 3.58–3.51 (m, 4H, OCHHCH2NH, H-5Man), 3.49–3.43 (m,
2H, OCH2CHHNH), 3.42–3.35 (m, 2H, OCH2CHHNH), 3.30 (br s,
2H, NHCH2CH2N), 3.13 (mc, 6H, NCH2CH2C(O)NH, NHCH2CH2N),
2.59 (br s, 4H, NCH2CH2C(O)NH) ppm; 13C NMR (125 MHz, CD3OD,
(NCH2CH2C(O)NH), 40.1 (OCH2CH2NH), 38.6 (NHCH2CH2N), 34.5
(NCH2CH2C(O)NH), 28.8 (C(CH3)3), 20.7, 20.7, 20.6, 20.6 (8
OC(O)CH3) ppm; MALDI-TOF MS (DHB): calcd for C45H71N4O24
m/z 1051.45 [M+H]+; found: m/z 1051.50 [M+H]+; calcd for
C45H70NaN4O24
m/z 1073.43 [M+Na]+; found: m/z 1073.49
[M+Na]+; ESI MS: calcd for C45H71N4O24: m/z 1051.445 [M+H]+;
found: m/z 1051.443 [M+H]+; calcd for C45H70NaN4O24
m/z
1073.427 [M+Na]+; found: m/z 1073.434 [M+Na]+; IR (ATR):
:
:
:

J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
29
300 K): d = 174.2 (NHC(O)), 101.6 (C-1Man), 74.9 (C-5Man), 72.6 (C-
4.14. 3-Cascade:N-(fluorenylmethoxycarbonyl)-S-(benzyl)-L-
3Man), 72.0 (C-2Man), 68.8 (C-4Man), 67.2 (OCH2CH2NH), 63.1 (C-
Man), 50.7 (NCH2CH2C(O)NH), 50.6 (NHCH2CH2N), 40.5
cysteinyl-aminomethane[3]: N0-[2-(2,3,4,6-tetra-O-
pyranosyloxy)ethyl] propionic acid amide (13)
a-D-manno-
6
(OCH2CH2NH), 37.2 (NHCH2CH2N), 32.2 (NCH2CH2C(O)NH) ppm;
+
MALDI-ToF MS (DHB): calcd for
C
24H47N4O14
:
m/z 615.31
According to the general procedure for Staudinger ligation man-
noside 1 (1.31 g, 3.02 mmol), triacid 12 (384 mg, 580 mol) and
HOBt (408 mg, 3.02 mmol) were dried for 1 h in vacuo and
dissolved in anhydrous THF (25 mL). DIC (468 L, 3.02 mmol)
and n-tributylphosphane (754 L, 3.02 mmol) were added and it
[M+H]+; found: m/z 615.31 [M+H]+; HR-ESI MS: calcd for
l
C
24H47N4O14
:
m/z 615.3083 [M+H]+; found: m/z 615.3087
[M+H]+; IR (ATR):
= 3294, 2934, 1669, 1562, 1428, 1344, 1138,
l
~
m
1127, 1092, 1054, 1028, 974, 915, 837, 799 cmꢂ1
.
l
was stirred overnight. After a standard work-up, the crude product
was obtained and subjected to MPLC (150 g, silica column, A: ethyl
acetate, B: cyclohexane, A: 90% ? A: 100%, 105 min; A: ethyl ace-
tate, B: methanol, A: 100% ? 85%, 135 min) followed by size exclu-
sion chromatography (LH-20, methanol) yielding the title
4.12. 3-Cascade:N-(fluorenylmethoxycarbonyl)-S-(benzyl)-L-
cysteinyl-aminomethane[3]:propionic acid tert-butyl ester (11)
Fmoc-Cys(Bn)-OH (728 mg, 1.68 mmol), the trivalent ester 10
(350 mg, 842
l
mol) and HBTU (637 mg, 1.68 mmol) were dried
compound (640 mg, 359 lmol, 62%) as a colourless foam; Rf 0.55
for 1 h in vacuo. Then dry DMF (20 mL) and DIPEA (577
lmol,
(methanol/ethyl acetate, 1:9); ½a D23
ꢀ
+32.6 (c 0.475, chloroform);
3.37 mmol) were added and it was stirred overnight at ambient
temperature. Solvents were removed under reduced pressure and
the crude product was dissolved in dichloromethane (25 mL). It
was washed twice with water and brine (25 mL each), volatile
compounds were removed under reduced pressure and the crude
product was subjected to column chromatography (ethyl acetate/
1H NMR (500 MHz, CDCl3, 300 K): d = 7.74 (d, 3J = 7.6 Hz, 2H, Haryl,Fmoc),
7.60 (mc, 2H, Haryl,Fmoc), 7.38 (dt, 4J = 2.1 Hz, 3J = 7.5 Hz, 2H,
Haryl,Fmoc), 7.32–7.25 (m, 6H, Haryl,Fmoc, Haryl,benzyl), 7.24–7.19 (m,
1H, Haryl,benzyl), 7.14 (br s, 1H, NH), 6.56 (br s, 3H, HNCH2CH2O),
5.94 (d, 3J = 6.7 Hz, 1H, NH), 5.31 (dd, 3J = 10.1 Hz, 3J = 3.5 Hz, 3H,
H-3Man), 5.26 (dd, 3J = 3.5 Hz, 3J = 1.7 Hz, 3H, H-2Man), 5.24 (ddꢁt,
3J = 10.1 Hz, 3J = 10.0 Hz, 3H, H-4Man), 4.80 (d, 3J = 1.7 Hz, 3H,
H-1Man), 4.40 (q, 3J = 10.6 Hz, 3J = 7.3 Hz, 1H, CHHFmoc), 4.26 (t,
3J = 7.3 Hz, 1H, CHHFmoc), 4.23 (dd, 2J = 12.3 Hz, 3J = 5.5 Hz, 3H,
cyclohexane, 1:4) yielding the title compound (609 mg, 733 lmol,
87%) as a colourless solid; Rf 0.34 (ethyl acetate/cyclohexane, 1:3);
1H NMR (500 MHz, CDCl3, 300 K) d = 7.76 (d, 3J = 7.5 Hz, 2H,
Haryl,Fmoc), 7.62–7.59 (m, 2H, Haryl,Fmoc), 7.39 (mc, 2H, Haryl,Fmoc),
H-6aMan), 4.22–4.18 (m, 2H, CHFmoc, HCys, ), 4.10 (mc, 3H, H-6bMan),
a
7.37–7.28 (m, 6H, Haryl,Fmoc, Haryl,benzyl), 7.26–7.22 (m, 1H, Haryl,benzyl),
6.32 (br s, 1H, NH), 5.61 (br s, 1H, NH), 4.44 (mc, 1H, CHHFmoc), 4.38
(br s, 1H, CHHFmoc), 4.23 (t, 3J = 7.1 Hz, 1H, CHFmoc), 4.12 (br s, 1H,
3.98 (m, 3H, H-5Man), 3.78–3.67 (m, 5H, CH2,benzyl, HNCH2CHHO),
3.52–3.44 (m, 6H, HNCH2CHHO, HNCHHCH2O), 3.33–3.25 (m, 3H,
HNCHHCH2O), 2.85–2.73 (m, 2H, CH2,Cys,b), 2.20 (mc, 6H,
CCH2CH2C(O)), 2.11,2.07 (s, 18H, OC(O)CH3), 2.01–1.94 (m, 6H,
CCH2CH2C(O)), 2.00, 1.95 (s, 18H, OC(O)CH3) ppm; 13C NMR
(125 MHz, CDCl3, 300 K): d = 173.5 (3 NHC(O)), 170.7, 170.2,
170.2 (9 OC(O)CH3), 169.9 (NHC(O)), 169.7 (3 OC(O)CH3), 157.9
(OC(O)NH), 143.8, 141.2 (Caryl,Fmoc), 137.8 (Caryl,benzyl), 129.0,
128.7, 127.3 (CHaryl,benzyl), 127.8, 127.1, 125.1 120.0 (CHaryl,Fmoc),
97.6 (C-1Man), 69.4 (C-3Man), 69.2 (C-2Man), 68.7 (C-5Man), 67.1
(CH2,Fmoc), 67.0 (HNCH2CH2O), 66.1 (C-4Man), 62.5 (C-6Man), 58.8
HCys, ), 3.82–3.72 (m, 2H, CH2,benzyl), 2.88–2.80 (m, 1H, CH2,Cys, ba),
a
2.71 (mc, 1H, CH2,Cys, bb), 2.20 (mc, 6H, CCH2CH2C(O)), 1.95 (mc, 6H,
CCH2CH2C(O)), 1.42 (s, 27H, C(CH3)3) ppm; 13C NMR (125 MHz,
CDCl3, 300 K): d = 172.7 (OC(O)), 169.0 (NHC(O)), 158.2 (OC(O)NH),
143.7 (Caryl,benzyl), 141.3, 141.3 (Caryl,Fmoc), 129.0, 128.7 (CHaryl,Fmoc),
127.7, 127.3, 127.1 (CHaryl,benzyl), 125.1, 120.0 (CHaryl,Fmoc), 80.7
(C(CH3)3), 67.2 (CH2,Fmoc), 58.0 (NHC(CH2CH2)3), 54.5 (CHCys, ),
a
47.5 (CHFmoc), 36.8 (CH2,benzyl), 34.2 (CH2,Cys,b), 29.9 (CCH2CH2C(O)),
29.7 (CCH2CH2C(O)), 28.1 (C(CH3)3)ppm; ESI MS: calcd for
(NHC(CH2CH2)3), 54.7 (CHCys, ), 47.1 (CHFmoc), 38.8 (HNCH2CH2O),
a
C
47H62N2NaO9S: m/z 854.073 [M+Na]+; found: m/z 854.452
36.5 (CH2,benzyl), 33.5 (CH2,Cys,b), 31.3 (CCH2CH2C(O)), 30.8
(CCH2CH2C(O)), 20.9, 20.7, 20.7, 20.7 (12 OC(O)CH3) ppm; MALDI-
ToF MS (DHB): calcd for C83H107N5NaO36S: m/z 1804.63 [M+Na]+;
found: m/z 1804.64 [M+Na]+; calcd for C83H107KN5O36S: m/z
~
m = 3326,
[M+Na]+.
4.13. 3-Cascade:N-(fluorenylmethoxycarbonyl)-S-(benzyl)-L-
cysteinyl-aminomethane[3]:propionic acid (12)
1820.60 [M+K]+; found: m/z 1820.61 [M+K]+; IR (ATR):
2942, 1743, 1652, 1531, 1451, 1368, 1218, 1136, 1083, 1044,
The cysteinylated triester 11 (609 mg, 733
lmol) was dis-
977, 762, 744 cmꢂ1
.
solved in formic acid (20 mL) and it was stirred overnight at
ambient temperature. The solvent was removed under reduced
pressure and it was codistilled three times with toluene (10 mL
each). Then it was lyophilised leading to the title compound
4.15. 3-Cascade:S-(benzyl)-L-cysteinyl-aminomethane[3]: N0-[2-
(2,3,4,6-tetra-O-
amide (14)
a-D-mannopyranosyloxy)ethyl] propionic acid
(485 mg, 733 l
mol, quant.) as a colourless lyophilisate. 1H NMR
(500 MHz, CD3OD, 300 K) d = 7.78 (d, 3J = 7.5 Hz, 2H, Haryl,Fmoc),
7.68–7.64 (m, 2H, Haryl,Fmoc), 7.37 (t, 3J = 7.4 Hz, 2H, Haryl,Fmoc),
The Fmoc-protected glycocluster 13 (629 mg, 353 lmol) was
dissolved in DMF (9 mL) and piperidine (1 mL) was added. The
reaction mixture was stirred at ambient temperature for 90 min.
All volatile compounds were removed under reduced pressure
and the crude product was subjected to MPLC (50 g, silica column,
A: ethyl acetate, B: methanol, A: 100% ? 90%) yielding the free
7.34–7.27 (m, 7H, Haryl,Fmoc
,
Haryl,benzyl), 4.43 (dd, 3J = 6.9 Hz,
2J = 10.3 Hz, 1H, CHHFmoc), 4.35 (dd, 3J = 7.3 Hz, 2J = 10.3 Hz, 1H,
CHHFmoc), 4.24–4.22 (m, 2H, HCys, , CHFmoc), 3.77 (s, 2H, CH2,benzyl),
a
2.74 (dd, 3J = 7.2 Hz, 2J = 13.5 Hz, 1H, CH2,Cys,ba), 2.60 (dd,
3J = 7.3 Hz, 2J = 13.5 Hz, 1H, CH2,Cys,bb), 2.32–2.29 (m, 6H,
CCH2CH2C(O)), 2.04–2.00 (m, 6H, CCH2CH2C(O)) ppm; 13C NMR
(125 MHz, CD3OD, 300 K): d = 177.0 (HOC(O)), 172.4 (NHC(O)),
158.2 (OC(O)NH), 145.4 (Caryl,benzyl), 145.2 (C-aryl), 142.6,
142.6 (Caryl,Fmoc), 130.1, 129.6 (CHaryl,Fmoc), 128.8, 128.2, 128.1
(CHaryl,benzyl), 126.2, 120.9 (CHaryl,Fmoc), 68.1 (CH2,Fmoc), 59.0
amine (541 mg, 347 lmol, 98%) as a colourless syrup; Rf 0.31
(methanol/ethyl acetate, 1:5); ½a D23
ꢀ
+41.7 (c 0.5, methanol); 1H
NMR (500 MHz, CDCl3, 300 K): d = 7.44 (br s, 1H, NH), 7.29–7.25
(m, 4H, Haryl,benzyl), 7.22–7.18 (m, 1H, Haryl,benzyl), 6.56 (t,
3J = 5.5 Hz, 3H, HNCH2CH2O), 5.27 (dd, 3J = 9.9 Hz, 3J = 3.4 Hz, 3H,
H-3Man), 5.25–5.20 (m, 6H, H-2Man, H-4Man), 4.79 (d, 3J = 1.5 Hz,
3H, H-1Man), 4.23 (dd, 2J = 12.3 Hz, 3J = 5.5 Hz, 3H, H-6aMan), 4.10–
4.05 (m, 3H, H-6bMan,), 3.96 (m, 3H, H-5Man), 3.75–3.67 (m, 5H,
(NHC(CH2CH2)3), 56.3 (CHCys, ), 48.4 (CHFmoc), 37.0 (CH2,benzyl),
a
33.8 (CH2,Cys,b), 30.5 (CCH2CH2C(O)), 29.2 (CCH2CH2C(O)) ppm;
HR-ESI MS: calcd for C35H39N2O9S: m/z 663.2376 [M+H]+; found:
m/z 663.2384 [M+H]+.
CH2,benzyl, HNCH2CHHO), 3.52–3.42 (m, 7H, HCys, , HNCH2CHHO,
a
HNCHHCH2O), 3.35–3.28 (m, 3H, HNCHHCH2O), 2.83 (dd,

30
J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
3J = 5.3 Hz, 2J = 13.4 Hz, CH2,Cys,ba), 2.73 (dd, 3J = 7.4 Hz, 2J = 13.4 Hz,
CH2,Cys,bb), 2.19 (t, 3J = 7.9 Hz, 6H, CCH2CH2C(O)), 2.11, 2.06 (each s,
18H, OC(O)CH3), 2.02–1.92 (m, 6H, CCH2CH2C(O)), 2.00, 1.94 (each
s, 18H, OC(O)CH3) ppm; 13C NMR (125 MHz, CDCl3, 300 K):
d = 173.5 (3 NHC(O)), 171.1 (NHC(O)), 170.7, 170.2, 170.2, 169.7
(12 OC(O)CH3), 137.9 (Caryl,benzyl), 128.9, 128.7, 127.3 (CHaryl,benzyl),
97.6 (C-1Man), 69.4 (C-3Man), 69.2 (C-2Man), 68.6 (C-5Man), 67.0
(HNCH2CH2O), 66.0 (C-4Man), 62.4 (C-6Man), 58.3 (NHC(CH2CH2)3),
lyophilisate; ½a D23
ꢀ
+45.2 (c 0.5, methanol); 1H NMR (500 MHz,
CD3OD, 300 K): d = 7.40–7.29 (m, 4H, Haryl,benzyl), 7.27–7.23 (m,
1H,
H
aryl,benzyl), 4.77 (d, 3J = 1.6 Hz, 3H, H-1Man), 3.85 (dd,
2J = 12.0 Hz, 3J = 2.3 Hz, 3H, H-6aMan), 3.84–3.81 (m, 5H, CH2,benzyl
,
H-2Man), 3.77–3.73 (m, 3H, HNCH2CHHO), 3.73–3.66 (m, 7H, H-
3
4
Man, H-6bMan, HCys, , 3.61 (ddꢁt, 3J = 9.7 Hz, 3J = 9.2 Hz, 3H, H-
a
Man), 3.57–3.51 (m, 6H, H5Man, HNCH2CHHO), 3.46–3.40 (m, 3H,
HNCHHCH2O), 3.38–3.32 (m, 3H, HNCHHCH2O), 2.89 (dd,
3J = 6.1 Hz, 2J = 13.6 Hz, CH2,Cys,ba), 2.75 (dd, 3J = 7.5 Hz,
2J = 13.4 Hz, CH2,Cys,bb), 2.27–2.17 (m, 6H, CCH2CH2C(O)), 2.01 (t,
3J = 8.3 Hz, 6H, CCH2CH2C(O)) ppm; 13C NMR (125 MHz, CD3OD,
300 K): d = 176.1 (NHC(O)), 176.0 (3 NHC(O)), 139.3 (Caryl,benzyl),
130.1, 129.7, 128.3 (CHaryl,benzyl), 101.7 (C-1Man), 74.8 (C-5Man),
72.6 (C-3Man), 72.1 (C-2Man), 68.7 (C-4Man), 67.3 (HNCH2CH2O),
53.9 (CHCys, ), 38.9 (HNCH2CH2O), 36.5 (CH2,benzyl), 36.1 (CH2,Cys,b),
a
31.2 (CCH2CH2C(O)), 30.7 (CCH2CH2C(O)), 20.8, 20.7, 20.7, 20.6 (12
OC(O)CH3) ppm; MALDI-ToF MS (DHB): calcd for C68H97N5NaO34S:
m/z 1582.56 [M+Na]+; found: m/z 1586.68 [M+Na]+; calcd for
C
68H97KN5O34S: m/z 1598.54 [M+K]+; found: m/z 1598.64 [M+K]+;
ESI MS: calcd for C68H98N5O34S: m/z 1560.581 [M+H]+; found: m/
z 1560.586 [M+H]+; IR (ATR):
= 3302, 2944, 1740, 1651, 1538,
63.0 (C-6Man), 59.6 (NHC(CH2CH2)3), 55.0 (CHCys, ), 40.4
(HNCH2CH2O),
~
m
a
1432, 1368, 1217, 1135, 1082, 1044, 978, 897, 692 cmꢂ1
.
37.3
(CH2,benzyl),
36.0
(CH2,Cys,b),
31.8
(CCH2CH2C(O)), 31.3 (CCH2CH2C(O)) ppm; MALDI-ToF MS (Cl-
CCA): calcd for C44H74N5O22S: m/z 1056.45 [M+H]+; found: m/z
1056.44 [M+H]+; calcd for C44H73N5NaO22S: m/z 1078.44
[M+Na]+; found: m/z 1078.45 [M+Na]+; ESI MS: calcd for
4.16. 3-Cascade:N-(acetyl)-S-(benzyl)-L-cysteinyl-aminometh-
ane[3]: N0-[2-
amide (15)
a-D-mannopyranosyloxy)ethyl] propionic acid
C
44H74N5O22S: m/z 1056.454 [M+H]+; found: m/z 1056.455
~
m
According to the general procedure for acetylation the amine 14
(124 mg, 79.5 mol) was treated with pyridine (3 mL) and acetic
anhydride (300 L) for 2 h at ambient temperature. Then volatile
[M+H]+; IR (ATR):
= 3292, 2929, 1634, 1424, 1244, 1202, 1132,
l
1056, 1028, 969, 806 cmꢂ1
.
l
compounds were removed and after co-distillation the crude prod-
uct was dissolved in dichloromethane (5 mL) and washed three
times with water (5 mL each). The organic layer was evaporated
and the crude product was subjected to de-O-acetylation according
to the general procedure using sodium in dry methanol (5 mL) for
2 h. After neutralisation, purification of the product by RP-MPLC
(120 g, RP-18 column, A: water, B: methanol, A: 25% ? 50%,
4.18. Biological assays
4.18.1. Media, buffer solutions, bacteria
LB-medium (+AMP,+CAM) (PKL1162): Tryptone (10.0 g), sodium
chloride (10.0 g) and yeast extract (5.00 g) were dissolved in bid-
est. water (1.00 L); after sterilisation, ampicillin (100 mg) and
chloramphenicol (50.0 mg) were added; PBS buffer solution (pH
7.2): Sodium chloride (8.00 g), potassium chloride (200 mg), so-
dium hydrogen phosphate-dihydrate (1.44 g) and potassium dihy-
drogen phosphate (200 mg) were dissolved in bidest. water
(1.00 L); PBST buffer solution (pH 7.2): PBS buffer + 0.05% v/v
TweenÒ 20. pH-values were adjusted with 0.1 M HCl or 0.1 M
NaOH, respectively. Carbonate buffer solution (pH 9.6): Sodium car-
bonate (10.6 g) and sodium hydrogen carbonate (8.40 g) were dis-
solved in bidest. water. Bacteria culture: E. coli bacteria
(PKL1162)38,40 were used and grown in LB-media + AMP + CAM
(100 mg ampicillin, 50 mg chloramphenicol/L) at 37 °C under
slight agitation.38
90 min) gave the pure title compound (82 mg, 74.7 lmol, 94% over
two steps) as a colourless lyophilisate; Rf 0.57 (RP-18, water/meth-
anol, 1:1); ½a 2D3
ꢀ
+43.8 (c 0.5, methanol); 1H NMR (500 MHz, CD3OD,
aryl,benzyl), 7.26–7.22 (m, 1H,
300 K): d = 7.37–7.27 (m, 4H,
H
H
aryl,benzyl), 4.77 (d, 3J = 1.5 Hz, 3H, H-1Man), 4.36 (t, 3J = 7.2 Hz,
1H, HCys, , 3.84 (dd, 2J = 11.9 Hz, 3J = 2.4 Hz, 3H, H-6aMan), 3.83–
a
3.80 (m, 5H, H-2Man, CH2,benzyl), 3.79–3.73 (m, 3H, HNCH2CHHO),
3.73–3.66 (m, 6H, H-3Man
,
H-6bMan
,
3.60 (ddꢁt, 3J = 9.8 Hz,
3J = 9.2 Hz, 3H, H-4Man), 3.57–3.50 (m, 6H, H-5Man, HNCH2CHHO),
3.46–3.39 (m, 3H, HNCHHCH2O), 3.38–3.32 (m, 3H, HNCHHCH2O),
2.80 (dd, 3J = 6.8 Hz, 2J = 13.5 Hz, CH2,Cys,ba), 2.67 (dd, 3J = 7.7 Hz,
2J = 13.5 Hz, CH2,Cys,bb), 2.25–2.19 (m, 6H, CCH2CH2C(O)), 2.01 (s,
3H, NHC(O)CH3), 2.00–1.96 (m, 6H, CCH2CH2C(O)) ppm; 13C NMR
(125 MHz, CD3OD, 300 K): d = 176.0 (3 NHC(O)), 172.2 (NHC(O)),
139.5 (Caryl,benzyl), 130.2, 129.6, 128.2 (CHaryl,benzyl), 101.7 (C-
4.18.2. Covalent surface functionalisation
Serial dilutions of the amino-functionalised carbohydrate in
carbonate buffer (100 mM, pH 9.6, 60 lL) were pipetted into 96
1
Man), 74.8 (C-5Man), 72.6 (C-3Man), 72.1 (C-2Man), 68.8 (C-4Man),
well Black Immobilizer Amino™ F96 MicroWell™ plates (Thermo
Fisher Scientific, Nunc). Immobilisation was carried out overnight
at room temperature under gentle agitation. The wells were
67.3 (HNCH2CH2O), 63.0 (C-6Man), 59.6 (NHC(CH2CH2)3), 55.2
(CHCys, ), 40.4 (HNCH2CH2O), 37.0 (CH2,benzyl), 36.0 (CH2,Cys,b),
a
31.9 (CCH2CH2C(O)), 31.3 (CCH2CH2C(O)), 22.6 (NHC(O)CH3) ppm;
MALDI-ToF MS (Cl-CCA): calcd for C46H75N5NaO23S: m/z 1120.45
[M+Na]+; found: m/z 1120.41 [M+Na]+; HR-ESI MS: calcd for
washed with PBST three times (150
bation with ethanolamine solution (10 mM in PBS, 100 mM pH 7.2;
100 L/well) for 2 h at room temperature under slight agitation.
lL/well) and blocked by incu-
l
C
46H75N5NaO23: m/z 1120.4466 [M+Na]+; found: m/z 1120.4476
+
~
m
[M+Na] ; IR (ATR):
= 3282, 2932, 1634, 1539, 1430, 1373, 1245,
EA: calcd for
46H75N5O23S ꢃ 4 H2O: C 47.21; H 7.15; N 5.98; found: C 47.20;
H 6.99; N 5.94.
4.18.3. Bacterial adhesion assay on microtiter plates
1132, 1055, 1032, 969, 881, 806 cmꢂ1
;
After washing the plate with PBST three times (150 lL/well) a
C
suspension of the bacteria (60
all wells and the plate was agitated for 60 min at 37 °C. The wells
were washed three times with PBS (150 L/well). PBS (100 L/
lL, 1 mg/mL in PBS) was added to
l
l
4.17. 3-Cascade:S-(benzyl)-L-cysteinyl-aminomethane[3]: N0-
[2- -D-mannopyranosyloxy)ethyl] propionic acid amide (16)
well) was added to the wells and the surface bound bacteria were
detected via fluorescence readout (excitation wavelength, 485 nm,
emission wavelength 535 nm).
a
According to the general procedure for deacetylation the amine
14 (186 mg, 119 mol) was treated with sodium in dry methanol
l
4.18.4. Mannan coating
(3 mL) overnight and it was neutralised. The crude product was
washed three times with dichloromethane (5 mL each) yielding
Black 96-well plates (Nunc Maxisorp) were filled with a solu-
tion of mannan from Saccharomyces cerevisiae (1.2 mg/mL in car-
the title compound (124 mg, 118
lmol, 99%) as a colourless
bonate buffer, pH 9.6; 120 lL solution per well) and allowed to

J. W. Wehner et al. / Carbohydrate Research 371 (2013) 22–31
31
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L/well). Then
l
l
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Funding of this work by the DFG (collaborative network
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Supplementary data
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