Trivalent α-D-mannoside clusters as inhibitors of type-1 fimbriae-mediated adhesion of Escherichia coli: Structural variation and biotinylation

Article abstract of DOI:10.1039/b009786l

Structural modifications of trivalent cluster mannosides are presented to further elucidate the ligand preferences of the type-1 fimbrial lectin of Escherichia coli. Two types of variations are performed, either regarding the aglycone part of cluster mann

Full text of DOI:10.1039/b009786l

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Trivalent ꢀ-D-mannoside clusters as inhibitors of type-1 fimbriae-
mediated adhesion of Escherichia coli: structural variation and
biotinylation
1
a
a
b
b
Thisbe K. Lindhorst, * Sven Kötter, Ulrike Krallmann-Wenzel and Stefan Ehlers
a
Institute of Organic Chemistry, Christiana Albertina University of Kiel, Otto-Hahn-Platz 4,
D-24098 Kiel, Germany. E-mail: tklind@oc.uni-kiel.de; Fax ϩ49 431 880 7410
Division of Molecular Infection Biology, Research Center Borstel, Parkallee 22,
D-23845 Borstel, Germany. E-mail: sehlers@fz-borstel.de; Fax ϩ49 4537 188 481
b
Received (in Cambridge, UK) 6th December 2000, Accepted 13th February 2001
First published as an Advance Article on the web 21st March 2001
Structural modifications of trivalent cluster mannosides are presented to further elucidate the ligand preferences
of the type-1 fimbrial lectin of Escherichia coli. Two types of variations are performed, either regarding the aglycone
part of cluster mannosides of type 2, leading to 27, or altering the spacer lengths of mannosyl clusters of type 1,
leading to clusters 20–22. Biotinylation of the cluster mannoside with the highest affinity to the type-1 fimbrial
lectin is also shown (33). Testing of the inhibitory potencies of the synthesised cluster glycosides as inhibitors of
mannose-specific (type-1 fimbriae-mediated) binding of E. coli to mannan in an enzyme-linked immunosorbent
assay (ELISA) suggests that a structural preorganisation as given in cluster 2a can be favourably combined with
greater spacer flexibility as in cluster 22.
Introduction
for ligand binding, we decided to vary the structures of cluster
mannosides 1 and 2 in order to obtain more information about
the investigated ligand–receptor system from additional data
on structure–activity relationships.
Bacteria often use their own specialised proteins, so-called
lectins, to interact with carbohydrate structures on the surface
of potential host cells. By establishing many of such
carbohydrate–protein contacts, bacteria accomplish adhesion
to the host cell surface, an event which is of crucial importance
for cell infection. Synthetic carbohydrate derivatives have been
designed to interfere in the microbial adhesion process by
competing for the carbohydrate-recognition domains of the
1
By combination of the synthetic strategies which led to 1 and
we planned to vary (i) the length of spacers and (ii) the
2
aglycone moieties of related glycoclusters. Furthermore, it was
of interest to utilise the architecture of the cluster mannosides
prepared, to introduce a bio-label, e.g. by biotinylation.
2
3
involved lectins. This has been demonstrated to work in vitro,
to allow the evaluation of such an approach towards an anti-
adhesion therapy to be used for certain in vivo applications.
Results and discussion
Synthetic part
4
As mannose-specific adhesion is among the most widely
5
distributed types of carbohydrate-specific bacterial adhesion,
To provide a collection of spacer-modified cluster mannosides,
we set out to synthesise a set of ‘mannoside donors’ with
ω-amino-functionalised aglycone moieties. Three different
ω-halogen-functionalised alkyl and ethoxyalkyl alcohols (3, 4,
and 5, respectively) were used as glycosyl acceptors in the
glycosylation reactions which eventually led to the desired
‘glycoside donors’ 16, 17, and 18 (Scheme 2). The mannosyl
our interest has been directed towards the development of
oligomannoside mimetics to serve as high-affinity inhibitors in
this system. Mannose-specific adhesion of bacteria is mediated
by so-called type-1 fimbriae, long proteinogenous appendages
on the bacterial surface, which contain several copies of the
relevant carbohydrate-recognition domains. The adhesin is
called the FimH protein, which has received much attention
10
trichloroactimidate 6 served as glycosyl donor in the Lewis
acid-catalysed reaction leading to the acetyl-protected glyco-
sides 7, 8, and 9 in excellent yields. In the next synthetic step
the halogen substituents in the aglycone moieties of these
glycosides could be substituted by azide in almost quantitative
reactions to give mannosides 10, 11, and 12, respectively, which
6
recently. It is encoded by the fimH gene within the gene cluster
7
responsible for type-1 fimbriae assembly.
As part of our former work we have synthesised a series of
trisaccharide mimetics to serve as ligands for the type-1 fimbrial
8
adhesin. These trivalent glycoclusters either were obtained by
11
mannosylation of a branched oligo-alcohol to yield cluster
mannosides such as compound 1 (Scheme 1) or were the result
of a peptide-coupling reaction of a triacid and a 6-amino-6-
deoxymannoside, which can be regarded as a ‘glycoside donor’
were in turn de-acetylated under Zemplén conditions
and subsequently reduced to the unprotected ω-amino-
functionalised mannosides 16, 17, and 18. These could be taken
through an 2-ethoxy-N-ethoxycarbonyl-1,2-dihydroquinoline
(EEDQ)-assisted peptide-coupling reaction with the triacid
(
Scheme 1). This approach led to cluster glycosides such as 2a
12
and allows us to vary the aglycone part to provide an array of
clusters such as 2b. Clusters 1 and 2b were shown to serve as
particularly successful inhibitors of type-1 fimbriae-mediated
19 (Scheme 3) without further protection and deprotection
steps to provide the trivalent cluster mannosides 20, 21, and 22,
which were tested as inhibitors of type-1 fimbriae-mediated
bacterial adhesion (vide supra).
9
adhesion of Escherichia coli to high mannose-type surfaces,
3
having up to 10 -fold better inhibitory potencies than mono-
In the second synthetic part, it was our goal to replace
the methyl aglycone groups in cluster mannoside 2a by p-
nitrophenyl moieties. This is of interest as aromatic moieties
can increase the affinity of a given carbohydrate ligand to its
valent methyl α--mannoside (MeMan). As the conformational
preorganisation as well as conformational flexibility of the
sugar epitopes have to be regarded as important parameters
DOI: 10.1039/b009786l
J. Chem. Soc., Perkin Trans. 1, 2001, 823–831
This journal is © The Royal Society of Chemistry 2001
823

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Scheme 1 Cluster glycosides can be obtained by glycosylation of branched oligo-alcohols with classical glycosyl donors (route to cluster 1) or by
peptide) coupling of functionalised glycosides, which can be regarded as ‘glycoside donors’ to suitable core molecules (route to clusters 2). The latter
route allows variation of the aglycone moiety RЈ.
(
Scheme 2 Reagents, conditions, and yields: i, BF ؒEt O (for 3), TMSOTf (for 4 and 5), CH Cl , rt, 96% (7), 92% (8), 89% (9); ii, NaN , DMF, 50–
3
2
2
2
3
6
9
0 ЊC, 98% (10), 99% (11), 97% (12); iii, NaOMe, MeOH, rt, 99% (13), 99% (14), 97% (15); iv, H , Pd–C (10%), 1 : 1 ethyl acetate–EtOH, 98% (16),
2
8% (17), 98% (18).
1
3
lectin receptor by hydrophobic interactions. The inhibitory
potency of p-nitrophenyl α--mannoside (pNPMan), for
example, is approximately 100 times higher than that of the
Finally, Staudinger reduction was the method of choice after
it was found that the intermediate triphenylphosphine which
was formed in the reaction of 25 with triphenylphosphine
could be readily hydrolysed using silica gel. Peptide coupling of
the unprotected amine 26 with the triacid 19 gave the cluster
mannoside 27 in excellent yield, carrying three p-nitrophenyl
α--mannosyl units.
3a,5
respective methyl mannoside.
As cluster glycoside 2a was
shown to be an excellent ligand for the type-1 fimbrial lectin
9
earlier by our group it could be expected that its pNP analogue
2
7 would perform even better.
For the preparation of 27 a 6-amino-6-deoxy-functionalised
In order to facilitate biological testing, the third part of
this study was dedicated to biotinylation of trivalent cluster
mannosides. This was exemplified using cluster 2a, as it is the
most potent inhibitor of mannose-specific bacterial adhesion
pNP mannoside was required, which was obtained starting
from pNPMan 23. Regioselective activation of the 6-position
followed by nucleophilic displacement of the sulfonate from 24
9
8a
led to the 6-azido-6-deoxymannoside 25 (Scheme 4). For the
in a series of synthetic trivalent cluster mannosides. To allow
reduction of the azide function in 25 catalytic hydrogenation
could not be used as the p-nitro group had to be kept intact.
biotinylation, we tried to reduce the nitro group in 2a to
generate an amino function; however, all our attempts led to
8
24
J. Chem. Soc., Perkin Trans. 1, 2001, 823–831

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1
4
unsatisfactory results. Therefore, the desired amino group
6-deoxy-α--mannoside 32 to yield the desired biotinylated
trivalent cluster mannoside 33 in a clean reaction. The struc-
tures of all prepared cluster mannosides could unequivocally be
confirmed by means of NMR spectroscopy. The obtained data
were supported by matrix-assisted laser desorption ionisation
time-of-flight (MALDI-TOF) mass spectrometry in all cases.
was introduced at an earlier stage of the synthesis. The tris(tert-
12
butyl ester) 28 was reduced to the γ-amino triacid 29 (Scheme
5
), followed by introduction of the biotin label to yield 30
in excellent yield. The tert-butyl esters were cleaved under acidic
conditions, followed by peptide coupling with methyl 6-amino-
Testing part
The prepared cluster mannosides were tested for their capacity
to inhibit mannose-specific adhesion of E. coli, using a bac-
terial strain, E. coli HB 101 (pPKl4), which expresses only
15
type-1 fimbriae on its surface. As assay a sandwich-type
enzyme-linked immunosorbent assay (ELISA) was used in9
which the microtitre plate was coated with yeast mannan.
Adhesion of bacteria and inhibition of adhesion were deter-
mined via an antibody–enzyme conjugate-based colour reac-
tion. These ELISAs allowed us to measure IC -values for
50
the inhibition of E. coli adhesion which reflect the inhibitor
concentration which causes 50% inhibition of bacterial binding
to yeast mannan. Duplicate results were used for the con-
struction of the inhibition curves for each individual ELISA
experiment. Typically, the intra-assay variation of an individual
ELISA is very small, whereas the IC -values obtained from
50
several independently performed ELISAs differ significantly.
However, when relative inhibition potencies are calculated
from independently obtained data, the results are highly
reproducible. The results obtained are listed in Table 1. IC -
50
Values are average values from five independent ELISA exper-
iments and are depicted together with the resulting standard
deviations.
The new clusters were compared with the known trivalent
cluster mannosides 1 and 2a (Scheme 1), which were shown
earlier to surpass the inhibitory potency of pNPMan without
containing an aromatic moiety. Unfortunately, the water solu-
bility of 27, the pNP analogue of 2a, was not good enough to
provide reliable data. The spacer-modified cluster mannosides
Scheme 3 Reagents, conditions, and yields: i, 3.3 equiv. amine (16, 17,
or 18, respectively), EEDQ, DMA, 60 ЊC, 4 d, 74% (20), 69% (21), 59%
2
0, 21, and 22, however, revealed interesting results. None of
these compounds reached the inhibitory potency of 1 or 2a.
(
22).
Scheme 4 Reagents, conditions, and yields: i, TsCl, pyridine, 0 ЊC → rt, 57%; ii, NaN , DMF, 80 ЊC, 91%; iii, Ph P, silica gel, 4 : 1 THF–water,
3
3
4
5 ЊC, 98%; iv, EEDQ, DMA, 5 d, 40 ЊC, 91%.
J. Chem. Soc., Perkin Trans. 1, 2001, 823–831
825

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assembly of mannosyl units as in 2a with a flexible spacer archi-
tecture as given in 22. Thus, our future work will be directed
towards the multiple, flexible assembly of glycocluster 2a in a
multivalent neoglycoconjugate, which can be provided by the
principal architecture as chosen in 22.
In summary, we showed the synthesis of spacer-modified
cluster mannosides 20, 21, and 22 in excellent yields and
demonstrated the utilisation of the contained nitro group for
biotinylation. Biotinylation of the cluster mannoside adds an
option for assaying biological functions. An aglycone-modified
analogue of cluster mannoside 2a could also be prepared
containing three p-nitrophenyl moieties, which, unfortunately,
showed limited water solubility and could, therefore, not be
tested for its affinity to the type-1 fimbrial lectin. The inhibitory
potencies of clusters 20, 21, and 22 did not surpass that of
‘lead’ cluster 2a. However, the most flexible cluster 22 showed
almost double the affinity of its counterparts 20 and 21. These
results suggest attempts should be made to combine the
structural characteristics of clusters 2a and 22, which will be
subject of our future work.
Experimental
Materials
Methyl α--mannoside was purchased from Merck, p-nitro-
phenyl α--mannoside was from SENN Chemicals. The
ω-halogen-substituted alcohols 3, 4, and 5 were purchased from
Aldrich. F-Shaped 96-well microtitre plates from Sarstedt were
used for ELISAs. Mannan from Saccharomyces cerevisiae
was purchased from Sigma and was used in 50 mM aq. Na CO
2
3
Ϫ3
(
1 mg cm ; pH 9.6). A polyclonal anti-fimA antibody was used
for ELISAs, and was produced at Borstel Research Centre. All
the other material needed for ELISA as well as the required
buffers was used as reported earlier. The recombinant type-1
1
5
fimbriated E. coli strain, E. coli HB101 (pPKl4), used was
3a,9
cultured as reported in the literature. Light petroleum refers
to the fraction with boiling range 40–60 ЊC.
General methods
Scheme 5 Reagents, conditions, and yields: i, H , T1 Raney-nickel,
2
Optical absorbance (A) was measured on a Dynatech 5000
ELISA reader at 405 nm with the reference red at 490 nm.
ELISA plates were incubated at 37 ЊC. For flash chrom-
atography Merck silica gel 60 (0.040–0.063 mm, 230–400 mesh)
EtOH, 3 bar, 2 d, 60 ЊC, 99%; ii, biotin, EDC–1-HOBT (1 : 2), DIPEA,
DMF, 0 ЊC → rt, 97%; iii, HCO H, 99%; iv, EDC–1-HOBT (1 : 2),
2
DIPEA, DMF, 0 ЊC → rt, 91%.

Table 1 IC -Values measured by ELISA are listed together with the
was used. TLC was performed on Alugram Sil G/UV254
50
relative values based on MeMan. IC -Values are average values from
plates, purchased from Macherey Nagel, or on Kieselgel 60 F254
plates from Merck. Detection was carried out under UV light
or by spraying with 20% ethanolic sulfuric acid or ninhydrin
solution (2% in water–propan-2-ol, 1 : 1) with subsequent
heating. NMR spectra were measured on a Bruker AMX 400
5
0
five independent ELISA experiments and the resulting standard
deviations are indicated
MeMan pNPMan
1
2a
20
21
22
1
13
(
(
400 MHz for H and 100.67 MHz for C NMR) or a DRX 500
1 13
a
IC (µM)
4250
1173
1
43
14
99
17
5
4
0.7
238 222 134
50
500 MHz for H and 125.84 MHz for C NMR). Chemical
b
S
90
18
65
19
59
32
c
shifts are in ppm, relative to internal SiMe (TMS) (0.00 ppm
4
1 13
RIC50
250 1063
for H and C NMR) or solvent peaks which were calibrated
a
b
c
Obtained by ELISA. Standard deviation. Relative IC -values
1
13
5
0
as follows: CDCl (δ 7.26 for H and 77.00 for C NMR), D O
[
(
3
2
based on methyl α--mannoside (MeMan) as standard.
1
13
δ 4.65 for H; for C NMR CD CN (δ 1.30) or D -acetone
3 6
1
δ 30.60) was added], D -methanol (δ 3.35 for H and 49.30
4
13
This suggests that the structural positioning of the α-mannosyl
units especially in 2a is advantageous over the other tested
analogues for receptor binding and was disrupted by increased
length of the spacers. While 20 and 21 performed almost
equally well, the most flexible cluster glycoside prepared so
far, 22, showed a significant increase in inhibitory potency
compared with its less flexible counterparts 20 and 21.
While it seems that more flexibility of the α-mannosyl
moieties, as presented in glycoclusters 20–22, is appreciated in
the molecular recognition process, the structural preorganis-
ation which is provided in the structurally very different cluster
mannosides 1 and 2a is of greater importance for lectin binding
in this case. These data suggested that we combine a structural
for C NMR). J-Values are in Hz. Where necessary, two-
1 1 1 13
dimensional H– H or H– C homonuclear/two-dimensional
correlation spectroscopy (COSY) experiments were performed
for complete signal assignments. Optical-rotation values were
obtained using a Perkin-Elmer polarimeter 341 or 243 (Na-
line, 589 nm, cell length 10 cm). [α] -Values are given in units
D
Ϫ1
2
Ϫ1
of 10 deg cm g . Mass spectra were measured on a VG
Analytical 70-250S (FAB MS) or a Finnigan MAT 95
(MALDI-TOF MS) instrument. ELISAs were performed as
described earlier. For ELISA controls, bacterial adhesion
to blocked, uncoated microtitre plates was checked, and the
reaction of antibodies with carbohydrate derivatives as well as
the reaction of the employed antibodies with yeast mannan was
8
26
J. Chem. Soc., Perkin Trans. 1, 2001, 823–831

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3
tested and found to be negligible. The low background obtained
from these control experiments was subtracted when calcu-
lating the IC -values. The percentage inhibition was calculated
(50 cm ) and the glycosylation reaction was started by the
addition of a solution of TMSOTf in dry CH Cl (0.02 M;
2
2
3
0.05 cm ) at 0 ЊC. After stirring of the mixture at rt for 1 h,
additional TMSOTf solution (0.3 cm ) was added and then
50
Ϫ1
3
as [A(nI) Ϫ A(I)] × 100 × [A(nI)] (nI: no inhibitor, I: with
inhibitor).
the reaction mixture was stirred for 12 h at rt. Triethylamine
3
(
0.2 cm ) was added, the mixture was concentrated under high
vacuum and the residue was purified by flash chromatography
2
-Bromoethyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-mannopyranoside 7
-Bromoethanol 3 (345 mg, 2.76 mmol) and the trichloro-
(
(
ethyl acetate–light petroleum, 1 : 1) to yield the title glycoside
1.78 g, 89%) as a colourless syrup (Found: C, 48.13; H, 6.28;
2
acetimidate 6 (1.63 g, 3.31 mmol) were dissolved in dry CH Cl
2
2
Cl, 7.05. C H ClO requires C, 48.15; H, 6.26; Cl, 7.11%);
2
0
31
12
3
(
50 cm ) and the glycosylation reaction was started by
20
[
α]D ϩ119.1 (c 0.30 in CHCl ); δH (400 MHz; CDCl ; Me Si)
3
3
4
3
the addition of BF ؒEt O (0.01 cm ) at rt. After stirring of the
3
2
5
5
.36 (1 H, dd, J2,3 3.6, J3,4 10.2, 3-H), 5.29 (1 H, dd ≈ t, 4-H),
.27 (1 H, dd, 2-H), 4.88 (1 H, d, J1,2 1.5, 1-H), 4.30 (1 H, dd,
3
mixture at rt for 15 h, triethylamine (0.2 cm ) was added, the
reaction mixture was concentrated under reduced pressure
and the residual syrup was purified by flash chromatography
J5,6 5.1, 6-H), 4.11 (1 H, dd, J5,6Ј 2.3, J6,6Ј 12.2, 6-HЈ), 4.07 (1 H,
ddd, J4,5 10.2, 5-H), 3.86–3.76 (3 H, m, OCHHCH OCH -
2
2
(
toluene–ethyl acetate, 4 : 1) to give the desired manno-
CH OCH CH Cl), 3.70–3.63 (9 H, m, OCHHCH OCH CH -
2
2
2
2
2
2
pyranoside 7 (1.21 g, 96%) as an amorphous white solid (Found:
C, 42.24; H, 5.12; Br, 17.45. C H BrO requires C, 42.21; H,
5
OCH CH Cl), 2.16, 2.11, 2.06, 1.99 (each 3 H, 4 s, 4 × COCH );
2
2
3
1
6
23
10
δ (100.67 MHz; CDCl ; Me Si) 170.65, 170.01, 169.87, 169.70
C
3
4
20
.09; Br, 17.55%); [α]D ϩ37.3 (c 0.75 in CHCl ); δH (400
3
(
4 × COCH ), 97.69 (C-1), 71.36 (OCH CH OCH CH OCH -
3 2 2 2 2 2
MHz; CDCl ; Me Si) 5.32 (1 H, dd, J 3.6, J3,4 9.7, 3-H), 5.25
3
4
2,3
CH Cl), 70.70, 70.63, 70.01 (OCH CH OCH CH OCH CH -
2
2
2
2
2
2
2
(
1
1 H, dd ≈ t, J4,5 10.2, 4-H), 5.25 (1 H, dd, 2-H), 4.85 (1 H, d, J1,2
.5, 1-H), 4.24 (1 H, dd, J5,6 6.1, 6-H), 4.11 (1 H, dd, J5,6Ј 2.04,
J6,6Ј 12.7, 6-H), 4.11 (1 H, m , 5-H), 3.91 (2 H, m , OCH -
Cl), 69.56 (C-2), 69.04 (C-3), 68.39 (C-5), 67.36 (OCH CH -
2
2
OCH CH OCH CH Cl), 66.13 (C-4), 62.39 (C-6), 42.73
2
2
2
2
c
c
2
(
(
OCH CH Cl), 20.87, 20.72, 20.67, 20.65 (4 × COCH ); m/z
2 2 3
ϩ
CH Br), 3.49 (2 H, t, J 6.1, OCH CH Br), 2.13, 2.08, 2.03, 1.97
2
2
2
MALDI-TOF) 521.82 (M ϩ Na. C H ClNaO requires
20
31
12
(
each 3 H, 4 s, 4 × COCH ); δ (100.67 MHz; CDCl ; Me Si)
ϩ
3
C
3
4
m/z 521.14), m/z (MALDI-TOF) 537.63 (M ϩ K. C H -
20
31
1
6
70.65, 170.04, 169.89, 169.78 (4 × COCH ), 97.73 (C-1), 69.42,
3
ClKO12 requires m/z 537.11).
9.00, 68.95, 68.48 (C-2, -3, -4, -5), 66.00 (OCH CH Br), 62.41
2
2
(
(
C-6), 29.60 (OCH CH Br), 20.84, 20.73,ϩ 20.68, 20.65
2
2
2
-Azidoethyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-mannopyranoside 10
4 × COCH ); m/z (MALDI-TOF) 455.25 (M ϩ H. C H -
3
16 24
The bromo-substituted mannoside 7 (1.2 g, 2.64 mmol) and
sodium azide (1.37 g, 21 mmol) were dissolved in dry DMF
BrO10 requires m/z 455.06), m/z (MALDI-TOF) 477.23
(
TOF) 493.18 (M ϩ K. C H BrKO requires m/z 493.01).
ϩ
M ϩ Na. C H BrNaO requires m/z 477.04), m/z (MALDI-
16 23 10
ϩ
3
(
50 cm ) and the reaction mixture was stirred at 60 ЊC for 15 h.
16
23
10
The solvent was removed under high vacuum and the residue
was dissolved in ethyl acetate (50 cm ) and filtered. The filtrate
was evaporated under reduced pressure and purified by flash
chromatography (light petroleum–ethyl acetate, 1 : 1) to yield
the title azide (1.08 g, 98%) as an amorphous solid (Found:
C, 46.09; H, 5.58; N, 10.02. C H N O requires C, 46.04; H,
3
2
-(2-Chloroethoxy)ethyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-manno-
pyranoside 8
The alcohol 4 (698 mg, 5.60 mmol) and the trichloroacet-
imidate 6 (3.31 g, 6.72 mmol) were dissolved in dry CH Cl
(
addition of a solution of trifluoromethyl trifluoromethane-
sulfonate (TMSOTf) in dry CH Cl (0.02 M; 0.2 cm ) at 0 ЊC.
After stirring of the mixture at rt for 4 h, additional TMSOTf
solution (0.4 cm ) was added and then the reaction mixture was
stirred for 40 h at rt. Triethylamine (0.2 cm ) was added, the
mixture was concentrated under high vacuum and the residue
was purified by flash chromatography (ethyl acetate–toluene,
3
syrup (Found: C, 47.57; H, 6.04; Cl, 7.72. C H ClO requires
C, 47.53; H, 5.98; Cl, 7.79%); [α]D ϩ120.5 (c 0.75 in CHCl );
δH (400 MHz; CDCl ; Me Si) 5.37 (1 H, dd, J 3.5, J3,4 9.8,
2
2
16
23
3
10
3
20
60 cm ) and the glycosylation reaction was started by the
5
.55; N, 10.07%); [α]D ϩ50.2 (c 0.75 in CHCl ); δ (400
3 H
MHz; CDCl ; Me Si) 5.37 (1 H, dd, J 3.1, J3,4 10.2, 3-H), 5.30
3
4
2,3
3
2
2
(1 H, dd ≈ t, J4,5 10.2, 4-H), 5.28 (1 H, dd, 2-H), 4.88 (1 H, d, J1,2
.5, 1-H), 4.30 (1 H, dd, J5,6 5.6, 6-H), 4.13 (1 H, dd, J5,6Ј 2.5,
J6,6Ј 12.2, 6-HЈ), 4.05 (1 H, ddd, 5-H), 3.88 (1 H, m , OCHH),
1
3
c
3
3
2
1
.68 (1 H, m , OCHH), 3.48 (2 H, m , CH N ), 2.22, 2.11, 2.06,
.00 (each 3 H, 4 s, 4 × COCH ); δC (100.67 MHz; CDCl3)
70.59, 169.98, 169.78, 169.73 (4 × COCH ), 97.74 (C-1), 69.38
c
c
2
3
3
3
: 1) to yield the title glycoside (2.35 g, 92%) as a colourless
(
C-2), 68.85 (C-3, C-5), 67.02 (OCH CH ), 66.01 (C-4), 62.45
2 2
18
27
11
(C-6), 50.34 (CH N ), 20.83, 20.70, 20.67, 20.62 (4 × COCH );
2 3 3
ϩ
20
3
m/z (MALDI-TOF) 418.36 (M ϩ H. C H N O requires
16 24 3 10
ϩ
3
4
2,3
m/z 418.15), m/z (MALDI-TOF) 440.37 (M ϩNa. C H N -
16 23 3
3
-H), 5.29 (1 H, dd ≈ t, J4,5 9.8, 4-H), 5.27 (1 H, dd, 2-H), 4.89
1 H, d, J1,2 1.6, 1-H), 4.29 (1 H, dd, J5,6 6.6, 6-H), 4.11 (1 H, dd,
J5,6Ј 2.2, J6,6Ј 12.6, 6-HЈ), 4.13–4.10 (1 H, m, 5-H), 3.86–3.81
1 H, m, OCHHCH OCH CH Cl), 3.77–3.75 (2 H, m, OCH -
NaO10 requires m/z 440.13), m/z (MALDI-TOF) 456.31
(M ϩ K. C H KN O requires m/z 456.10).
ϩ
(
16
23
3
10
(
2
2
2
2
2-(2-Azidoethoxy)ethyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-manno-
pyranoside 11
CH OCH CH Cl), 3.71–3.69 (3 H, m, OCHHCH OCH -
CH Cl), 3.63 (2 H, t, J 5.7, OCH CH OCH CH Cl), 2.16, 2.11,
2
2
2
2
2
2
2
2
2
2
2
The chloro-substituted mannoside 8 (1.60 g, 3.52 mmol) and
sodium azide (1.83 g, 28.15 mmol) were dissolved in dry DMF
.04, 1.99 (each 3 H, 4 s, 4 × COCH ); δ (100.67 MHz; CDCl ;
3
C
3
Me Si) 170.64, 170.02, 169.89, 169.73 (4 × COCH ), 97.61
4
3
3
(
60 cm ) and the reaction mixture was stirred at 60 ЊC for 16 h.
(
C-1), 71.37 (OCH CH OCH CH Cl), 70.05 (OCH CH -
2 2 2 2 2 2
The solvent was removed under high vacuum, the residue was
dissolved in ethyl acetate (50 cm ) and the solution was filtered
to remove inorganic salts. The filtrate was evaporated under
reduced pressure and purified by flash chromatography (light
petroleum–ethyl acetate, 1 : 1) to yield the title azide (1.62 g,
OCH CH Cl), 69.52 (C-2), 69.06 (C-3), 68.39 (C-5), 67.13
2
2
3
(
(
(
OCH CH OCH CH Cl), 66.13 (C-4), 62.43 (C-6), 42.80
2 2 2 2
OCH CH Cl), 20.85, 20.72, 20.65, 20.64 (4 × COCH ); m/z
2
2
3
ϩ
MALDI-TOF) 477.04 (M ϩ Na. C H ClNaO requires
18
27
11
ϩ
m/z 477.11), m/z (MALDI-TOF) 493.02 (M ϩ K. C H -
ClKO11 requires m/z 493.09).
18
27
9
9%) as a colourless syrup (Found: C, 46.87; H, 5.96; N, 9.10.
20
D
C H N O requires C, 46.85; H, 5.90; N, 9.11%); [α]
18
27
3
11
ϩ105.7 (c 0.60 in CHCl ); δH (400 MHz; CDCl ; Me Si)
3
3
4
2
-[2-(2-Chloroethoxy)ethoxy]ethyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-
5
.38 (1H, dd, J2,3 3.6, J3,4 10.2, 3-H), 5.29 (1 H, dd ≈ t, J4,5 10.2,
mannopyranoside 9
4
-H), 5.28 (1 H, dd, 2-H), 4.89 (1 H, d, J1,2 1.5, 1-H), 4.29 (1 H,
The alcohol 5 (673 mg, 3.99 mmol) and the trichloroacet-
dd, J5,6 5.1, 6-H), 4.11 (1 H, dd, J5,6Ј 2.6, J6,6Ј 12.2, 6-HЈ), 4.08
(1 H, ddd, 5-H), 3.88–3.81 (1 H, m, OCHHCH OCH CH Cl),
imidate 6 (2.36 g, 4.79 mmol) were dissolved in dry CH Cl2
2
2
2
2
J. Chem. Soc., Perkin Trans. 1, 2001, 823–831
827

View Online
3
.69–3.67 (5 H, m, OCHHCH OCH CH Cl), 3.40 (2 H, t, J 5.1,
J1,2 1.5, 1-H), 3.91 (1 H, dd, J2,3 3.6, J3,4 9.2, 3-H), 3.88 (1 H,
2
2
2
OCH CH OCH CH Cl), 2.16, 2.11, 2.04, 2.00 (each 3 H, 4 s,
dd ≈ t, J4,5 9.2, 4-H), 3.87 (dd, 1 H, 2-H), 3.75 (1 H, dd, J5,6Ј 2.5,
J6,6Ј 12.2, 6-HЈ), 3.73 (1 H, dd, J5,6 5.1, 6-H), 3.74–3.58 (6 H, m,
3 × OCH ), 3.61 (1H, ddd, 5-H), 3.43–3.39 (2 H, m, CH N ); δ
2
2
2
2
4
1
× COCH ); δ (100.67 MHz; CDCl ; Me Si) 170.64, 170.02,
3 C 3 4
69.89, 169.74 (4 × COCH ), 97.71 (C-1), 70.17 (OCH CH -
3
2
2
2
2
3
C
OCH CH Cl), 70.04 (OCH CH OCH CH Cl), 69.52 (C-2),
(100.67 MHz; CD OD) 102.11 (C-1), 74.90, 72.85, 72.39, 68.86
2
2
2
2
2
2
3
6
9.06 (C-3), 68.42 (C-5), 67.28 (OCH CH OCH CH Cl), 66.13
(C-2, -3, -4, -5), 71.55, 71.47 (2 × OCH ), 68.16 (OCH CH N ),
2
2
2
2
2
2
2
3
(
2
C-4), 62.43 (C-6), 50.71 (OCH CH Cl), 20.85, 20.70, 20.65,
63.20 (C-6), 52.03 (CH N ); m/z (MALDI-TOF) 316.18
2 3
ϩ
2
2
ϩ
0.64 (4 × COCH ); m/z (MALDI-TOF) 484.05 (M ϩ Na.
(M ϩ Na. C H N NaO requires m/z 316.11), m/z (MALDI-
10 19 3 7
ϩ
3
C H N NaO requires m/z 484.15), m/z (MALDI-TOF)
5
TOF) 332.14 (M ϩ K. C H N NaO requires m/z 332.09).
10 19 3 7
18
27
3
11
ϩ
00.02 (M ϩ K. C H N KO requires m/z 500.13).
18 27 3 11
2
-[2-(2-Azidoethoxy)ethoxy]ethyl ꢀ-D-mannopyranoside 15
2
-[2-(2-Azidoethoxy)ethoxy]ethyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-
The protected mannoside 12 (650 mg, 1.29 mmol) was dissolved
in dry MeOH (80 cm ), and sodium methanolate solution (1 M
mannopyranoside 12
3
Mannoside 9 (1.02 g, 2.05 mmol) was dissolved in dry DMF (70
cm ), sodium azide (1.06 g, 16.36 mmol) was added, and the
in MeOH) was added until pH 9 was reached. Then the reac-
tion mixture was stirred at rt until the deprotection reaction
was complete (TLC n-PrOH–water, 7 : 3, ϩ1% NH ; R 0.60,
3
reaction mixture was stirred at 50 ЊC for 48 h. Then, the solvent
was removed under high vacuum and the residue was passed
through a silica gel column (light petroleum–ethyl acetate, 1 : 1)
to yield the title compound (1.02 g, 2.02 mmol, 97%) as a colour-
less syrup (Found: C, 47.55; H, 6.21; N, 8.29. C H N O
3
f
H SO ). It was neutralised with ion-exchange resin (Levatit
2
4
ϩ
1080 H ), filtered, and the solvent removed in vacuo. After flash
chromatography (i-PrOH–water, 7 : 3, ϩ1% NH ) the depro-
3
tected title compound (423 mg, 97%) was obtained as colourless
2
0
31
3
12
2
0
requires C, 47.52; H, 6.18; N, 8.31%); [α] ϩ61.5 (c 0.22 in
syrup (Found: C, 42.75; H, 6.90; N, 12.42. C H N O requires
D
12 23
3
8
CHCl ); δ (400 MHz; CDCl ; Me Si) 5.37 (1 H, dd, J 3.6, J3,4
C, 42.73; H, 6.87; N, 12.46%); δH (400 MHz; CD OD) 4.84
3
H
3
4
2,3
3
1
0.2, 3-H), 5.29 (1 H, dd ≈ t, J4,5 9.7, 4-H), 5.27 (1 H, dd, 2-H),
(1 H, d, J1,2 1.5, 1-H), 3.89 (1 H, dd, J2,3 3.6, J3,4 10.7, 3-H), 3.89
(1 H, dd ≈ t, J4,5 9.2, 4-H), 3.85 (1 H, dd, 2-H), 3.79–3.63 (12 H,
m, J6,6Ј 11.2, 6-H, 6-HЈ, 5 × CH ), 3.60 (1 H, ddd, J 5.6, J5,6Ј
4
.88 (1 H, d, J1,2 1.5, 1-H), 4.30 (1 H, dd, J5,6 4.6, 6-H), 4.10
(
3
1 H, dd, J5,6Ј 2.6, J6,6Ј 12.2, 6-HЈ), 4.07 (1 H, ddd, 5-H), 3.86–
.76 (1 H, m, OCHHCH OCH CH OCH CH N ), 3.71–3.65
2
5,6
2.0, 5-H), 3.43–3.39 (2 H, m, CH N ); δ (100.67 MHz;
2
2
2
2
2
3
2 3 C
(
9 H, m, OCHHCH OCH CH OCH CH N ), 3.40 (2 H, t,
CD OD) 102.05 (C-1), 74.90 (C-5), 72.87 (C-2), 72.41 (C-3),
2
2
2
2
2
3
3
J 5.1, OCH CH N ), 2.16, 2.11, 2.05, 1.99 (each 3 H, 4 s,
72.01, 71.85, 71.74, 71.49 (4 × OCH ), 68.90 (C-4), 68.09
2
2
3
2
4
1
× COCH ); δ (100.67 MHz; CDCl ) 170.61, 169.97, 169.82,
(COCH ), 63.23 (C-6), 52.06 (CH N ); m/z (MALDI-TOF)
3
C
3
2
2
3
ϩ
69.67 (4 × COCH ), 97.65 (C-1), 70.72 (OCH CH OCH -
338.34 (M ϩ H. C H N O requires m/z 338.16), m/z
12 24 3 8
ϩ
3
2
2
2
CH OCH CH N ), 70.62, 70.02, 69.99 (OCH CH OCH -
CH OCH CH N ), 69.498 (C-2), 69.00 (C-3), 68.34 (C-5),
(MALDI-TOF) 360.32 (M ϩ Na. C H N NaO requires
12 23 3 8
ϩ
2
2
2
3
2
2
2
m/z 360.14), m/z (MALDI-TOF) 376.40 (M ϩ K. C H -
2
2
2
3
12 23
6
7.33 (OCH CH OCH CH OCH CH N ), 66.08 (C-4), 62.339
KN O requires m/z 376.11).
2
2
2
2
2
2
3
3
8
(
C-6), 50.59 (OCH CH OCH CH OCH CH N ), 20.82,
2 2 2 2 2 2 3
2
0.67, 20.62, 20.61 (4 × COCH ); m/z (MALDI-TOF) 528.18
3
ϩ
2-Aminoethyl ꢀ-D-mannopyranoside 16
(
M ϩ Na. C H N NaO requires m/z 528.18), m/z (MALDI-
20 31 3 12
ϩ
The azide 13 (420 mg, 1.69 mmol) was dissolved in ethyl
TOF) 544.11 (M ϩ K. C H KN O requires m/z 544.15).
20
31
3
12
3
acetate–ethanol (10 cm ; 1 : 1), palladium catalyst (10% Pd-C,
5
0 mg) was added and the reaction mixture was hydrogenated
2
-Azidoethyl ꢀ-D-mannopyranoside 13
at 1 bar until the reduction was complete (TLC i-PrOH–water,
7 : 3, ϩ1% NH ; R 0.04, H SO ). Then, it was filtered through
a Celite bed, washed with ethyl acetate–ethanol (1 : 1) and the
filtrate was concentrated under low pressure. Purification by
flash chromatography (i-PrOH–water, 6 : 2) gave the title amine
(370.2 mg, 98%) as a colourless syrup; [α]D ϩ84.5 (c 1.20 in
MeOH); δH (400 MHz; D O) 4.72 (1 H, d, J 1.5, 1-H), 3.82
(1 H, dd, 2-H), 3.74 (1 H, dd, J5,6Ј 1.5, 6-HЈ), 3.68 (1 H, dd, J2,3
3.6, J3,4 9.7, 3-H), 3.61 (1 H, dd ≈ t, J4,5 10.7, 4-H), 3.61 (1 H,
dd, J5,6 6.1, J6,6Ј 12.2, 6-H), 3.50 (2 H, m , OCH CH NH ), 3.38
The protected mannoside 10 (810 mg, 1.94 mmol) was dissolved
in dry MeOH (80 cm ) and sodium methanolate solution (1 M
in MeOH) was added until pH 9 was reached. Then the reac-
tion mixture was stirred at rt until the deprotection reaction
was complete (TLC n-PrOH–water, 7 : 3, ϩ1% NH ; R 0.61,
3
f
2
4
3
20
3
f
H SO ). Then it was neutralised with ion-exchange resin
2
4
2
1,2
ϩ
(
Levatit 1080 H ), filtered, and the solvent was removed in
vacuo. The deprotected title compound (481.3 mg, 99%) was
obtained as a white amorphous solid and used without further
purification; [α] ϩ139.2 (c 0.50 in CHCl ); δH (400 MHz;
c
2
2
2
2
0
(1 H, ddd, 5-H), 2.74–2.63 (2 H, m, OCH CH NH ); δ (100.67
D
3
2 2 2 C
CD OD) 4.82 (1 H, d, J 1.5, 1-H), 3.88 (1 H, dd, J2,3 3.6, J3,4
MHz; D O) 100.23 (C-1), 73.08, 70.90, 70.38, 67.16 (C-2, -3,
3
1,2
2
1
3
6
0.7, 3-H), 3.86 (1 H, dd ≈ t, J4,5 9.2, 4-H), 3.84 (1 H, dd, 2-H),
.74 (1 H, dd, J5,6Ј 2.1, J6,6Ј 12.2, 6-HЈ), 3.71 (1 H, dd, J5,6 5.6,
-H), 3.62–3.57 (3 H, m, OCH CH , 5-H), 3.40–3.36 (2 H, m,
-4, -5), 67.16 (OCH CH NH ), 61.31 (C-6), 40.29 (OCH -
2
2
2
2
ϩ
CH NH ); m/z (MALDI-TOF) 224.33 (M ϩ H. C H NO
2
2
8
ϩ
18
6
requires m/z 224.11), m/z (MALDI-TOF) 246.25 (M ϩ Na.
C H NNaO requires m/z 246.10), m/z (MALDI-TOF) 262.18
2
2
OCH N ); δ (100.67 MHz; CD OD) 102.03 (C-1), 74.88,
2
3
C
3
8
17
6
ϩ
7
2.86, 72.39, 68.88 (C-2, -3, -4, -5), 68.07 (OCH CH ), 63.21
(M ϩ K. C H KNO N requires m/z 262.07).
2
2
8 17 6
(
C-6), 52.03 (CH N ).
2 3
2
-(2-Aminoethoxy)ethyl ꢀ-D-mannopyranoside 17
2
-(2-Azidoethoxy)ethyl ꢀ-D-mannopyranoside 14
The azide 14 (521 mg, 1.78 mmol) was dissolved in ethyl
acetate–ethanol (10 cm ; 1 : 1), palladium catalyst (10% Pd-C,
50 mg) was added and the reaction mixture was hydrogenated
at 1 bar until the reduction was complete (TLC n-PrOH–water,
7 : 3, ϩ1% NH ; R 0.07, H SO ). Then, it was filtered through a
3
The protected mannoside 11 (1.42 g, 3.077 mmol) was dissolved
in dry MeOH (80 cm ) and sodium methanolate solution (1 M
in MeOH) was added until pH 9 was reached. Then the reaction
mixture was stirred at rt until the deprotection reaction was
3
3
f
2
4
complete (TLC n-PrOH–water, 7 : 3, ϩ1% NH ; Rf 0.59,
Celite bed, washed with ethyl acetate–ethanol (1 : 1), and the
filtrate was concentrated under reduced pressure. Purification
by flash chromatography (i-PrOH–water, 8 : 2) gave the title
3
H SO ). It was neutralised with ion-exchange resin (Levatit
2
4
ϩ
1
080 H ), filtered, and the solvent was removed in vacuo. The
20
deprotected title compound (891.3 mg, 99%) was obtained as a
amine (465 mg, 98%) as a colourless syrup; [α] ϩ57.2
D
20
colourless syrup and used without further purification; [α]D
ϩ62.9 (c 1.05 in MeOH); δ (400 MHz, CD OD) 4.84 (1 H, d,
(c 0.75 in H O); δ (400 MHz; D O) 4.74 (1 H, d, J 1.5, 1-H),
2
H
2
1,2
3.81 (1 H, dd, J2,3 3.6, 2-H), 3.75–3.54 (6 H, m, 3-, 4-, 5-H, 6-H2,
H
3
8
28
J. Chem. Soc., Perkin Trans. 1, 2001, 823–831

View Online
OCHHCH NH ), 3.52–3.49 (3 H, m, OCHHCH O), 3.47–3.43
and stirred at 60 ЊC for 4 days. Then the solvent was removed
2
2
2
(
2 H, m, OCH ), 2.66 (2 H, t, J 5.6, OCH CH NH ); δ (100.67
by lyophilisation and the residue was taken up in water
2
2
2
2
C
3
MHz; D O) 100.31 (C-1), 73.15, 70.88, 70.34, 67.12 (C-2,
-
(50 cm ). Impurities were extracted three times with Et O (each
2
2
3
3, -4, -5), 72.23 (OCH CH O), 69.69 (OCH CH NH ), 66.81
40 cm ), then the aqueous phase was set under slight vacuum
2
2
2
2
2
(
(
2
OCH CH O), 61.30 (C-6), 40.16 (OCH CH NH ); m/z
to remove traces of Et O. The residual solution was diluted to
2
2
2
2
2
2
ϩ
MALDI-TOF) 268.29 (M ϩ H. C H NO requires m/z
three-fold its volume with water and the resulting solution
was freeze-dried. The obtained lyophilisate was subjected to
gel-permeation chromatography (Biogel P2 extra fine; 15 mM
aq. NH HCO as the eluent) to yield the pure title cluster (38
1
0
22
7
ϩ
68.14), m/z (MALDI-TOF) 290.25 (M ϩ Na. C H NNaO
10 21 7
ϩ
requires m/z 290.12), m/z (MALDI-TOF) 306.20 (M ϩ K.
C H KNO requires 306.10).
1
0
21
7
4
3
mg, 69%)asawhitelyophilisate;TLCn-PrOH–water, 7 : 3, ϩ 1%
2
0
NH (R 0.14, H SO ); [α] ϩ20.0 (c 0.90 in MeOH); δH (400
2
-[2-(2-Aminoethoxy)ethoxy]ethyl ꢀ-D-mannopyranoside 18
3
f
2
4
D
b
MHz; CD OD) 4.85 (3 H, d, J 1.9, 1-H), 3.88 (3 H, dd, 6-H ),
.86 (3 H, dd, 2-H), 3.75 (3 H, dd, J6a,6b 11.7, 6-H ), 3.745 (3 H,
dd, J2,3 3.5, J3,4 9.5, 3-H), 3.70–3.66 (12 H, m, CONHCH2-
CH OCH ), 3.65 (3 H, dd ≈ t, J 9.8, 4-H), 3.60 (3 H, ddd, J5,6a
3
1,2
The azide 15 (380 mg, 1.13 mmol) was dissolved in ethyl
acetate–ethanol (10 cm ; 1 : 1), palladium catalyst (10% Pd-C,
6
1
7
a Celite bed, washed with ethyl acetate–ethanol (1 : 1) and the
filtrate was concentrated under reduced pressure. Purification
by flash chromatography (i-PrOH–water, 7 : 2) gave the title
a
3
3
0 mg) was added and the reaction mixture was hydrogenated at
bar until the reduction was completed (TLC i-PrOH–water,
: 3, ϩ1% NH ; R 0.05, H SO ). Then, it was filtered through
2
2
4,5
5
.7, J5,6b 2.2, 5-H), 3.60–3.58 (6 H, m, manOCH ), 3.41 (6 H, t,
2
q
3
f
2
4
J 5.4, CONHCH ), 2.31–2.26 (12 H, m, O NC CH CH -
2
2
2
2
q
CONH); δ (100.67 MHz; CD OD) 174.62 (C , C᎐O), 101.98
C-1), 94.63 (C NO ), 74.94 (C-5), 72.87 (C-3), 72.41 (C-2),
1.50 (CONHCH CH OCH ), 70.81 (CONHCH CH -
2 2 2 2 2
OCH ), 69.00 (C-4), 68.05 (manOCH ), 63.31 (C-6), 40.84
CONHCH ), 32.44 (O NC CH CH ), 31.48 (O NC CH -
CH ); m/z (MALDI-TOF) 1047.55 (M ϩ Na. C H N NaO
requires m/z 1047.43), m/z (MALDI-TOF) 1063.55 (M ϩ K.
C H KN O requires m/z 1063.41).
C
3
q
(
2
7
20
amine (345 mg, 98%) as a colourless syrup; [α]D ϩ32.6
2
2
(
c 0.60 in MeOH); δ (400 MHz; CD OD) 4.87 (1 H, d, J 1.5,
q
q
H
3
1,2
(
2
2
2
2
ϩ
2
2
1-H), 3.89 (1 H, dd, J2,3 3.6, J3,4 9.2, 3-H), 3.88 (1 H, dd ≈ t,
4-H), 3.85 (1 H, dd, 2-H), 3.79–3.62 (12 H, m, J6,6Ј 12.2, 6-H2,
5 × CH ), 3.58 (1 H, ddd, J 10.2, J5,6 5.1, J5,6Ј 2.5, 5-H), 3.40
2
40 72
4
ϩ
26
2
4,5
40
72
4
26
(
(
2 H, m, OCH CH NH ); δ (100.67 MHz; CD OD) 102.00
C-1), 74.94 (C-5), 73.23 (OCH ), 72.85 (C-2), 72.40 (C-3),
2 2 2 C 3
2
Tris{2٣-[(2ٞ-{2Љ-[2Ј-(ꢀ-D-mannopyranosyloxy)ethoxy]-
ethoxy}ethyl)carbamoyl]ethyl}nitromethane 22
7
6
1.82, 71.61, 70.88 (3 × OCH ), 68.93 (C-4), 67.85 (OCH CH ),
2
2
2
3.23 (C-6), 41.68 (CH NH ); m/z (MALDI-TOF) 312.23
2
2
ϩ
A solution of the amino-functionalised mannoside 18 (41.3 mg,
.133 mmol) and the triacid 19 (10.2 mg, 0.037 mmol) in dry
DMA (15 cm ) was treated with EEDQ (39.4 mg, 0.214 mmol)
and stirred at 60 ЊC for 4 days. Then the solvent was removed by
lyophilisation and the residue was taken up in water (50 cm ).
Impurities were extracted three times with Et O (each 40 cm ),
then the aqueous phase was set under slight vacuum to remove
traces of Et O. The residual solution was diluted to three
times its volume with water and the resulting solution was
freeze-dried. The obtained lyophilisate was subjected to gel-
permeation chromatography (Biogel P2 extra fine; 15 mM aq.
NH HCO as the eluent) to yield the pure title cluster (25.3 mg,
9%) as a white lyophilisate; TLC n-PrOH–water, 7 : 3, ϩ 1%
NH (R 0.13, H SO ); [α] ϩ23.4 (c 0.60 in H O); δ (400
(
M ϩ H. C H NO requires m/z 312.17), m/z (MALDI-
12 26 8
ϩ
0
TOF) 334.21 (M ϩ Na. C H NNaO requires m/z 334.15),
12
25
ϩ
8
3
m/z (MALDI-TOF) 350.17 (M ϩ K. C H KNO requires
m/z 350.12).
12
25
8
3
3
2
Tris(2Љ-{[2Ј-(ꢀ-D-mannopyranosyloxy)ethyl]carbamoyl}ethyl)-
nitromethane 20
2
A solution of the amino-functionalised mannoside 16 (29 mg,
0
.130 mmol) and the triacid 19 (10.9 mg, 0.039 mmol) in dry
3
dimethylacetamide (DMA) (10 cm ) was treated with EEDQ
26.7 mg, 0.108 mmol) and stirred at 60 ЊC for 4 days. Then the
(
4
3
solvent was removed by lyophilisation and the residue was
taken up in water (50 cm ). Impurities were extracted three
times with Et O (each 40 cm ), then the aqueous phase was
set under slight vacuum to remove dissolved Et O, and was
subsequently diluted to the three-fold volume with water. The
resulting solution was freeze-dried and the obtained lyophil-
isate was subjected to gel-permeation chromatography (Biogel
P2 extra fine; 15 mM aq. NH HCO as the eluent) to yield
the pure title cluster (26 mg, 74%) as a white lyophilisate;
TLC n-PrOH–water, 7 : 3, ϩ 1% NH (R 0.11, H SO ); [α]
ϩ43.6 (c 0.70 in MeOH); δ (400 MHz; CD OD) 4.86 (3 H, d,
J1,2 1.5, 1-H), 3.92 (3 H, dd, 2-H), 3.89 (3 H, dd, J
1
J3,4 9.2, 3-H), 3.77 (3 H, dd, J5,6a 5.6, 6-H ), 3.67 (3 H, dd ≈ t, J
9
OCHH), 2.30 (12 H, br s, O NC CH CH CO); δ (100.67
MHz; CD OD) 175.13 (C᎐O), 101.32 (C-1), 94.55 (C NO ),
5
3
20
D
3
f
2
4
2
H
3
2
MHz; D O) 4.74 (3 H, d, J 1.5, 1-H), 3.81 (3 H, dd, 2-H), 3.73
2 1,2
a
2
(3 H, dd, J5,6a 6.6, 6-H ), 3.72 (3 H, dd, J 3.6, J3,4 9.1, 3-H),
2,3
3
3
1
.66 (3 H, ddd, J4,5 9.1, 5-H), 3.62–3.56 (6 H, m, spacer-CH ),
.60 (3 H, dd, J5,6b 3.1, J6a,6b 11.2, 6-H ), 3.55–3.53 (15 H, m,
2 spacer-CH , 4-H), 3.51–3.46 (12 H, m, spacer-CH ), 3.24
2
b
2
2
4
3
(6 H, t, J 5.6, spacer-CH ), 2.13 (12 H, br s, core-CH ), 1.10
2
2
and 1.03 (together 3 H, t, J 7.1, NH); δ (100.67 MHz; D O)
174.81 (NHC᎐O), 100.30 (C-1), 94.43 (C NO ), 73.13 (C-5),
70.891 (C-3), 70.35 (C-2), 70.02 (CONHCH CH OCH ), 69.88
C
2
2
0
q
3
f
2
4
D
2
H
3
2
2
2
5,6b
2.2, J6a,6b
(manOCH CH OCH ), 69.79 (manOCH CH OCH ), 69.15
2 2 2 2 2 2
b
2.2, 6-H ), 3.88–3.77 (3 H, m, OCHH), 3.79 (3 H, dd, J 3.6,
2,3
a
(CONHCH CH OCH ), 67.12 (C-4), 66.71 (manOCH CH ),
2 2 2 2 2
q
4,5
61.31 (C-6), 39.39 (CONHCH CH ), 30.90 (O NC CH -
2 2 2 2
q
.7, 4-H), 3.62–3.58 (6 H, m, CH N), 3.52–3.37 (6 H, m, 5-H,
2
q
CH CO), 30.38 (O NC CH CH CO); m/z (MALDI-TOF)
2 2 2 2
ϩ
2
2
2
C
1179.54 (M ϩ Na. C H N NaO requires m/z 1179.51), m/z
46 84 4 29
ϩ
q
3
2
(MALDI-TOF) 1195.54 (M ϩ K. C H KN O requires
46 84 4 29
7
4.49 (C-5), 72.29 (C-3), 71.77 (C-2), 68.35 (C-4), 67.16
m/z 1195.49).
(
(
OCH CH NH), 62.57 (C-6), 40.48 (OCH CH NH), 32.19
CH CH CONH), 31.41 (CH CH CONH); m/z (MALDI-
2
2
2
2
2
2
2
2
p-Nitrophenyl 6-amino-6-deoxy-ꢀ-D-mannopyranoside 26
ϩ
TOF) 915.48 (M ϩ Na. C H N NaO requires m/z 915.35),
m/z (MALDI-TOF) 931.46 (M ϩ K. C H KN O requires
m/z 931.33).
34
60
ϩ
4
23
9
To a solution of the 6-azido-6-deoxymannopyranoside 25
334.5 mg, 1.025 mmol) in tetrahydrofuran (THF)–water (4 : 1;
0 cm ) was added PPh (349.6 mg, 1.33 mmol) at rt and, after
34
60
4
23
(
5
3
3
3
0 min of stirring, silica gel (1 g) was added and the reaction
Tris[2ٞ-({2Љ-[2Ј-(ꢀ-D-mannopyranosyloxy)ethoxy]ethyl}-
carbamoyl)ethyl]nitromethane 21
mixture was stirred at 45 ЊC for 12 h. The silica gel was then
filtered off, the solvents were removed in vacuo, and the residue
was purified by flash chromatography (using two gradients:
ethyl acetate–toluene, 19 : 1, ϩ3% pyridine; →ethyl acetate–
MeOH, 2 : 1, ϩ3% pyridine; →ethyl acetate–MeOH, 4 : 1,
A solution of the amino-functionalised mannoside 17 (47.6 mg,
0
.178 mmol) and the triacid 19 (14.96 mg, 0.054 mmol) in dry
3
DMA (15 cm ) was treated with EEDQ (52.84 mg, 0.214 mmol)
J. Chem. Soc., Perkin Trans. 1, 2001, 823–831
829

View Online
ϩ 3% pyridine) to give the title glycoside (302.4 mg, 98%) as
(CMe ), 27.94 (CH CH CH CH CONH); m/z (MALDI-TOF)
3 2 2 2 2
ϩ
20
a slightly yellow amorphous solid; [α]D ϩ82.8 (c 0.50 in
664.28 (M ϩ Na. C H N NaO S requires m/z 664.36), m/z
32 55 3 8
ϩ
3
MeOH); δH (400 MHz; CD OD) 8.27 (2 H, d, J 9.2, m-ArH),
7
(MALDI-TOF) 680.17 (M ϩ K. C H KN O S requires
32 55 3 8
m/z 680.33).
3
3
.32 (2 H, d, J 9.2, o-ArH), 5.71 (1 H, d, J1 1.5, 1-H), 4.09
,2
(
1 H, dd, J2,3 3.6, 2-H), 3.93 (1 H, dd, 3-H), 3.68 (1 H, dd ≈ t, J3,4
.2, J4,5 9.7, 4-H), 3.49 (1 H, ddd, 5-H), 2.97 (1 H, dd, J5,6Ј 3.1,
J6,6Ј 13.2, 6-HЈ), 2.82 (1 H, dd, J 7.1, 6-H); δ (100.67 MHz;
9
Biotinamidotris(2-carboxyethyl)methane 31
5,6
C
The tert-butyl triester 30 (200 mg, 0.312 mmol) was stirred in
q
q
CD OD) 162.92 (i-aryl-C ), 144.17 ( p-aryl-C ), 127.07 (m-aryl-
3
3
formic acid (98%; 10 cm ) for 5 h at rt. Then the solvent was
CH), 117.93 (o-aryl-CH), 100.18 (C-1), 76.18 (C-5), 72.41
removed in vacuo with repeated co-evaporation with toluene to
yield the title triacid (146 mg, 99%) as a colourless syrup which
(
C-3), 71.86 (C-2), 69.83 (C-4), 43.87 (C-6); m/z (MALDI-
ϩ
TOF) 301.14 (M ϩ H. C H N O requires m/z 301.10), m/z
12
17
2
7
was not further purified; TLC n-PrOH–water, 7 : 3, ϩ 1% NH3
ϩ
(
MALDI-TOF) 323.12 (M ϩ Na. C H NaN O requires
12 16 2 7
ϩ
(
R 0.35, H SO ); δ (500 MHz; CD OD) 8.63 (1 H, br s,
f
2
4
H
3
m/z 323.09), m/z (MALDI-TOF) 339.10 (M ϩ K. C H K-
12
16
3
3
NHCO), 4.53 (1 H, dd, J 4.4, J
dd, J 4.4, J
2
8.2, CHCH S), 4.36 (1 H,
7.6, CHCHRS), 3.25 (1 H, ddd, CHCHRS),
cis
trans
2
N O requires m/z 339.06).
2
7
3
3
cis
trans
3
2
.97 (1 H, dd, J 5.0, J 12.6, CHCHHS), 2.74 (1 H, dd ≈ d,
Tris{2Ј-[(6-deoxy-1-O-p-nitrophenyl-ꢀ-D-mannopyranos-6-
yl)carbamoyl]ethyl}nitromethane 27
CHCHHS), 2.32 (6 H, m , CH CH COOH), 2.24 (2 H, m ,
c
2
2
c
CH CH CH CH CONH), 2.06 (6 H, m , CH CH COOH),
2
2
2
2
c
2
2
Mannoside 26 (33.3 mg, 0.11 mmol) and triacid 19 (7.9 mg,
1.77 (1 H, m , CHHCH CH CH CONH), 1.68 (3 H, m ,
CHHCH CH CH CONH), 1.50 (2 H, m , CH CH CH CH -
2 2 2 c 2 2 2 2
c 2 2 2 c
3
0
.029 mmol) were dissolved in dry DMA (12 cm ) and treated
with EEDQ (32.9 mg, 0.133 mmol, 1.2 eq.) for 5 days. Then,
the solvent was removed by lyophilisation and the resulting
CONH); δ (125.77 MHz; CD OD) 177.45 (CH CH COOH),
C
3
2
2
q
176.01 (CH CONH), 166.47 (NHCONH), 91.92 (C ), 63.55
2
lyophilisate was dissolved in Et O–light petroleum–MeOH–
water (2 : 1 : 1 : 1; 20 cm ) to remove an excess of EEDQ and
(CHCH S), 61.94 (CHCHRS), 58.89 (CHCHRS), 41.34
2
2
3
(CHCH S), 37.67 (CH CH CH CH CONH), 32.08 (CH -
2
2
2
2
2
2
any formed quinoline. The aqueous phase was separated, again
concentrated by lyophilisation, and the residue was purified on
silica gel (ethyl acetate–methanol, 9 : 1) to yield the title cluster
mannoside 27 (29.2 mg, 91%) as a white amorphous solid,
CH COOH),
30.78
(CH CH CH CH CONH),
30.03
2
2
2
2
2
(CH CH COOH), 29.57 (CH CH CH CH CONH), 27.26
2
2
2
2
2
2
ϩ
(CH CH CH CH CONH) m/z (MALDI-TOF) 496.17 (M ϩ
2
2
2
2
Na. C H NaN O S requires m/z 496.17), m/z (MALDI-TOF)
20
31
ϩ
3
8
20
[
α] ϩ78.6 (c 0.40 in MeOH); δH (400 MHz; CD OD) 8.26,
512.13 (M ϩ K. C H KN O S requires m/z 512.15).
D
3
20 31 3 8
3
7
.31 (each 6 H, 2 × d, J 9.7, ArH), 5.69 (3 H, d, J1 1.5, 1-H),
,2
4
.09 (3 H, dd, J2,3 3.6, 2-H), 3.93 (3 H, dd, J3,4 9.2, 3-H), 3.65
Biotinamidotris{2Ј-[(6-deoxy-1-O-methyl-ꢀ-D-mannopyranos-6-
yl)carbamoyl]ethyl}methane 33
(
3 H, dd ≈ t, J4 9.2, 4-H), 3.62 (3 H, dd, J
3.6, J6a,6b
,5
5,6b
b
a
1
2.7, 6-H ), 3.60 (3 H, dd, J5 6.1, 6-H ), 3.59–3.55 (3 H, m,
,6a
The biotinylated triacid 31 (67.29 mg, 0.1421 mmol) and the
amino-functionalised glycoside 32 (87.85 mg, 0.455 mmol) were
5
-H), 2.12–1.98 (12 H, m, CH CH CO); δ (100.67 MHz;
2
2
C
q
CD OD) 174.57 (NHCO), 162.84 (i-aryl-C ), 144.21 (p-aryl-
C ), 127.09 (m-aryl-CH), 118.00 (o-aryl-CH), 100.16
3
3
3
dissolved in dry DMF (50 cm ). Then DIPEA (0.5 cm ) was
q
added, the mixture was cooled to 0 ЊC, and a solution of EDC
q
(
C-1), 94.49 (C -NO ), 74.35 (C-5), 72.24 (C-3), 71.85 (C-2),
2
(
27.24 mg, 0.1421 mmol) and 1-HOBT (38.4 mg, 0.284 mmol)
6
9.66 (C-4), 41.67 (C-6), 32.19 (CH CH CO), 31.39 (CH -
2 2 2
ϩ
3
in dry DMF (10 cm ) was added dropwise over a period of
CH CO); m/z (MALDI-TOF) 1146.34 (M ϩ Na. C H N -
2
46 57
7
2
4
0 min. The reaction mixture was stirred at 0 ЊC for 1 h and for
8 h at rt. Then, the solvent was removed under high vacuum
NaO26 requires m/z 1146.325), m/z (MALDI-TOF) 1162.33
ϩ
(
M ϩ K. C H KN O requires m/z 1162.299).
46 57 7 26
and the residue was purified by gel chromatography (first on
Sephadex LH-20 with MeOH as eluent, followed by Sephadex
G-15 with 15 mM aq. NH HCO as eluent) to yield the title
Biotinamidotris[2-(tert-butoxycarbonyl)ethyl]methane 30
4
3
To a solution of (ϩ)-biotin (307.6 mg, 1.26 mmol) and diiso-
compound (129 mg, 91%) as a slightly yellow amorphous solid
3
propylethylamine (DIPEA) (0.3 cm ) in dry dimethylformamide
after lyophilisation; TLC n-PrOH–water, 7 : 3, ϩ 1% NH (R
3
f
3
16
20
(
DMF) (12 cm ) was added a solution of the amine 29 (561
0.27, H SO ); [α] ϩ96.7 (c 0.45 in water); δH (400 MHz;
2 4 D
3
mg, 1.26 mmol) in dry DMF (10 cm ). Then the mixture
was cooled to 0 ЊC and 1-[3-(dimethylamino)propyl]-3-ethyl-
carbodiimide (EDC) (241.3 mg, 1.26 mmol) and N-hydroxy-
benzotriazole (1-HOBT) (340.2 mg, 2.52 mmol) were added.
The reaction mixture was stirred at 0 ЊC for 1 h followed by
stirring at rt for 12 h. Then the solvent was removed in vacuo
and the residue was purified by flash chromatography (ethyl
acetate–MeOH, 4 : 1) to yield the biotinylated title compound
CDCl ; Me Si) 8.33 (6 H, br s, NHCO), 4.61 (3 H, d, 1-H), 4.47
3
4
3
3
3
(1 H, dd, J 4.6, J
Jcis 4.6, CHCHRS), 3.79 (3 H, dd, J1,2 1.5, J2,3 3.6, 2-H), 3.60
(3 H, dd, J3,4 9.7, 3-H), 3.51–3.46 (6 H, m, 5-H, 6-H ), 3.42 (3 H,
dd ≈ t, J4,5 9.7, 4-H), 3.30 (3 H, dd, J5,6a 5.1, J6a,6b 12.7, 6-H ),
7.6, CHCH S), 4.29 (1 H, dd, J
7.6,
cis
trans
2
trans
3
b
a
3.24 (9 H, s, OCH ), 3.23–3.05 (1 H, m, CHCHRS), 2.87 (1 H,
3
3
2
dd, J 4.6, J 13.2, CHCHHS), 2.65 (1 H, dd ≈ d, CHCHHS),
2.22–2.05 (8 H, m, 3 × CH CH CONHCH , CH CH CH -
2
2
2
2
2
2
(
780 mg, 97%) as a white amorphous solid; δH (400 MHz;
CH CONH), 1.90–1.73 (8 H, m, CH CH CH CH CONH,
2 2 2 2 2
CDCl ; Me Si) 6.69 (1 H, br s, NHCONH), 6.52 (1 H, br s,
3 × CH CH CONHCH ), 1.62–1.41 (2 H, m, CH CH CH -
3
4
2
2
2
2
2
2
q
3
NHCONH), 6.21 (1 H, br s, CONHC ), 4.45 (1 H, dd ≈ t, J
CH CONH), 1.31–1.24 (2 H, m, CH CH CH CH CONH);
cis
2
2
2
2
2
3
3
3
4
.7, J
7.6, CHCH S), 4.30 (1 H, dd ≈ t, J 4.7, J
7.3,
δC (100.67 MHz; CDCl ; Me Si) 176.74 (CONHCH man),
3 4 2
trans
2
cis
trans
3
CHCHRS), 3.12 (1 H, m , CHCHRS), 2.88 (1 H, dd, J 4.7,
176.47 (NHCO-biotinamide), 165.73 (NHCONH-biotin),
c
2
q
J 12.6, CHCHHS), 2.73 (1 H, dd ≈ d, CHCHHS), 2.20 (6 H,
101.17 (C-1), 92.92 (C , CONHC), 71.02 (C-5), 70.73 (C-3),
t
m , CH COOBu ), 2.15 (2 H, m , CH CH CH CH CONH),
70.24 (C-2), 68.49 (C-4), 62.42 (CONHCHCHRS), 60.61
(CONHCHCH S), 55.69 (OCH ), 54.99 (CHCHRS), 40.44
c
2
c
2
2
2
2
t
1
.97 (6 H, m , CH CH COOBu ), 1.71 (1 H, m , CHHCH -
c 2 2 c 2
2
3
CH CH CONH), 1.62 (3 H, m , CHHCH CH CH CONH),
(C-6), 40.09 (SCH ), 36.61 (CH CH CH CH CONH), 31.84
(CH CH CONHCH ), 30.38 (CH CH CH CH CONH), 28.40
2 2 2 2 2 2 2
2
2
c
2
2
2
2 2 2 2 2
t
1
1
9
6
.42 (29 H, m , CH CONH, 3 × Bu ); δ (100.67 MHz; CDCl )
c
2
C
3
72.96 (NHCO-biotinamide),q 162.73 (NHCONH-biotin),
(CH CH CONHCH ),
28.04
(CH CH CH CH CONH),
2
2
2
2 2 2 2
q
1.92 (C , CONHC), 82.07 (C Me ), 61.86 (CONHCHCH S),
25.61 (CH CH CH CH CONH); m/z (MALDI-TOF) 999.41
3
2
2
2
2
2
ϩ
0.38 (CONHCHCHRS), 57.32 (CONHCHCHRS), 40.52
(M ϩ H. C H N O S requires m/z 999.44), m/z (MALDI-
41 71 6 20
ϩ
(
(
(
CONHCHCH S), 36.76 (CH CH CH CH CONH), 32.08
TOF) 1021.40 (M ϩ Na. C H NaN O S requires m/z
41 70 6 20
ϩ
2
2
2
2
2
CH CH COOtBu), 30.70 (CH CH CH CH CONH), 30.21
1021.43), m/z (MALDI-TOF) 1037.52 (M ϩ K. C H KN -
41 70 6
2
2
2
2
2
2
CH CH COOtBu), 29.82 (CH CH CH CH CONH), 28.68
O S requires m/z 1037.40).
2
2
2
2
2
2
20
8
30
J. Chem. Soc., Perkin Trans. 1, 2001, 823–831

View Online
J. Finne and G. Magnusson, J. Am. Chem. Soc., 1997, 119, 6974; (c)
L. E. Strong and L. L. Kiessling, J. Am. Chem. Soc., 1999, 121, 6193;
(d) P. I. Kitov, J. M. Sadowska, G. Mulvey, G. D. Armstrong,
H. Ling, N. S. Pannu, R. J. Read and D. R. Bundle, Nature, 2000,
Acknowledgements
In memoriam of Professor Dr Göran Magnusson, who has
been one of those who has kindly supported the academic
career of T. K. L.
4
03, 669; (e) D. Zanini and R. Roy, J. Am. Chem. Soc., 1997, 119,
088.
2
This work was performed in the collaborative network SFB
4
(a) Toward Anti-adhesion Therapy for Microbial Diseases, Adv. Exp.
Med. Biol., 1996, 408; (b) K. J. Yarema and C. R. Bertozzi,
Curr. Opin. Chem. Biol., 1998, 2, 49.
4
70 (project A6). It has also been supported by the Fonds der
Chemischen Industrie (FCI).
5
6
N. Sharon, FEBS Lett., 1987, 217, 145.
D. Choudhury, A. Thompson, V. Stojanoff, S. Langermann,
J. Pinkner, S. J. Hultgreen and S. D. Knight, Science, 1999, 285,
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