Bi- and trivalent glycopeptide mannopyranosides as inhibitors of type 1 fimbriae-mediated bacterial adhesion: Variation of valency, aglycon and scaffolding

Article abstract of DOI:10.1016/j.carres.2011.04.023

In order to test relevant structural parameters for effective inhibition of mannose-specific bacterial adhesion, bi- and trivalent glycopeptide α-d-mannopyranosides were synthesized that differ in their conformational properties as well as in the spatial

Full text of DOI:10.1016/j.carres.2011.04.023

Carbohydrate Research 346 (2011) 1519–1526
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Bi- and trivalent glycopeptide mannopyranosides as inhibitors of type 1
fimbriae-mediated bacterial adhesion: variation of valency, aglycon and
scaffolding
⇑
Alexander Schierholt, 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:
Available online 24 April 2011
In order to test relevant structural parameters for effective inhibition of mannose-specific bacterial adhe-
sion, bi- and trivalent glycopeptide a-D-mannopyranosides were synthesized that differ in their confor-
mational properties as well as in the spatial arrangement of attached mannosyl residues. They were
tested in an inhibition adhesion assay with fluorescent Escherichia coli bacteria and testing results were
Dedicated to Professor András Lipták on the
occasion of his 75th birthday
referenced to the inhibitory potency of methyl
ure of the mannoside aglycon moiety, scaffolding of
a
-
D
-mannopyranoside. It was shown, that besides the nat-
-mannopyranosides on a peptide backbone was
a
-D
Keywords:
important for the performance of the synthesized glycopeptides as inhibitors of bacterial adhesion.
Glycopeptides
Mannopyranosides
Bacterial adhesion
Type 1 fimbriae
Antiadhesives
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
tive FimH antagonists that can be employed as specific
antiadhesives.8–11 Antiadhesive therapy could eventually be uti-
Adhesion of bacteria to the surface of their eukaryotic target
cells is mediated by adhesive organelles projecting from the bacte-
rial surface. These proteinaceous hair-like appendages are called
fimbriae or, somewhat inaccurately, pili.1 Mostly, bacterial adhe-
sion is a prerequisite for infection such as in the case of uropatho-
genic Escherichia coli (UPEC), which cause urinary tract infections,
one of the most common infections with millions of infected
patients every year.2
lized to treat infections such as urinary tract infections.12,13
Design of ligands for the type 1 fimbrial lectin FimH should con-
sider a number of known structural features as well as a number of
unknowns: It is known from X-ray studies that the bacterial lectin
FimH accommodates one single
a-D-mannosyl residue with the
aglycon portion sticking out of the carbohydrate binding site.14–
16
Furthermore, it has been clearly elucidated that the entrance
of the FimH carbohydrate binding site is flanked by two aromatic
tyrosine residues, those of Tyr48 and Tyr137, forming what has
been called a ‘tyrosine gate’.17 Therefore, synthetic mannopyrano-
sides with an aromatic aglycon show increased affinity for FimH
E. coli bacteria possess
a-mannoside-specific type 1 fimbriae,
which have been identified as a major virulence factor in UPEC
infections.3,4 They have been thoroughly investigated, however,
type 1 fimbriae-mediated bacterial adhesion in an in vivo-environ-
ment has not been fully understood until today.5 Therefore, for
many years, it has been our goal to synthesize and employ syn-
thetic mannopyranosides as well as mannosidic glycomimetics to
improve our understanding of type 1 fimbriae-mediated bacterial
adhesion.6,7 In addition, it has been appealing to design high-affin-
ity ligands for the fimbrial lectin FimH in an attempt to find effec-
due to
p–p interactions of the mannosidic aglycon with the tyro-
sine gate at the entrance of FimH. This knowledge can be utilized
when FimH antagonists are designed.7,11 In addition, mannopyran-
osides with a rather extended aglycon moiety have often shown
enhanced binding affinity,18–20 but in this case no conclusive inter-
pretation of structure–activity relationships has been achieved.
The same is true for multivalency effects that have frequently been
observed with miscellaneous cluster mannopyranosides as ligands
for type 1 fimbriated bacteria, especially with bi- and trivalent gly-
coclusters.21–25 A possible explanation, why multivalent mannopy-
ranosides perform well as inhibitors of FimH, can be found in a
statistical effect (basically an elevated local mannoside concentra-
tion),26 on the other hand, it has been speculated that secondary
Abbreviations: Fmoc, fluorenylmethoxycarbonyl; Gly,
L-glycine; Ser, L-serine;
Lys,
L-lysine; DPr, -diaminopropionic acid; Ala, -alanine; HATU, O -(7-aza-
L
L
benzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; DIPEA, N-
ethyl-N-diisopropylamine; GFP, green fluorescent protein.
⇑
Corresponding author. Tel.: +49 431 8802023; fax: +49 431 8807410.
E-mail address: tklind@oc.uni-kiel.de (T. K. Lindhorst).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.04.023

1520
A. Schierholt et al. / Carbohydrate Research 346 (2011) 1519–1526
binding sites on the fimbrial lectin might be occupied by multiva-
lent mannnosides, hence resulting in an increased affinity of such
ligands.27
On this account, a number of open questions about carbohy-
drate recognition of type 1 fimbriated E. coli bacteria have formed
the basis of a study, in which bi- and trivalent low-molecular
weight glycopeptides have been synthesized and tested as inhibi-
tors of type 1 fimbriae-mediated bacterial adhesion to a mannan-
coated surface.
glycopeptides having mannopyranoside 5 attached. Both car-
boxy-functionalized mannopyranosides, 5 as well as 6, could be
obtained from mannose in four easy steps according to litera-
ture-known procedures.28–30
For the preparation of the target glycopeptides, HATU-mediated
peptide coupling was employed. First pentapeptide 1 was reacted
with the aliphatic carboxylic acid 5, as well as with its benzylic
analogue 6 to yield the bivalent glycopeptides 7 and 8, respectively
(Scheme 1). Likewise pentapeptide 2, the di-lysine analogue of
pentapeptide 1, furnished the respective bivalent glycopeptides 9
and 10. In all peptide coupling reactions a three-fold excess of
the carboxy-functionalized mannoside was employed to drive the
reaction to completion. Nevertheless, mono-coupling products
were formed as side products in all cases, in addition to other side
products, according to MS analysis. Unfortunately, purification of
the obtained product mixtures was extremely tedious. Only a com-
bination of MPLC, followed by gel chromatography (GPC) to re-
move low molecular weight impurities, and final HPLC
purification gave pure products, in considerably lowered yields.
Here, yields were not optimized, but the pure structurally varied
glycopeptide mannopyranosides were employed in biological tests.
2. Results and discussion
A collection of six mannosidic glycopeptides were prepared in
order to vary three different structural parameters, which might
influence their affinity as ligands for the bacterial lectin FimH: (i)
carbohydrate valency, (ii) the glycoside aglycon, and particularly
(iii) the spatial arrangement of the ligated mannopyranosides.
Hence, the peptide backbone functions as a scaffold molecule
and, in addition, may add favourably to glycopeptide–FimH inter-
actions when employed in adhesion assays (Fig. 1).
2.1. Synthesis of glycopeptides
2.2. Adhesion inhibitions assays
Four different peptides, pentapeptides 1–3 and the heptapep-
tide 4 were designed as oligoamines to allow attachment of car-
The synthesized glycopeptide mannopyranosides 7–12 (Scheme
1) were investigated as inhibitors of type 1 fimbriae-mediated bac-
terial adhesion to the polysaccharide mannan on mannan-coated
96-well microtiter plates.31 A GFP-tagged uropathogenic E. coli
strain was employed, which expresses exclusively type 1 fimbriae.
Serial dilutions of the respective inhibitor were incubated with
fluorescent E. coli cells and inhibition curves were determined from
which IC50 values were deduced for each tested glycopeptide. As
the tested glycopeptides could never effect 100% inhibition of bac-
terial adhesion, even at their maximal concentrations employed
(Table 1), IC50 values were defined as the inhibitor concentration,
which causes 50% of the maximally achieved inhibition. On each
boxy-functionalized
a-mannopyranosides via peptide coupling
(Fig. 2). Peptides 1–4 were readily prepared by a standard SPPS (so-
lid phase peptide synthesis) Fmoc protocol and obtained in good
yields and high purity. Amino functional groups were either intro-
duced as
nonproteinogenic amino acid
e
-amino groups of lysine (Lys) (2–4) or by employing the
-diaminopropionic acid (Dpr). When
L
Dpr was used in SPPS a significantly less flexible scaffold peptide
was obtained than when lysine was employed for the synthesis
of analogous peptides. Consequently, glycopeptides derived from
pentapeptide 1 might exhibit quite different properties than anal-
ogous peptides derived from 2.
Carboxy-functionalized mannopyranosides 5 and 6 were se-
lected as principal ligands for the FimH carbohydrate binding site
(Fig. 2). They vary with respect to their aglycon moieties. In light
of the structure of the fimbrial lectin FimH, it was anticipated that
glycopeptides decorated with mannopyranoside 6, carrying the
aromatic aglycon moiety, would perform better as inhibitors of
type 1 fimbriae-mediated bacterial adhesion than corresponding
individual test plate the standard inhibitor methyl a-D-mannopy-
ranoside (MeMan) was tested in parallel. Then, the inhibitory
potencies of tested glycopeptides were referenced to that deter-
mined for MeMan on the same plate, leading to relative inhibitory
potencies (RIP values) for every tested compound. Thus, RIP values
allow to compare the potencies of all tested glycopeptides as inhib-
itors of type 1 fimbriae-mediated bacterial adhesion, because they
are consistently referenced.
Testing results are collected in Table 1. In addition to the mea-
sured IC50 values and deduced RIP values, valency-corrected RIP
values, RIPvc, are provided, in order to assess the inhibitory potency
of a compound irrespective of its mannoside valency.
From the testing results collected in Table 1 it can be seen that
phenyl mannopyranoside residues make a big difference when
compared to alkyl mannopyranosides ligated to the same peptide.
Hence, glycopeptides 8, and 10–12 show enhanced inhibitory
potencies in comparison to the standard glycoside for this assay,
MeMan. Glycopeptides 7 and 9 on the other hand, do not signifi-
cantly exceed the inhibitory potency of MeMan. Glycopeptide 9,
varied
C=O
NH
C=O
NH
varied
however, having two
a-D-mannosyl residues attached on longer
side chains than in 7, has advantages (RIP = 2) over the less flexible
molecule 7 (RIP = 1). When a phenyl moiety is included in the man-
noside aglycone portion, the inhibitory potency of the respective
glycopeptides is directly enhanced. This effect is more pronounced
when 7 is changed into 8, than when 9 is substituted by 10. Strik-
ingly, variation of the spatial exposition of mannoside ligands
makes a big difference as well, as it can be seen by comparison
of 10 (RIP = 20) and 11 (RIP = 8). This result is somewhat unex-
pected but quite instructive as it exemplifies the importance of
the peptide scaffold that is chosen for an equal number of equal
Glycopeptide
Figure 1. Mannopyranoside ligands for the bacterial lectin FimH with diverse
aglycon moieties can be scaffolded on small peptides in order to vary the valency of
the resulting glycopeptides, and the spatial arrangement of the ligated saccharides.

A. Schierholt et al. / Carbohydrate Research 346 (2011) 1519–1526
Gly Dpr
1521
Dpr
O
AcHN-Ala
O
Ala
O
H
N
H
N
OH
N
N
N
H
O
H
H
O
O
NH2
NH2
1
NH2
Lys
Gly
Lys
O
Lys
O
AcHN-Ala
O
Ala
Gly
AcHN-Lys
O
Ala
Ala
O
O
H
H
H
N
H
N
N
N
OH
OH
N
H
N
N
N
N
N
H
H
H
O
H
H
O
O
O
O
O
2
3
NH2
NH2
Gly
NH2
Gly
Lys
Lys
Lys
AcHN-Ala
O
Ala
O
O
O
H
N
H
N
H
N
OH
N
N
H
N
H
N
+
H
O
H
O
O
O
4
NH2
NH2
NH2
bi- and trivalent
glycopeptides
OH
OH
OH
O
OH
O
HO
HO
HO
HO
O
5
6
O
O
OH
O
OH
Figure 2. Pentapeptides 1, 2, and 3, and the heptapeptide 4 were prepared by standard Fmoc SPPS and used as scaffold molecules for the synthesis of bi- and trivalent
glycopeptide mannopyranosides by peptide coupling. Carboxy-functionalized -mannopyranosides, 5 and 6 were employed in peptide coupling reactions in solution.
a-D
mannopyranosides. The best inhibitor tested was the trivalent gly-
copeptide 12, exceeding the inhibitory potency of MeMan by 43-
fold. On a valency-corrected basis, 12 (RIPvc = 14) ranges together
with the bivalent glycopeptide 8 (RIPvc = 13.5).
because intramolecular p–p stacking is hampered. In addition it
should be stated that earlier on, lysine-based glycodendrimers
have been shown to effectively inhibit adhesion of type 1 fimbri-
ated adhesion of E. coli.25
In future studies we will expand this work on the development
of potent inhibitors of type 1 fimbriae-mediated bacterial adhesion
by combining small peptide scaffold molecules with advanced
mannoside residues.34 This approach could lead to very effective
antagonists of the bacterial lectin FimH, also useful in an in vivo-
scenario.
3. Conclusions
From the results obtained in this account it can be concluded
that the glycopeptide approach to inhibitors of type 1 fimbriae-
mediated bacterial adhesion is a promising one. The structurally
versatile system of mannopyranoside scaffolding on a peptide
backbone32 allows to optimize valency (cf. 12), conformational
flexibility (cf. 8), and the spatial arrangement of mannoside ligands
(cf. 10 vs 11). Apparently, it is fundamental for optimization of
inhibitory potencies of mannoside derivatives, to choose manno-
pyranosides with an aromatic aglycon portion, as this leads to
4. Experimental
4.1. Synthesis
4.1.1. General methods
favourable
p
–
p
interactions with the tyrosine gate at the entrance
Thin layer chromatography was performed on silica gel plates
(GF 254, Merck). Detection was effected by UV irradiation and sub-
sequent 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) using dis-
tilled solvents. Optical rotations were measured on a Perkin–Elmer
241 polarimeter (Na-D-line: 589 nm, length of cell 1 dm). MALDI-
MS measurements were recorded on a MALDI-ToF-MS-Biflex III
(Bruker) instrument. Preparative MPLC was performed on an
of the fimbrial lectin FimH. This result is in accordance with pub-
lished work16–20,24 and again confirmed with the herein synthe-
sized, so far unknown glycopeptides.
It can be assumed that intramolecular interactions, such as p–p
stacking interactions of phenyl mannopyranoside residues within
one glycopeptide, might lead to diminished inhibitory potencies.
This has been suggested earlier, after molecular dynamic studies
which have shown, that clustering of phenyl mannopyranosides
on a multivalent scaffold is not necessarily a successful approach
to high affinity ligands for FimH.33 However, here it was also seen,
that a rather narrow assembly of phenyl mannopyranosides such
as in case of glycopeptide 8 can be relatively successful, probably
apparatus of BÜCHI Labortechnik GmbH using
a LiChroprep
RP-18 (40–60 m, Merck) column for reversed-phase silica gel
l
chromatography. Preparative HPLC was performed on a Shimadzu
instrument with diode array detection and a LiChrosorb RP-8

1522
A. Schierholt et al. / Carbohydrate Research 346 (2011) 1519–1526
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
O
HO
HO
HO
HO
HO
HO
HO
HO
O
O
O
O
O
O
O
O
OH
O
NH
NH
7
NH
NH
O
O
O
H
H
H
N
N
N
9
N
H
N
H
OH
O
O
OH
H
H
N
O
O
O
AcHN
N
5
6
N
H
N
H
O
5
6
OH
OH
O
O
a
OH
O
a
OH
O
1
2
OH
HO
HO
HO
HO
OH
O
OH
O
HO
HO
HO
HO
O
O
O
O
O
O
8
10
NH
O
NH
NH
NH
O
O
O
H
N
H
H
N
N
N
N
OH
H
H
O
O
O
O
O
O
H
N
H
N
AcHN
N
H
N
H
OH
O
O
OH
OH
OH
OH
OH
O
OH
O
OH
O
OH
O
HO
HO
HO
HO
HO
HO
HO
HO
O
O
O
O
O
O
NH
NH
O
NH
O
NH
O
O
O
O
O
O
O
6
6
a
H
N
H
H
H
N
H
N
H
4
N
OH
N
N
3
N
N
H
N
H
N
N
N
OH
a
H
H
H
H
O
O
O
O
O
O
O
11
12
NH
O
O
OH
OH
O
OH
OH
Scheme 1. Reagents and conditions: (a) peptide coupling: HATU, DIPEA, DMF, 0 °C to rt, overnight; 7: 21%; 8: 32%; 9: 15%; 10: 16%; 11: 24%; 12: 25%.
(7
l
m) silica gel column (Merck). 1H and 13C spectra were recorded
Fmoc-Ala-Wang resin was swollen in DMF (4 mL) for 2 h before
the synthesis and then deprotected with 20% piperidine solution
in DMF (2 ꢀ 15 min). The next Fmoc-protected amino acid
(4 equiv), HBTU (3.6 equiv) and HOBt (4 equiv) were dissolved in
DMF (3 mL), it was shaken for 5 min, then DIPEA (4 equiv) was
added and this mixture shaken for another 2 min before it was
transferred to the reaction vessel which contained the resin. The
reaction mixture was shaken for 4 h or overnight at rt, filtered
and washed with DMF (5 ꢀ 5 mL). For diaminopropionic acid cou-
pling, Fmoc-Dpr(Boc)-OH (2 equiv) and HATU (2 equiv) were dis-
solved in DMF (3 mL), shaken for 5 min and then DIPEA (2 equiv)
was added. This mixture was shaken for another 2 min and then
on Bruker DRX-500 and AV-600 spectrometers. 2D NMR tech-
niques (1H–1H-COSY, 1H–13C-HSQC and 1H–13C-HMBC) were used
for full assignment of the spectra. Chemical shifts were reported
relative to internal tetramethylsilane (d 0.00 ppm) or D2O
(d 4.76 ppm). Air- and/or moisture-sensitive reactions were carried
out under an atmosphere of nitrogen. Commercial reagents were
used without purification unless otherwise noted.
4.1.2. General procedure for Fmoc SPPS
The glycopeptide was assembled manually by using a fritted
glass reaction vessel according to a standard Fmoc SPPS protocol.

A. Schierholt et al. / Carbohydrate Research 346 (2011) 1519–1526
1523
Table 1
The potencies of tested glycopeptides as inhibitors of E. coli adhesion to mannan were referenced to MeMan, which was tested on the same microtitre plate.
Glycopeptide
Structure of scaffolded
mannopyranoside
moiety (valency)
Maximal concentration
applied on test plate (mM)
Maximal inhibition
achieved (%)
RIP (SD)
RIPvc
OH
OH
O
HO
7
31.0
26.2
30.9
32.8
69
75
61
66
1 (0.2)
0.5
13.5
1
HO
O
O
OH
(2)
OH
OH
O
HO
HO
8
27 (2)
2 (0.3)
20 (7)a
O
O
OH
(2)
OH
OH
O
HO
9
HO
O
O
OH
(2)
OH
OH
O
HO
HO
10
10
O
O
OH
OH
OH
(2)
(2)
(3)
OH
OH
O
HO
HO
11
12
22.8
16.7
78
48
8 (0.8)
4
O
O
OH
OH
O
HO
HO
43 (14)a
14
O
O
RIP values are listed together with their standard deviations (SD).
a
Standard deviations in these cases are rather high, as one experiment gave outlier values.
transferred to the reaction vessel. The reaction mixture was shaken
overnight at rt, filtered and washed with DMF (5 ꢀ 5 mL). Any
unreacted amino groups are capped as acetamides by treatment
rt. The solvent was removed in vacuo and the crude product puri-
fied by MPLC (acetonitrile/water, 1:1), followed by GPC on Sepha-
dex LH-20 (eluent: methanol) and final RP-HPLC employing a
LiChrosorb RP-8 column at a flow rate of 10 mL/min (linear gradi-
ent of 20% acetonitrile to 80% acetonitrile over 100 min).
of the resin with a solution of Ac2O (160 lL) and DIPEA (280 lL)
in 3 mL DMF (1 ꢀ 60 min, 1 ꢀ 30 min). After coupling of the N-ter-
minal amino acid, the peptide was treated first with piperidine to
remove the terminal Fmoc-group and capped afterwards. Cleavage
of the peptide from the resin was achieved with TFA/CH2Cl2 (5:1)
(1 ꢀ 15 min, 1 ꢀ 10 min), followed by washing with EtOH. The sol-
vent was removed in vacuo and the crude product subjected to
lyophilization in order to yield a fluffy product, which can be easily
handled.
4.1.4. N-Acetyl-
diaminopropionyl-
The glass reaction vessel was loaded with Fmoc-Ala-Wang resin
(220 mg, 176 mol, 1 equiv). According to the general procedure
L
-alanyl-L-diaminopropionyl-glycyl-L-
L-alanine (1)
l
for Fmoc SPPS, Fmoc-Dpr(Boc)-OH (150 mg, 0.351 mmol, 2 equiv),
Fmoc-Gly-OH (210 mg, 0.706 mmol, 4 equiv), Fmoc-Dpr(Boc)-OH
(150 mg, 0.351 mmol, 2 equiv), and Fmoc-Ala-OH (220 mg,
0.707 mmol, 4 equiv) were reacted successively and cleaved from
the resin to yield the title compound as a white lyophilisate
4.1.3. General procedure for peptide coupling to achieve target
glycopeptides
The carboxy-functionalized mannoside (3 equiv for each amino
function) and HATU (2.7 equiv for each amino function) were dis-
solved in dry DMF under nitrogen atmosphere and the solution
cooled to 0 °C. DIPEA (3 equiv for each amino group) was added
and the reaction mixture shaken for 2 min. Then, the glycopeptide
was dissolved in dry DMF and added dropwise at 0 °C. After 4 h the
cooling was removed and the reaction mixture stirred overnight at
(62 mg, 0.14 mmol, 82%). 1H NMR (600 MHz, D2O): d = 4.80 (m,
3
2H, CHCH2, hidden under water peak), 4.40 (q, 1H, JH-
a
,H-b
3
7.2 Hz, H-
H- (Gly)), 3.51 (mc, 2H, CHCHH), 3.34–3.26 (m, 2H, CHCHH), 2.02
(s, 3H, NHAc), 1.42 (d, 3H, JH- 7.3 Hz, H-b(Ala)), 1.39 (d,
a
(Ala)), 4.26 (q, 1H, JH-
7.2 Hz, H-a(Ala)), 4.02 (s, 2H,
a
,H-b
a
a
(Ala),H-b(Ala)
3H, JH-
7.3 Hz, H-b(Ala)
)
ppm. 13C NMR (150 MHz,
D2O): d = 178.1–166.8 (COOH, 4CONH, COCH3), 52.4 (2C-CHCH2)
a
(Ala),H-b(Ala)

1524
A. Schierholt et al. / Carbohydrate Research 346 (2011) 1519–1526
52.0, 50.4 (2C-
a
(Ala)), 44.1 (C-
a
(Gly)), 41.8 (2C–CH2), 23.4 (COCH3),
172.8 (COOH, 4CONH, COCH3), 55.6, 55.2, 51.3, 50.5 (5C-
43.0 (C- (Gly)), 41.2 (3C- ), 32.3 (2C-b(Lys)), 28.2 (2C-d), 24.0
(COCH3), 23.6 (2C-
a(Lys,Ala)),
18.0 (C-b(Ala)) ppm. MALDI-ToF-MS: calcd for [C16H29N7O7+H]+:
432.22; found m/z 433.22; calcd for [C16H29N7O7+Na]+: 454.20;
found m/z 454.21.
a
e
c
), 18.0, 17.8 (C-b(Ala)) ppm. MALDI-ToF-MS:
calcd for [C30H56N10O9+H]+: 701.43; found m/z 702.08; calcd for
[C30H56N10O9+Na]+: 723.41; found m/z 724.05.
4.1.5. N-Acetyl-
The glass reaction vessel was loaded with Fmoc-Ala-Wang resin
(220 mg, 176 mol, 1 equiv). According to the general procedure
for Fmoc SPPS, Fmoc-Lys(Boc)-OH (330 mg, 0.704 mmol, 4 equiv),
Fmoc-Gly-OH (210 mg, 0.706 mmol, 4 equiv), Fmoc-Lys(Boc)-OH
(330 mg, 0.704 mmol, 4 equiv), and Fmoc-Ala-OH (220 mg,
0.707 mmol, 4 equiv) were reacted successively and cleaved from
the resin to yield the title compound as a white lyophilisate
(83 mg, 0.16 mmol, 91%). 1H NMR (600 MHz, DMSO-d6): d = 8.30–
8.26 (m, 1H, NH), 8.15–8.09 (m, 1H, NH), 8.06–8.02 (m, 1H, NH),
L-alanyl-L-lysyl-glycyl-L-lysyl-L-alanine (2)
4.1.8. N-Acetyl-
mannopyranosyloxy)-acetamido}-glycyl-
Nb-{(
-mannopyranosyloxy)-acetamido}-
According to the general procedure for the peptide coupling in
solution, mannoside (98 mg, 0.41 mmol, 6 equiv), HATU
(141 mg, 0.370 mmol, 5.4 equiv), DIPEA (70 L, 0.41 mmol,
6 equiv) and peptide 1 (30 mg, 69 mol, 1 equiv) were reacted in
DMF and purified. The title compound was obtained as a white lyo-
philisate (13 mg, 14 mol, 21%). HPLC: tR = 9.3 min [A = waterf,
B = acetonitrile + 1% TFA, 20% B?80% B, 100 min, 10 mL/min].
+24.5 (c 0.6, MeOH). 1H NMR (600 MHz, D2O/H2O, 1:11):
L
-alanyl-
L
-diaminopropionyl-Nb-{(
-diaminopropionyl-
-alanine (7)
a-D-
l
L
a-
D
L
5
l
l
l
7.96–7.92 (m, 1H, NH), 4.31–4.14 (m, 4H, H-
2H, H- (Gly)), 2.78–2.70 (m, 4H, H- ), 1.84 (s, 3H, NHAc), 1.72–
1.61 (m, 2H, H-baLys), 1.58–1.47 (m, 6H, H-bb(Lys), H-d), 1.36–1.29
a(Lys,Ala)), 3.72 (mc,
a
e
½ ꢁ
a 2D0
d = 8.44–8.32 (m, 3H, NH), 8.28–8.26 (m, 1H, NH), 8.17–8.12 (m,
(m, 4H, H-
c
), 1.27 (d, 3H, JH-
7.4 Hz, H-b(Ala)), 1.18 (d,
1H, NH), 8.05–7.96 (m, 2H, NH), 4.80 (d, 2H, J1,2 1.5 Hz, H-1),
4.46–4.40 (m, 2H, CHCH2), 4.27–4.25 (m, 2H, H-a(Ala)), 4.13–4.11
a
(Ala),H-b(Ala)
3H, JH-
7.4 Hz, H-b(Ala)
)
ppm. 13C NMR (150 MHz,
a
(Ala),H-b(Ala)
DMSO-d6): d = 174.0–168.5 (COOH, 4CONH, COCH3), 52.4–47.5
(4C- (Lys,Ala)), 42.0 (C- (Gly)), 38.6 (2C- ), 30.8 (2C-b(Lys)), 26.5
(m, 2H, OCHH), 3.98–3.96 (m, 2H, OCHH), 3.80 (dd, 2H, J5,6a
2.2 Hz, J6a,6b 12.3 Hz, H-6a), 3.66–3.62 (m, 2H, H-6b), 3.62–3.52
a
a
e
(2C-d), 22.5 (COCH3), 22.0 (2C-
c
), 17.9, 17.0 (C-b(Ala)) ppm. MAL-
(m, 8H, H-2, H-4, H-5, H-
9.7 Hz, H-3), 3.07 (mc, 4H, CHCH2), 1.92 (s, 3H, NHAc), 1.30 (t,
a(Gly)), 3.45 (dd, 2H, J2,3 3.9 Hz, J3,4
DI-ToF-MS: calcd for [C22H41N7O7+H]+: 516.31; Found m/z
516.96; calcd for [C22H41N7O7+Na]+: 538.29; found m/z 538.92.
3H, J 7.2 Hz, H-b(Ala)), 1.22 (d, 3H, JH-
7.2 Hz, H-b(Ala)) ppm.
,H-b
a
13C NMR (150 MHz, D2O/H2O, 1:11): d = 174.4, 174.2, 172.5,
172.3, 171.9, 171.2 (6CONH, COCH3) 101.1 (2C-1), 73.2 (2C-5),
72.5 (2C-2), 71.3 (2C-3), 68.3 (OCH2) 66.6 (2C-4), 60.9 (2C-6),
4.1.6. N-Acetyl-
The glass reaction vessel was loaded with Fmoc-Ala-Wang resin
(220 mg, 176 mol, 1 equiv). According to the general procedure
L-lysyl-L-lysyl-glycyl-L-alanyl-L-alanine (3)
l
52.5 (CHCH2) 50.0, 49.0 (2C-a(Ala)), 46.6 (CHCH2) 39.9 (C-a(Gly)),
for Fmoc SPPS, Fmoc-Ala-OH (220 mg, 0.707 mmol, 4 equiv),
Fmoc-Ala-OH (220 mg, 0.707 mmol, 4 equiv), Fmoc-Gly-OH
(210 mg, 0.706 mmol, 4 equiv), Fmoc-Lys(Boc)-OH (330 mg,
21.8 (COCH3), 16.5, 16.3 (C-b(Ala)) ppm. MALDI-ToF-MS: calcd for
[C32H53N7O21+Na]+: 894.31; found m/z 894.91; calcd for
[C32H53N7O21+K]+: 910.29; found m/z 910.90.
0.704 mmol,
4 equiv),
and
Fmoc-Lys(Boc)-OH
(330 mg,
0.704 mmol, 4 equiv) were reacted successively and cleaved from
the resin to yield the title compound as a white lyophilisate
(81 mg, 0.15 mmol, 89%). 1H NMR (600 MHz, DMSO-d6): d = 8.31–
8.26 (m, 1H, NH), 8.17–8.10 (m, 1H, NH), 8.07–8.02 (m, 1H, NH),
4.1.9. N-Acetyl-
mannopyranosyloxy)-phenyl]-acetamido}-glycyl-
diaminopropionyl-Nb-{4-[(
-mannopyranosyloxy)-phenyl]-
acetamido}- -alanine (8)
According to the general procedure for the peptide coupling in
solution, mannoside (130 mg, 0.413 mmol, 6 equiv), HATU
(141 mg, 0.370 mmol, 5.4 equiv), DIPEA (70 L, 0.41 mmol,
6 equiv) and peptide 1 (30 mg, 69 mol, 1 equiv) were reacted in
DMF and purified. The title compound was obtained as a white lyo-
philisate (23 mg, 22 mol, 32%). HPLC: tR = 12.1 min [A = water,
B = acetonitrile + 1% TFA, 20% B?80% B, 100 min, 10 mL/min].
+11.8 (c 0.5, MeOH). 1H NMR (600 MHz, D2O/H2O, 1:11):
L-alanyl-L a-D-
-diaminopropionyl-Nb-{4-[(
L
-
a-D
L
7.95–7.90 (m, 1H, NH), 4.29–4.15 (m, 4H, H-
2H, H- (Gly)), 2.77–2.69 (m, 4H, H- ), 1.84 (s, 3H, NHAc), 1.72–
1.61 (m, 2H, H-ba(Lys)), 1.57–1.48 (m, 6H, H-bb(Lys), H-d), 1.38–
a(Lys,Ala)), 3.72 (mc,
a
e
6
l
1.29 (m, 4H, H-
1.19 (d, 3H, JH-
c), 1.27 (d, 3H, JH-
7.2 Hz, H-b(Ala)),
l
a
(Ala),H-b(Ala)
7.0 Hz, H-b(Ala)
)
ppm. 13C NMR
a
(Ala),H-b(Ala)
(150 MHz, DMSO-d6): d = 174.0–168.5 (COOH, 4CONH, COCH3),
52.5, 52.4, 47.7, 47.5 (4C- (Lys,Ala)), 42.1 (C- (Gly)), 38.6 (2C- ),
31.0 (2C-b(Lys)), 26.5 (2C-d), 22.5 (COCH3), 22.1 (2C- ), 17.9, 17.0
l
a
a
e
c
½ ꢁ
a 2D0
(C-b(Ala)
)
ppm. MALDI-ToF-MS: calcd for [C22H41N7O7+H]+:
d = 8.36–8.12 (m, 3H, NH), 8.06–7.98 (m, 1H, NH), 7.92–7.79 (m,
3H, NH), 7.14 (d, 4H, J 8.7 Hz, H-Ar), 7.00 (d, 4H, J 8.7 Hz, H-Ar),
516.31; found m/z 517.07; calcd. for [C22H41N7O7+Na]+: 538.29;
found m/z 539.05.
5.48 (br s, 2H, H-1), 4.40–4.31 (m, 2H, H-
6H, CHCH2, H-2, H-3), 3.74–3.69 (m, 4H, CH2-Ar), 3.67–3.57 (m,
8H, H-4, H-5, H-6a, H-6b), 3.55 (bs, 2H, H- (Gly)), 3.43–3.31 (m,
a(Ala)), 4.16–3.88 (m,
4.1.7. N-Acetyl-
L
-alanyl-
L
-lysyl-glycyl-
L
-lysyl-glycyl-
L-lysyl-
L
-
a
alanine (4)
4H, CHCH2), 1.83 (s, 3H, NHAc), 1.24–1.13 (m, 6H, H-b(Ala)) ppm.
13C NMR (150 MHz, D2O/H2O, 1:11): d = 173.5, 173.3, 171.8,
171.6, 171.0, 170.9 (6CONH, COCH3) 154.4, 130.7, 130.5, 117.3
(12C-Ar), 98.2 (2C-1), 73.3 (2C-5), 70.5 (2C-2), 70.0 (2C-3), 66.7
The glass reaction vessel was loaded with Fmoc-Ala-Wang resin
(220 mg, 176 mol, 1 equiv). According to the general procedure
for Fmoc SPPS, Fmoc-Lys(Boc)-OH (330 mg, 0.704 mmol, 4 equiv),
l
Fmoc-Gly-OH (210 mg, 0.706 mmol, 4 equiv), Fmoc-Lys(Boc)-OH
(2C-4), 60.7 (2C-6), 54.4 (2C-
a(Ala)), 50.1 (2CHCH2), 42.6 (2CHCH2),
(330 mg,
0.704 mmol,
4 equiv),
Fmoc-Gly-OH
(210 mg,
41.4 (2CH2-Ar), 40.1 (C-a(Gly)), 21.7 (COCH3), 16.3, 16.2 (C-b(Ala))
0.706 mmol, 4 equiv), Fmoc-Lys(Boc)-OH (330 mg, 0.704 mmol,
4 equiv), and Fmoc-Ala-OH (220 mg, 0.707 mmol, 4 equiv) were re-
acted successively and cleaved from the resin to yield the title
compound as a white lyophilisate (92 mg, 0.13 mmol, 74%). 1H
ppm. MALDI-ToF-MS: calcd for [C44H61N7O21+Na]+: 1046.38; found
m/z 1046.22; calcd for [C38H65N7O21+K]+: 1062.35; found m/z
1062.20.
e
NMR (600 MHz, D2O): d = 4.34–4.23 (m, 3H, H-
4.11 (m, 2H, H- (Lys,Ala)), 4.00–3.86 (mc, 4H, H-
(m, 6H, H- ), 1.97 (s, 3H, NHAc), 1.81–1.68 (m, 4H, H-ba(Lys)),
1.68–1.59 (m, 8H, H-bb(Lys), H-d), 1.46–1.39 (m, 6H, H- ), 1.38 (d,
3H, JH- 7.3 Hz, 3H, H-b(Ala)), 1.34 (d, 3H, JH-
a
(Lys,Ala)), 4.23–
4.1.10. N-Acetyl-
acetamido}-glycyl-
acetamido}- -alanine (9)
According to the general procedure for the peptide coupling in
solution, mannoside (125 mg, 0.525 mmol, 6 equiv), HATU
(179 mg, 0.470 mmol, 5.4 equiv), DIPEA (91 L, 0.525 mmol,
L
-alanyl-
L
-lysyl-N -{(
a
-
D
-mannopyranosyloxy)-
e
a
a
(Gly)), 2.97–2.90
L
-lysyl-N -{(
a-D-mannopyranosyloxy)-
e
L
c
5
a
(Ala),H-b(Ala)
a(Ala),H-b(Ala)
7.2 Hz, 3H, H-b(Ala)) ppm. 13C NMR (150 MHz, D2O): d = 178.2–
l

A. Schierholt et al. / Carbohydrate Research 346 (2011) 1519–1526
1525
6 equiv) and peptide 2 (45 mg, 87
DMF and purified. The title compound was obtained as a white lyo-
philisate (13 mg, 13 mol, 15%). HPLC: tR = 10.6 min [A = water,
B = acetonitrile + 1% TFA, 20% B?80% B, 100 min, 10 mL/min].
+6.3 (c 0.4, MeOH). 1H NMR (600 MHz, D2O/H2O, 1:11):
d = 4.94 (d, 2H, J1,2 1.7 Hz, H-1), 4.43–4.31 (m, 4H, H- (Lys,Ala)),
l
mol, 1 equiv) were reacted in
3H, NHAc), 1.92–1.85 (m, 2H, H-ba(Lys)), 1.78–1.72 (m, 2H, H-
bb(Lys)), 1.70–1.62 (m, 4H, H-d), 1.49–1.43 (m, 7H, H-c, H-b(Ala)),
l
1.42 (mc, 3H, H-b(Ala)) ppm. 13C NMR (150 MHz, D2O/H2O, 1:11):
d = 179.9 (COOH), 176.0, 175.8, 175.5, 174.2, 173.4 (5CONH,
COCH3) 154.4, 130.4, 128.1, 117.2 (12C-Ar), 99.3 (2C-1), 73.3 (2C-
5), 70.5 (2C-3), 70.0 (2C-2), 66.7 (2C-4), 60.7 (2C-6), 54.3, 50.7
½ ꢁ
a 2D0
a
4.26 (d, 2H, JOCHH,OCHH 15.1 Hz, OCHH), 4.15 (d, 2H, JOCHH,OCHH
15.1 Hz, OCHH), 4.10 (dd, 2H, J1,2 1.7 Hz, J2,3 3.4 Hz, H-2), 3.99 (br
(4C-
(2C-b(Lys)), 26.2 (2C-d), 21.9 (2C-
b(Ala)
ppm. MALDI-ToF-MS: calcd for [C50H73N7O21+Na]+:
a
(Lys,Ala)), 43.2 (C-
a
(Gly)), 42.5 (2CH2–C-Ar), 39.4 (2C-
e), 30.4
c
), 21.6 (COCH3), 16.5, 16.2 (C-
s, 2H, H-
J5,6b 5.9 Hz, J6a,6b 12.2 Hz, H-6b), 3.72 (ddꢂt, 2H, J 9.8 Hz, H-4),
3.68–3.64 (m, 2H, H-5), 3.30 (ddꢂt, 4H, J 6.7 Hz, H- ), 2.07 (s, 3H,
NHAc), 1.93–1.85 (m, 2H, H-ba(Lys)), 1.84–1.75 (m, 2H, H-bb(Lys)),
1.64–1.57 (m, 4H, H-d), 1.49–1.43 (m, 7H, H- , H-b(Ala)), 1.42 (d,
3H, JH-
7.2 Hz, H-b(Ala)) ppm. 13C NMR (150 MHz, D2O/
a(Gly)), 3.94–3.90 (m, 4H, H-3, H-6a), 3.81 (dd, 2H, HH,
)
1130.47; found m/z 1130.82; calcd for [C50H73N7O21+K]+:
1146.44; found m/z 1146.78.
e
e
c
4.1.13. N-Acetyl-
L
-alanyl-
L
-lysyl-N -{4-[(
a
-D
-
e
mannopyranosyloxy)-phenyl]-acetamido}-glycyl-
[( -mannopyranosyloxy)-phenyl]-acetamido}-glycyl-
N -{4-[( -mannopyranosyloxy)-phenyl]-acetamido}-
alanine (12)
L
-lysyl-N -{4-
a
(Ala),H-b(Ala)
H2O, 1:11): d = 174.9, 174.1, 173.6, 173.0, 170.9, 170.5 (5CONH,
COCH3) 99.5 (2C-1), 72.7 (2C-5), 69.9 (2C-3), 69.2 (2C-2), 66.1
(2C-4), 65.4 (OCH2), 60.3 (2C-6), 53.9, 53.3, 53.1, 52.9 (4C-a(Lys,A-
a
-D
L
-lysyl-
e
a-
D
L-
la)), 41.9 (C-a(Gly)), 38.2 (2C-
e
), 29.8 (2C-b(Lys)), 27.4 (2C-d), 21.8
According to the general procedure for the peptide coupling in
(2C-
c
), 21.1 (COCH3), 16.0, 15.8 (C-b(Ala)) ppm. MALDI-ToF-MS:
solution, mannoside 6 (134 mg, 0.426 mmol, 10 equiv), HATU
calcd for [C38H65N7O21+Na]+: 978.41; found m/z 978.95; calcd for
[C38H65N7O21+K]+: 994.38; found m/z 994.97.
(148 mg, 0.389 mmol, 9 equiv), DIPEA (73
and peptide 4 (30 mg, 42 mol, 1 equiv) were reacted in DMF and
purified. The title compound was obtained as a white lyophilisate
lL, 0.42 mmol, 10 equiv)
l
e
4.1.11. N-Acetyl-
L
-alanyl-
L
-lysyl-N -{4-[(
a-
D-
(17 mg, 10 lmol, 25%). HPLC: tR = 11.6 min [A = water, B = acetoni-
mannopyranosyloxy)-phenyl]-acetamido}-glycyl-
[( -mannopyranosyloxy)-phenyl]-acetamido}- -alanine (10)
According to the general procedure for the peptide coupling in
solution, mannoside (164 mg, 0.522 mmol, 6 equiv), HATU
(179 mg, 0.470 mmol, 5.4 equiv), DIPEA (89 L, 0.522 mmol,
6 equiv) and peptide 2 (45 mg, 87 mol, 1 equiv) were reacted in
DMF and purified. The title compound was obtained as a white lyo-
philisate (16 mg, 14 mol, 16%). HPLC: tR = 11.3 min [A = water,
B = acetonitrile + 1% TFA, 20% B?80% B, 100 min, 10 mL/min].
+14.0 (c 0.5, MeOH). 1H NMR (600 MHz, D2O/H2O, 1:11):
L
-lysyl-N -{4-
trile + 1% TFA, 20% B?80% B, 100 min, 10 mL/min]. ½a D20
ꢁ
+5.5 (c 0.4,
e
a-D
L
MeOH). 1H NMR (600 MHz, D2O/H2O, 1:11): d = 8.47–8.42 (m, 1H,
NH), 8.36–8.26 (m, 2H, NH), 8.25–8.20 (m, 1H, NH), 8.12–8.02 (m,
1H, NH), 7.99–7.89 (m, 3H, NH), 7.24 (d, 6H, J 8.4 Hz, H-Ar), 7.11 (d,
6H, J 8.4 Hz, H-Ar), 5.58 (br s, 3H, H-1), 4.32–4.20 (m, 5H, H-a(Lys,A-
la)), 4.16 (dd, 3H, J1,2 1.7 Hz, J2,3 3.3 Hz, H-2), 4.04 (dd, 3H, J2,3
6
l
l
3.4 Hz, J3,4 9.3 Hz, H-3), 3.91 (mc, 4H, H-
9.4 Hz, H-4), 3.76–3.67 (m, 9H, H-5, H-6a, H-6b), 3.51 (s, 6H, CH2CAr),
3.19–3.12 (m, 6H, H- ), 2.00 (s, 3H, NHAc), 1.84–1.75 (m, 3H, H-
ba(Lys)), 1.74–1.64 (m, 3H, H-bb(Lys)), 1.47 (mc, 6H, H-d), 1.41–1.36
(m, 6H, H- ), 1.34 (d, 6H, JH-
7.1 Hz, H-b(Ala)) ppm. 13C
a
(Gly)), 3.77 (ddꢂt, 3H, J
l
e
½ ꢁ
a 2D0
d = 8.40–8.36 (m, 1H, NH), 8.35–8.28 (m, 2H, NH), 8.26–8.22 (m,
2H, NH), 8.02–7.93 (m, 2H, NH), 7.32–7.24 (m, 4H, H-Ar), 7.18–
7.11 (m, 4H, H-Ar), 5.60 (br s, 2H, H-1), 4.34 -4.24 (m, 4H, H-a(Ly-
s,Ala)), 4.20–4.16 (m, 2H, H-2), 4.07–4.04 (m, 2H, H-3), 3.93 (mc,
c
a
(Ala),H-b(Ala)
NMR (150 MHz, D2O/H2O, 1:11): d = 177.1, 176.3, 175.8, 175.1,
173.1 (9CONH, COCH3) 156.2, 132.3, 131.3, 119.0 (18C-Ar), 99.9
(3C-1), 75.1 (3C-5), 72.3 (3C-3), 71.7 (3C-2), 68.4 (3C-4), 62.5 (3C-
2H, H-
3.17 (mc, 4H, H-
1.75–1.66 (m, 2H, H-bb(Lys)), 1.54–1.46 (m, 4H, H-d), 1.41–1.37 (m,
4H, H-
), 1.35 (d, 6H, JH- (Ala),H-b(Ala) 7.2 Hz, H-b(Ala)) ppm. 13C NMR
a
(Gly)), 3.81–3.70 (m, 8H, H-4, H-5, H-6), 3.52 (s, 4H, CH2CAr),
6), 55.7, 51.5 (5C-
a(Lys,Ala)), 44.3 (2C-a(Gly)), 43.4 (3CH2–C-Ar), 40.9
e
), 2.01 (s, 3H, NHAc), 1.84–1.76 (m, 2H, H-ba(Lys)),
(3C-e), 32.1 (3C-b(Lys)), 29.5 (3C-d), 23.9 (3C-c), 23.4 (COCH3), 18.3,
18.1 (C-b(Ala)) ppm. MALDI-ToF-MS: Calcd for [C72H104N10O30+Na]+:
1611.38; found m/z 1611.91.
c
a
(150 MHz, D2O/H2O, 1:11): d = 178.9 (COOH), 177.1, 176.3, 175.8
(5CONH, COCH3) 156.2, 132.0, 131.3, 130.5, 119.0 (12C-Ar), 99.9
(2C-1), 75.1 (2C-5), 72.2 (2C-3), 71.7 (2C-2), 68.4 (2C-4), 62.5
4.2. Adhesion inhibition assay
(2C-6), 55.7, 51.5 (4C-
a
(Lys,Ala)), 43.4 (C-
a(Gly)), 41.6 (2CH2-C-Ar),
4.2.1. GFP-based bacterial adhesion assay27
40.9 (2C- ), 32.0 (2C-b(Lys)), 29.5 (2C-d), 23.9 (2C-
e
c
), 23.4 (COCH3),
Black 96-well plates were filled with a solution of mannan from
Saccharomyces cerevisiae (1.2 mg/mL in carbonate buffer, pH 9.5;
18.3, 18.0 (C-b(Ala)) ppm. MALDI-ToF-MS: calcd for [C50H73N7O21+-
Na]+: 1130.47; found m/z 1130.70.
100
The plates were washed with PBST (3 ꢀ 150
4 °C. Before use, the wells were blocked with BSA (5% in PBS,
120 L/well) for 2 h at 37 °C and then washed with PBST
(3 ꢀ 150 L/well). Serial dilutions of the examined glycopeptide
lL solution per well) and allowed to dry in at 37 °C overnight.
lL/well) and stored at
e
4.1.12. N-Acetyl-
phenyl]-acetamido}-
phenyl]-acetamido}-glycyl-
According to the general procedure for the peptide coupling in
solution, mannoside (164 mg, 0.522 mmol, 6 equiv), HATU
(179 mg, 0.470 mmol, 5.4 equiv), DIPEA (89 L, 0.51 mmol,
6 equiv) and peptide 3 (45 mg, 87 mol, 1 equiv) were reacted in
DMF and purified. The title compound was obtained as a white lyo-
philisate (23 mg, 20 mol, 24%). HPLC: tR = 12.6 min [A = water,
B = acetonitrile + 1% TFA, 20% B?80% B, 100 min, 10 mL/min].
+16.5 (c 0.5, MeOH). 1H NMR (600 MHz, D2O, 1:11):
L
-lysyl-N -{4-[(
a
-
D
-mannopyranosyloxy)-
-mannopyranosyloxy)-
-alanine (11)
e
L
-lysyl-N -{4-[(
a
L
-
D
l
L
-alanyl-
l
inhibitor were prepared in the mannan-coated, BSA-blocked 96-
6
well plates. The bacteria suspension (2 mg/mL in PBS buffer, pH
l
7.2; 50
tated (80 rpm) and incubated for 45 min at 37 °C. After washing
with PBS (3 ꢀ 150 L/well), the wells were filled with PBS
(100 L/well) and the fluorescence intensity (485 nm/535 nm)
lL solution per well) was added and the plates were agi-
l
l
l
l
was determined. All assays were performed, using at least duplicate
samples of each well. LB: lysogeny broth; rpm: revolutions per min-
ute; PBS: phosphate buffered saline; PBST: PBS + 0.05% Tween 20.
½ ꢁ
a 2D0
d = 8.57–8.54 (m, 2H, NH), 8.41–8.37 (m, 1H, NH), 8.28–8.24 (m,
2H, NH), 8.02–7.94 (m, 1H, NH), 7.25 (d, 4H, J 8.5 Hz, H-Ar), 7.11
(d, 4H, J 8.5 Hz, H-Ar), 5.59 (bs, 2H, H-1), 4.39–4.25 (m, 4H, H-a(-
Lys,Ala)), 4.19–4.16 (m, 2H, H-2), 4.06 (dd, 2H, J2,3 3.1 Hz, J3,4
8.9 Hz, H-3), 3.82–3.71 (m, 8H, H-4, H-5, H-6), 3.70–3.65 (m, 2H,
4.2.2. Bacteria culture
E. coli bacteria of strain PKL1162 were grown in LB-med-
ia + AMP + CAM (100 mg ampicillin, 50 mg chloramphenicol/L) at
37 °C under slight agitation.
H-a(Gly)), 3.54 (s, 4H, CH2–C-Ar), 3.16, 3.00 (bs, 4H, H-e), 2.02 (s,

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A. Schierholt et al. / Carbohydrate Research 346 (2011) 1519–1526
14. Hung, C. S.; Bouckaert, J.; Hung, D.; Pinkner, J.; Widberg, C.; Defusco, A.;
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Microbiol. 2002, 44, 903–918.
Financial support by Christina Albertina University of Kiel is
gratefully acknowledged. We thank Thermo Fisher Scientific for a
generous gift of microtitre plates (Nunc Maxisorp).
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