Trivalent cluster mannosides with aromatic partial structure as ligands for the type 1 fimbrial lectin of Escherichia coli

Article abstract of DOI:10.1071/ch02025

Mannose-specific adhesion of E. coli bacteria to their host cells is mediated by so-called type 1 fimbriae containing lectin domains present on the type 1 fimbrial FimH protein. The crystal structure of a FimH-FimC(chaperone) protein complex revealed a nu

Full text of DOI:10.1071/ch02025

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Full Papers
Aust. J. Chem. 2002, 55, 87–93
Trivalent Cluster Mannosides with Aromatic Partial Structure
as Ligands for the Type 1 Fimbrial Lectin of Escherichia coli
Niels Röckendorf,A Oliver SperlingA and Thisbe K. Lindhorst A,B
A Institute of Organic Chemistry, Christiana-Albertina-University of Kiel, Otto-Hahn-Platz 4,
D-24098 Kiel, Germany.
B Author to whom correspondence should be addressed (e-mail: tklind@oc.uni-kiel.de).
Mannose-specific adhesion of E. coli bacteria to their host cells is mediated by so-called type 1 fimbriae containing
lectin domains present on the type 1 fimbrial FimH protein. The crystal structure of a FimH–FimC(chaperone)
protein complex revealed a number of amino acids in the carbohydrate binding site with aromatic side chains. This
finding is in keeping with earlier results showing high inhibitory potencies of aryl mannosides when tested as
inhibitors of type 1 fimbriae-mediated bacterial adhesion. In addition, clustering of mannosyl moieties also led to
favourable effects, as in the case of trivalent cluster mannosides such as (1). In order to combine both, i.e. the
clustering approach and the advantage of an aromatic moiety, the herein presented study has emphasized the
synthesis of three cluster mannosides (2), (3), and (4), as ligands for the type 1 fimbrial lectin, which contain a
phenyl partial structure in different proximity to the core of the molecule. The inhibitory potencies of the new
cluster mannosides were determined in enzyme-linked immunosorbent assays (ELISAs).
Manuscript received: 30 January 2002.
Accepted without change.
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Introduction
protein FimC. This crystal structure revealed a binding site
at the tip of FimH of the size of a monosaccharide unit.[6]
However, it can be assumed that there are additional binding
sites on the FimH protein which are also of importance for
ligand binding.[7]
Bacteria often use their own specialized 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 can accomplish
adhesion to the host cell surface, an event of crucial
importance for cell infection.[1] Synthetic carbohydrate
derivatives have been designed to interfere in the microbial
adhesion process by competing for the carbohydrate-
recognition domains of the involved lectins.[2] This has been
demonstrated to work in vitro,[3] and might be evaluated into
an in vivo anti-adhesion therapy to prevent bacterial
infection.
Mannose-specific adhesion is among the most widely
distributed types of carbohydrate-dependent bacterial
adhesion. It is mediated by so-called type l fimbriae, long
proteinogenous appendages on the bacterial surface, which
contain several copies of the mannose-specific adhesin,
which is called FimH protein.[4] FimH is encoded by the
fimH gene within the gene cluster which is responsible for
type 1 fimbriae assembly. The structure of the carbohydrate
recognition domain of FimH has long been unknown; a
model, based on testing results with several α-D-mannosides
and mannose-containing oligosaccharides, has attributed the
size of a trisaccharide to the binding pocket with a
hydrophobic domain in or close to it.[5] More recently, FimH
has been crystallized in conjugation with the chaperone
Two tyrosine residues are close to the carbohydrate-
binding domain identified by X-ray analysis,[6] reasoning the
finding that mannosides with aromatic aglycon moieties
resemble especially potent ligands for type 1 fimbriae.[5] In
our group, a series of trivalent cluster mannosides, such as
compound (1) (Fig.1), have been synthesized,[8] several of
which were found to be potent ligands of type 1 fimbriae.
Tested by ELISA, glycocluster (1) has shown a 200-fold
higher inhibitory potency than methyl α-D-mannoside
(MeMan), which was used as a monovalent standard. To test
if incorporation of a phenyl partial structure into
glycoclusters such as (1) can further improve their potency in
inhibiting type 1 fimbriae-mediated bacterial adhesion,
cluster mannosides (2), (3), and (4) (each having a phenyl
ring in different proximity to the core) became the target
structures of this study.
Results and Discussion
Synthesis
Cluster glycosides (2), (3), and (4) (Fig.1) are formally based
on C3-elongated tris(hydroxymethyl) methane derivatives in
which the C3-linker may be regarded as a spacer for one
© CSIRO 2002
10.1071/CH02025
0004-9425/02/010087

88
N. Röckendorf, O. Sperling and T. K. Lindhorst
into the bicyclic orthoacetate (6).[9] Orthoester (6) is known
to polymerize easily at higher temperatures and is therefore
difficult to purify. The remaining free hydroxy functional
group in (6) was etherified with 1-bromo-3-phenylpropane
in a Williamson synthesis, followed by acidic cleavage of the
orthoester protective group in ‘one pot’ to give (7). The low
overall yield in this step is due to the lability of the orthoester
and the limited reactivity of the alkyl bromide employed.
To obtain the triols for the synthesis of (3) and (4) in
which the phenyl partial structure is located closer to the core
region than in (2), aliphatic aldehydes were selected to serve
as starting materials in a Tollens condensation.[10] A Tollens
condensation resembles a hydroxymethylation followed by a
Cannizzaro-type reaction, analogous to the synthesis of
pentaerytritol from acetaldehyde and formaldehyde.
Following this approach, a large variety of functionalized
tris(hydroxymethyl) methane derivatives such as (10) and
(11) is accessible in one step, due to the availability of a wide
range of different aldehydes.
All three triols (7), (10), and (11), thus obtained were
equipped with C3 spacers by an allylation-hydroboration-
oxidation sequence leading to their elongated analogues
(18), (19), and (20) (Scheme 2). First, conversion with allyl
bromide under phase-transfer conditions gave the allyl ethers
(12), (13), and (14), which were subjected to hydroboration
using 9-BBN (9-borabicyclo[3.3.1]nonane). Oxidative
workup of the intermediate hydroboranes, with H2O2 in
aqueous sodium hydroxide solution, gave the primary triols
as the anti-Markownikov products. To facilitate purification
and characterization of the polar products, they were purified
at the stage of their acetylated derivatives (15), (16), and
(17), which were prepared in pyridine using acetic
anhydride. Eventually, deprotection according to
Zemplén[11] led to the desired core triols (18), (19), and (20).
For mannosylation of these three core triols (O-(2,3,4,6-
tetra-O- benzoyl-α -D-mannopyranosyl ) - trichloroacetimi-
Fig. 1. The known cluster mannoside (1) and the target molecules
of this study. (1) is a 200-fold more potent ligand for the type 1
fimbrial lectin than methyl α-D-mannoside (MeMan). Incorporation
of a phenyl moiety in varied proximity to the mannosyl residues
(glycoclusters (2), (3), and (4)) might further improve its binding
potency.
mannosyl unit. Mannosylation of the respective triols would
eventually give the target glycoclusters. In order to vary the
maximal distance between the phenyl moiety and the
mannoside residues of the respective glycoclusters, two
synthetic strategies were employed (Scheme 1).
The core triol, which was needed for the synthesis of (2),
was derived from pentaerythritol (5), which can be converted

Trivalent Cluster Mannosides with Aromatic Partial Structure
89
because the contaminating orthoesters had to be hydrolysed
in this step too. Amberlite IR 120 was suitable to destroy any
glycosyl orthoester impurities by acidic cleavage and thus
the deprotected cluster mannosides (2)–(4) were obtained as
pure products and in high yield after gel-permeation
chromatography (GPC). They were eventually tested for
their inhibitory properties with type
1
fimbriated
Escherichia coli in an ELISA (enzyme-linked immuno-
sorbent assay).
Biology
The trivalent cluster mannosides (2)–(4) were tested for their
capacity to inhibit mannose-specific adhesion of E. coli using
a recombinant strain, E. coli HB 101 (pPK14), which
expresses only type 1 fimbriae on its surface.[14] A sandwich-
type ELISA was employed using microtiter plates that were
coated with yeast mannan.[15] E. coli readily bind to the
mannan surface on the microtiter plates by their mannose-
specific lectins. Binding of bacteria can be inhibited by α-
mannosyl-containing molecules at different concentrations,
thus reflecting their inhibitory potency. As adhesion of
bacteria can be quantified by a peroxydase-based colour
reaction, an ELISA allows us to measure IC50 values for each
molecule that can inhibit E. coli adhesion in a concentration-
dependent manner. IC50 values inthe ELISAemployedreflect
the inhibitor concentration which causes 50% inhibition of
bacterial binding to yeast mannan. Average IC50 values from
three independently performed ELISAs are collected in
Table 1. Based on methyl α-D-mannoside as the reference,
relative IC50 values (RIC50) were deduced. Typically, relative
inhibition potencies are highly reproduceable.
date[12] was used as the glycosyl donor (Scheme 3). Benzoyl
protecting groups were chosen to prevent 1,2-O-glycosyl-
orthoester formation during the glycosylation step.[8] In
order to minimize the amount of mono- and di-mannosylated
side products, reactions were carried out in highly
concentrated solution and a seven-fold excess of the glycosyl
donor was used per hydroxy group. The Lewis acid TMSOTf
(trimethyl-silylmethyl triflate) was employed as a promotor
of the glycosylation reaction, and is also known to catalyse
isomerization of glycosyl orthoesters, which are formed as
intermediates of the reaction. In spite of this, orthoester
formation could not be prevented totally. Traces of glycosyl
orthoesters were detected by proton nuclear magnetic
resonance (NMR) and could not be removed during the
purification steps on silica gel and Sephadex LH-20. A
methanol/dichloromethane mixture was used as the eluent to
increase the solubility of the benzoylated glycoclusters and
to reduce adsorption of the products on the gel. Thus, the
benzoylated cluster mannosides (21), (22), and (23) were
obtained, which were deprotected under modified Zemplén
conditions.[13] Due to the low solubility of the benzoylated
glycoclusters in methanol, elongated reaction times of up to
3 days were necessary to complete the reaction. As usual,
acidic ion exchange resin was used to neutralize the reaction
mixture after Zemplén deprotection. In this case, however,
the right choice of ion exchange resin was of importance,
Table 1. Antiadhesive properties of the synthesized cluster
mannosides obtained in ELISA inhibition assays, in comparison to
methyl α-D-mannopyranoside (MeMan) and p-nitrophenyl α-D-
mannopyranoside (pNPMan). Inhibition values obtained in three
independently performed assays were averaged
IC50 [µMl]
MeMan pNPMan
(2)
(3)
(4)
IC50A (assay 1)
IC50A (assay 2)
IC50A (assay 3)
Average IC50
3010
5070
2660
3580
1302
1
23
40
23
29
10
198
130
159
162
34
155
87
86
109
40
33
120
85
125
110
22
Standard deviation
B
RIC50
125
22
33
A
B
IC50: 50% of binding inhibited.
RIC50: Relative IC50 based on
MeMan (≡ 1).
As expected, methyl α-D-mannoside (MeMan) inhibited
the adhesion of E. coli HB 101 (pPK14) at millimolar
concentrations, whereas p-nitrophenyl α-D-mannoside
(pNPMan), due to its aromatic aglycone, has an inibitory
potency which is approximately two orders of magnitude
better.[5,16] Looking at the values obtained for the new cluster
mannosides (2), (3), and (4) it is obvious that an effect such
as that obtained with pNPMan is not achieved. The
inhibitory potency of all three cluster mannosides with

90
N. Röckendorf, O. Sperling and T. K. Lindhorst
distilled under reduced pressure (125°C, 0.4 Torr). The colourless
crystals that formed in the distillate were collected and recrystallized
from toluene/ethyl acetate (10 : 1).
aromatic partial structure is approximately in the same
range, reflecting a 20- to 30-fold higher RIC50 than MeMan.
Moreover, cluster mannosides (2), (3), and (4) do not even
reach the inhibitory potency of (1), which was shown earlier
to possess a 200-fold higher RIC50 than MeMan.[8]
General Procedure used for the O-Allylation of Triols
The triol was suspended in an aqueous 30% sodium hydroxide solution
(0.2 mol/L), TBABr (tetrabutylammonium bromide, 0.5 equiv.) was
added, the mixture was heated to 40°C and then allyl bromide (4 equiv.)
was added at this temperature. The reaction mixture was stirred
overnight at room temperature, then water was added, the phases were
separated and the aqueous phase was extracted three times with
CH2Cl2. The combined organic phases were dried over MgSO4, then
they were filtered and the solvent was removed under reduced pressure.
The residual oil was purified on silica gel (n-pentane/ethyl acetate,
20 : 1).
This is a disappointing and somewhat unexpected,
nevertheless interesting result, which might be explained on
the basis of more structural data about FimH, which will be
eventually obtained. It is expected, on the basis of testing
results obtained earlier, that in addition to the small binding
site at the tip of FimH,[6] other regions of the FimH protein
contribute to ligand binding.[7] It is apparent that the design
of glycoclusters (2), (3), and (4) is not suitable for
positioning the different partial structures of the cluster in a
favourable way. It might rather be the case that the
interaction of the phenyl moiety with the aromatic amino
acid side chains prevents the α-D-mannosyl units from
important interactions with sugar-binding sites on FimH and,
therefore, the effect of (1) is not reached. It is our goal to
further investigate the structure–activity relationships in this
system, based on the results obtained herein.
General Procedure used for Hydroboration
The allylated derivative was dissolved in dry THF (0.05 mol/L), 9-BBN
(0.5 M solution in THF, 1.1-fold excess per allyl group) was added
under an argon atmosphere and the reaction mixture was stirred under
reflux for 3 h. An excess of aqueous 3 M sodium hydroxide solution
was added and the mixture was cooled to 0°C. An excess of hydrogen
peroxide was then added dropwise and the mixture was stirred
overnight at room temperature. The aqueous layer was saturated with
potassium carbonate, the organic layer was separated and dried over
MgSO4. After filtration, the solvent was removed under reduced
pressure. For acetylation, the residual oil was dissolved in pyridine, a
twofold excess of acetic anhydride per hydroxy group was added, and
the reaction mixture was stirred for 4 h at room temperature. The
solution was co-evaporated with toluene to yield an oil which was
purified on silica gel (n-pentane/ethyl acetate, 3 : 2).
Experimental
General
For silica gel chromatography, Merck silica gel 60 (0.040–0.063 mm,
230–400 mesh) was used on an MPLC from Büchi. Thin-layer
chromatography (TLC) was performed on Kieselgel 60 F254 plates from
Merck. Detection was carried out under ultraviolet light or by spraying
with 20% ethanolic sulfuric acid followed by heating. Size exclusion
chromatography (or GPC) was performed on Sephadex LH-20 from
Pharmacia. NMR spectra were measured on a Bruker ARX 300
(300 MHz for 1H and 75.47 MHz for 13C NMR) or on a Bruker DRX
500 (500 MHz for 1H and 125.47 MHz for 13C NMR). Chemical shifts
are given in ppm relative to internal tetramethylsilane (0.00 ppm
for 1H and 13C NMR). Wherever necessary, two-dimensional 1H–1H or
1H–13C correlation spectroscopy (COSY) experiments were carried out
for complete signal assignments. Matrix-assisted laser desorption
ionization time-of-flight (MALDI-TOF) mass spectra were measured
on a Bruker Biflex III 19 kV instrument. The matrix used was
norharmane (9H-pyrido-[3,4-b]-indol, from Sigma) in tetrahydrofuran
(THF). Elemental analyses of the target glycoclusters were measured at
the Institute of Inorganic Chemistry of CAU, Germany; however, they
did not lead to accurate values, possibly due to water content. High-
performance liquid chromatography (HPLC) analysis was performed
using a SP 250/10 Nucleosil 100-7 C18 column from Machery–Nagel
and a CH3CN/H2O gradient to prove purity of the target compounds.
A recombinant type 1 fimbriated E. coli strain, E coli HB101
(pPKl4)[14] was used in an ELISA, which was carried out as described
earlier.[15] F-shaped 96-well microtiter plates from Sarstedt were used.
Mannan from Saccharomyces cerevisae was purchased from Sigma and
was used in 50 mM aq. Na2CO3 (1 mg/mL; pH 9.6). The polyclonal
anti-fimA antibody was a kind gift from Professor Dr S. Ehlers
(FZ Borstel) and peroxidase-conjugated goat anti-rabbit antibody
(IgG, H+L) was purchased from Dianova. Skimmed milk was from
Ulzena, Tween 20 from Roth, ABTS [2,2´-azidobis(3-ethylbenzo-
thiazoline-6-sulfonic acid)] from Sigma and thimerosal {2-[(ethyl-
mercurio) thio]benzoic acid, sodium salt} from Merck.
General Procedure used for Deacetylation
The acetyl-protected derivative was dissolved in dry MeOH, sodium
was added (0.1 equiv. per ester group) and the reaction mixture was
stirred for 4 h at room temperature. When the reaction was complete,
ion exchange resin (Amberlite IR 120 H+) was added, and then filtered
off and the filtrate was concentrated under reduced pressure. The
resulting oil was used without further purification.
General Procedure used for Mannosylation of Triols
The acceptor triol was dissolved in dry CH2Cl2 (1 mmol/mL) under an
argon atmosphere and the mannosyl donor (O-(2,3,4,6-tetra-O-
benzoyl-α-D-mannopyranosyl)trichloroactemidate,[12]
7 equiv. per
hydroxy group) was added. The glycosylation reaction was then started
by adding a catalytic amount of TMSOTf (freshly prepared 5% solution
in dry CH2Cl2) at 0°C. The reaction mixture was stirred overnight at
room temperature, and then an excess of solid sodium hydrogen
carbonate was added, it was filtered off and the filtrate concentrated
under reduced pressure. The residue was purified on silica gel (n-
pentane/ethyl acetate, 3 : 1 → 1 : 1) followed by GPC on Sephadex LH-
20 (CH2Cl2/MeOH, 1 : 1).
General Procedure used for Debenzoylation of Cluster Glycosides
The benzoyl-protected cluster mannoside was dissolved in dry MeOH
(1.5 mmol/L), sodium methanolate (1 M in MeOH, 0.2 equiv. per
benzoylester function) was added and the reaction mixture was stirred
at room temperature for 56 h. Ion-exchange resin (Amberlite IR
120 H+) was then added for neutralization and cleavage of glycosyl
orthoester side products. After stirring for 10 min, the resin was filtered
off and the filtrate was concentrated under reduced pressure. The
residue was purified by GPC on Sephadex LH-20 (MeOH) to obtain the
product as a white foam after lyophylization. Further purification was
achieved by reverse phase HPLC.
General Procedure used in the Tollens Condensation
The aldehyde was dissolved in THF (1.5 mol/L), a fivefold excess of
paraformaldehyde and calcium hydroxide (1 equiv.) were added and the
suspension was stirred at 60–65°C for 5 days. After cooling, the
reaction mixture was filtered through a bed of Celite and the filtrate was
concentrated under reduced pressure to yield a dark syrup, which was
4-(Hydroxymethyl)-1-methyl-2,6,7-trioxabicyclo[2.2.2]octane (6)
Triethylorthoacetate (61 mL, 0.38 mol) and p-TsOH (166 mg) were
added to a suspension of pentaerythritol (5) (45.3 g, 0.38 mol) in 40 mL

Trivalent Cluster Mannosides with Aromatic Partial Structure
91
of toluene. The resulting mixture was heated slowly to 80°C and the
ethanol formed was distilled off over a period of 4 h. The temperature
was raised to 125°C and toluene was evaporated until a clear solution
was obtained. The residue was cooled to 40°C and concentrated under
reduced pressure. The resulting solid was sublimed (130°C, 0.5 Torr) to
yield a white solid (49.58 g, 0.31 mol, 39%, lit.[9] 94%), which was used
without further purification. Melting point (m.p.) 106°C; lit.[9] 110–
112°C. 1H NMR δ (CDCl3, 300 MHz) 4.02, s, OCH2C; 3.49, s,
CCH2OH; 1.61, br s, OH; 1.48, s, CCH3.
compound as a yellow oil (6.27 g, 19.8 mmol, 66%). 1H NMR (CDCl3,
300 MHz) δ 7.23, m, aryl H; 5.92, dddd, Jcis 10.4, Jtrans 17.4 Hz,
OCH2CHCH2; 5.21, ddd, Jgem 1.7 Hz, OCH2CHCH2; 3.97, ddd, J 5.3
Hz, OCH2CHCH2; 3.24, s, CCH2O; 2.73, s, CCH2aryl. 13C NMR δ
(CDCl3, 75.47 MHz) 137.79, aryl C; 135.19, OCH2CHCH2; 130.71,
127.79, 125.87, aryl C; 116.15, OCH2CHCH2; 72.05, OCH2CHCH2;
69.63, CCH2O; 44.5, CCH2O; 35.3, CCH2aryl.
Tris(allyloxymethyl)phenylmethane (14)
The triol (11) (2.22 g, 0.01 mol) was treated with allyl bromide (4 mL,
40 mmol) according to the general procedure to yield the allylated
product as a yellow oil (2.48 g, 8.2 mmol, 82%). 1H NMR (CDCl3, 300
MHz) δ 7.31, mc, aryl H; 5.86, dddd, Jcis 10.4, Jtrans 17.4 Hz,
OCH2CHCHH; 5.23, ddd ≈ dd, Jgem 1.7 Hz, OCH2CHCHH; 5.13, ddd
≈ dd, OCH2CHCHH; 3.96, dddd ≈ d, J 5.3 Hz, OCH2CHCH2; 3.77, s,
CCH2Oallyl. 13C NMR (CDCl3, 75.47 MHz) δ 141.9, arylC; 135.02,
OCH2CHCH2; 127.92, 127.18, 126.35, aryl C; 116.4, OCH2CHCH2;
72.31, 72.17, CCH2O, OCH2CHCH2; 48.08, CCH2O.
(5-Phenyl-2-oxapentyl)tris(hydroxymethyl)methane (7)
Powdered sodium hydroxide (15 g, 0.27 mol) was suspended in dimethyl
sulfoxide (150 mL) and stirred at room temperature for 5 min. The
orthoester (6) (15 g, 0.092 mol) was then added in one portion, quickly
followed by the addition of 1-bromo-3-phenylpropane (17.3 mL, 0.098
mol). The reaction mixture was stirred for another 30 min and was
subsequently diluted with water (400 mL). The solution was extracted
twice with CH2Cl2 (100 mL), the organic phase was washed with brine
(50 mL) and water (50 mL), dried over MgSO4, filtered and the filtrate
wasconcentratedunderreducedpressure. Theresultingoil wastriturated
with MeOH (50 mL) and 0.01 M hydrochloric acid (200 mL) was added.
Sodium hydrogen carbonate (7 g) was added and the resulting mixture
was stirred for 1 h at room temperature followed by concentration under
reduced pressure. The residue was purified on silica gel (n-pentane/ethyl
acetate, 1 : 3)toyield anoil (4.83g, 19.03mmol, 21%). 1HNMR (CDCl3,
300 MHz) δ 7.23, m, aryl H; 3.71, s, CCH2OH; 3.47, s, CCH2O; 3.44,
t, J 6.4 Hz, OCH2CH2CH2CH2; 3.28, br s, OH; 2.65, mc, aryl
CH2CH2CH2,1.87;m,arylCH2CH2CH2.13CNMR(CDCl3,75.47MHz)
(5-Phenyl-2-oxapentyl)tris(3-acetoxypropyloxymethyl)methane (15)
The allylated precursor (12) (981 mg, 2.62 mmol) was subjected to a
hydroboration-oxidation-acetylation sequence according to the general
procedure to yield a colourless oil (438 mg, 0.85 mol 33%). 1H NMR
(CDCl3, 300 MHz) δ 7.19, mc, aryl H; 4.14, t, J 6.6 Hz,
OCH2CH2CH2OAc; 3.46, t, J 6.1 Hz, OCH2CH2CH2OAc; 3.37, mc,
CCH2O, OCH2CH2CH2aryl; 2.66, mc, OCH2CH2CH2aryl; 1.98, s,
C(O)CH3; 1.87, mc, OCH2CH2CH2OAc, OCH2CH2CH2aryl. 13C NMR
(CDCl3, 75.47 MHz) δ 171.14, C=O; 142.15, 142.15, 128.47, 128.28,
125.71, aryl C; 70.35, OCH2CH2CH2aryl; 69.56, CCH2O; 67.6,
OCH2CH2CH2O; 61.79, OCH2CH2CH2O; 45.34, CCH2O; 32.32,
OCH2CH2CH2 aryl; 31.27, OCH2CH2CH2 aryl; 28.86, OCH2CH2-
CH2O; 20.99, C(O)CH3.
δ
141.49, 128.45, 125.91, aryl C; 73.41, OCH2C; 71.05,
OCH2CH2CH2aryl; 63.92, CCH2OH; 44.92 C(CH2)4; 32.22, aryl
CH2CH2CH2; 30.97, CH2CH2CH2 aryl.
Tris(hydroxymethyl)benzylmethane (10)
Tris(3-acetoxypropyloxymethyl)benzylmethane (16)
3-Phenylpropionaldehyde (8) (98 g, 0.75 mol) was dissolved in THF
(500 mL), and paraformaldehyde (141.1 g, 4.7 mol) and calcium
hydroxide (44 g, 0.6 mol) were added according to the general
procedure to yield the title compound as colourless crystals (43.41 g,
0.22 mol, 30%). 1H NMR (CDCl3, 300 MHz) δ 7.25, mc, arylH; 3.13,
s, CCH2OH; 3.04, br s, OH; 2.61, s, CCH2aryl. 13C NMR (CDCl3,
75.47 MHz) δ 136.86, 130.17, 128.09, 126.28, aryl C; 65.30, CCH2OH;
44.04, CCH2OH; 36.13, CCH2aryl.
The allylated precursor (13) (3.03 g, 9.49 mmol) was dissolved in dry
THF (200 mL) and 9-BBN (0.5 M solution in THF, 70 mL, 35 mmol)
was added. According to the general procedure for the hydroboration-
oxidation-acetylation sequence, the title compound was obtained as a
colourless oil (1.65 g, 3.32 mmol, 35%). 1H NMR (CDCl3, 300 MHz)
δ 7.21, m, aryl H; 4.18, t, J 6.6 Hz, OCH2CH2CH2OAc; 3.46, t, J 6.1
Hz, OCH2CH2CH2OAc; 3.17, s, CCH2O; 2.66, s, CCH2aryl; 2.07, s,
C(O)CH3; 1.91, mc, OCH2CH2CH2OAc. 13C NMR (MHz, CDCl3,
75.47 MHz) δ 171.15 (C=O); 137.71, 130.6, 127.84, 125.93, aryl C;
70.07, CCH2O; 67.33, OCH2CH2CH2OAc; 61.85, OCH2CH2CH2OAc;
44.4, CCH2O; 35.33, CCH2aryl; 28.94, OCH2CH2CH2OAc; 20.98,
C(O)CH3.
Tris(hydroxypropyl)phenylmethane (11)
Phenylacetaldehyde (9) (84 g, 0.69 mol) was dissolved in THF (500 mL)
and paraformaldehyde (131.3 g, 3.9 mol) and calcium hydroxide (40.6 g,
0.55 mol) were added according to the general procedure to yield the
productascolourlesscrystals(34.93g,0.19mol,48%).1HNMR(CDCl3,
300 MHz) δ 7.32, mc, aryl H; 4.06, s, CCH2OH; 2.47, br s, OH. 13C NMR
(CDCl3, 75.47 MHz) δ 139.59, 128.56, 128.41, 126.79, 126.7, 126.62,
aryl C; 65.84, CH2OH; 48.62, CCH2OH.
Tris(3-acetoxypropyloxymethyl)phenylmethane (17)
The allylated precursor (14) (2.1 g, 6.39 mmol) was dissolved in dry
THF (200 mL) and 9-BBN (0.5 M solution in THF, 70 mL, 35 mmol)
was added. According to the general procedure for the hydroboration-
oxidation-acetylation sequence, the title compound was obtained as a
colourless oil (2.49 g, 5.13 mmol, 80%). 1H NMR (CDCl3, 300 MHz)
δ 7.3, mc, aryl H; 4.08, t, J 6.6 Hz, OCH2CH2CH2Oac; 3.69, s, CCH2O;
3.46, t, J 6.1 Hz, OCH2CH2CH2Oac; 1.84, quin., OCH2CH2CH2Oac.
13C NMR (CDCl3, 75.47 MHz) δ 171.11 (C=O); 141.88, 127.94,
127.16, 126.39, aryl C; 72.7, CCH2O; 67.67, OCH2CH2CH2O; 61.78,
OCH2CH2CH2O; 48, CCH2O; 28.78, OCH2CH2CH2O; 20.99,
C(O)CH3.
(5-Phenyl-2-oxapentyl)tris(allyloxymethyl)methane (12)
The triol (7) (0.8 g, 3.15 mmol) was treated with allyl bromide (1 mL,
11.76 mmol) according to the general procedure to yield a yellow oil
(0.98 g, 2.61 mmol, 69%). 1H NMR (CDCl3, 300 MHz) δ 7.23, mc, aryl
H; 5.87, dddd, Jcis 10.4 Hz, Jtrans 17.4 Hz, OCH2CHCH2; 5.25, ddd ≈
dd, Jgem 1.7 Hz, OCH2CHCHH; 5.16, ddd ≈ dd, OCH2CHCHH; 3.96,
dddd ≈ d, J 5.3 Hz, OCH2CHCH2; 3.46, s, CCH2Oallyl; 3.41, s, CCH2O;
3.40, t, aryl CH2CH2CH2O; 2.67, m, J 6.4 Hz, aryl CH2CH2CH2O; 1.85,
m, aryl CH2CH2CH2O. 13C NMR δ (CDCl3, 75.47 MHz) 142.20, aryl
C; 135.25, OCH2CHCH2; 128.52, 128.27, 125.71, aryl C; 116.11,
OCH2CHCH2; 72.26, OCH2CHCH2; 70.24, aryl CH2CH2CH2O; 69.53,
CCH2O; 45.39, CCH2O; 32.30, aryl CH2CH2CH2O; 31.28, aryl
CH2CH2CH2O.
(5-Phenyl-2-oxapentyl)tris(3-hydroxypropyloxymethyl)methane (18)
The acetylated precursor (15) (406 mg, 0.79 mmol) was deacetylated
according to the general procedure to yield a slightly yellow oil (304
mg, 0.78 mmol, 99%). 1H NMR (CDCl3, 300 MHz) δ 7.23, mc, aryl H;
3.81, br s, OH; 3.76, dd, J 5.4 Hz, OCH2CH2CH2OH; 3.58, dd, J 5.3 Hz,
OCH2CH2CH2OH; 3.43, s, CCH2O; 3.39, t, CCH2OCH2CH2CH2aryl;
2.66, m, J 6.4 Hz, CCH2OCH2CH2CH2aryl; 1.84, m, CCH2OCH2-
CH2CH2aryl, OCH2CH2CH2OH. 13C NMR (CDCl3, 75.47 MHz) δ
Tris(allyloxymethyl)benzylmethane (13)
The triol (10) (5.02 g, 0.03 mol) was treated with allyl bromide (12 mL,
120 mmol) according to the general procedure to yield the title

92
N. Röckendorf, O. Sperling and T. K. Lindhorst
141.99, 128.45, 128.3, 125.76, arylC; 71.09, OCH2CH2CH2aryl; 70.57,
CCH2O; 70.09, OCH2CH2CH2O; 61.51, OCH2CH2CH2O; 45.75,
CCH2O; 32.27, OCH2CH2CH2aryl; 31.69, OCH2CH2CH2O; 31.07,
OCH2CH2CH2aryl.
Phenyltris[3-(2,3,4,6-tetra-O-benzoyl-α-D-
mannopyranosyloxy)propyloxymethyl]methane (23)
The triol (20) (107 mg, 0.31 mmol) and the mannosyl donor (5.1 g, 7.31
mmol) were dissolved in dry CH2Cl2 (6 mL) and treated with TMSOTf
solution (0.1 mL) according to the general mannosylation procedure to
yield the benzoylated cluster mannoside as a slightly yellow foam (617
mg, 0.29 mmol, 93%). 1H NMR (CDCl3, 500 MHz) δ 8.09, 8.05, 7.94,
7.83, mc, o-aryl H; 7.61–7.24, m, m- and p-aryl H; 6.12, dd, J3,4 10.1
Hz, H4; 5.91, dd, J2,3 3.4 Hz, H3; 5.68, dd, J1,2 1.6 Hz, H2; 5.02, d, H1;
4.67, dd, J5,6´ 2.6 Hz, H6´; 4.45, dd, J6,6´ 12.2, J5,6 4.1 Hz, H6; 4.38, mc
≈ ddd, J4,5 1.5 Hz, H5; 3.80, mc, OCH2CH2CHHOman; 3.79, mc,
CCH2O; 3.57, mc, OCH2CH2CHHOman; 1.93, mc, OCH2CH2-
CHHOman. 13C NMR (CDCl3, 125.47 MHz) δ 166.18, 165.49, 165.38,
C=O; 133.39, 133.11, 133.02, 129.92, 129.84, 129.79, 129.73, 129.41,
129.14, 129.01, 128.56, 128.44, 128.28, 127.95, 127.14, arylC; 97.73,
C1; 72.81, CCH2O; 70.53, C2; 70.19, C3; 68.75, C5; 67.86,
OCH2CH2CH2Oman; 66.94, C4; 65.71, OCH2CH2CH2Oman; 62.83,
C6; 48.14, CCH2O; 29.65, OCH2CH2CH2Oman. MALDI-TOF m/z
2113.7 [M + Na]+ (Calc. for C122H112O32: 2090.18).
Benzyltris(3-hydroxypropyloxymethyl)methane (19)
The acetylated precursor (16) (0.39 g, 0.78 mmol) was dissolved in dry
MeOH (150 mL) and deacetylated according to the general procedure
1
to yield a slightly yellow oil (281 mg, 0.76 mmol, 97%). H NMR δ
(CDCl3, 300 MHz) 7.21, mc, aryl H; 3.79, mc, OCH2CH2CH2OH; 3.59,
mc, OCH2CH2CH2OH; 3.24, s, CCH2O; 3.11, br s, OH; 2.65, s,
CCH2aryl; 1.84, mc, OCH2CH2CH2OH. 13C NMR (CDCl3,
75.47 MHz) δ 137.04, 130.48, 128.2, 126.14 aryl C; 71.3, CCH2O;
70.02, OCH2CH2CH2OH; 61.63, OCH2CH2CH2OH; 43.88, CCH2O;
35.96, CCH2aryl; 31.74 OCH2CH2CH2OH.
Tris(3-hydroxypropyloxymethyl)phenylmethane (20)
The acetylated precursor (17) (2.49 g 5.13 mmol) was dissolved in dry
MeOH (150 mL) and deacetylated according to the general procedure
to yield a slightly yellow oil (1.81 g, 5.03 mmol, 98%). 1H NMR
(CDCl3, 300 MHz) δ 7.30, m, aryl H; 3.75, s, CCH2O; 3.69, mc,
OCH2CH2CH2OH; 3.61, mc, OCH2CH2CH2OH; 1.78, mc,
OCH2CH2CH2OH. 13C NMR (CDCl3, 75.47 MHz) δ 141.07, 128.37,
126.77, 126.51, aryl C; 73.29, CCH2O; 70.39, OCH2CH2CH2OH;
61.42, OCH2CH2CH2OH; 47.71, CCH2O; 31.70, OCH2CH2CH2OH.
Tris[3-(α-D-mannopyranosyloxy)propyloxymethyl](5-phenyl-2-
oxapentyl)methane (2)
The benzoyl-protected cluster mannoside (21) (314 mg, 0.15 mmol)
was dissolved in dry MeOH (100 mL), sodium methanolate (1 M in
MeOH, 1 mL) was added and the reaction mixture was stirred for 56 h
at room temperature. The deprotected product (112 mg, 0.12 mmol,
83%) was obtained according to the general procedure as a white
lyophilisate. 1H NMR (CDCl3, 500 MHz) δ 7.32–7.17, mc, aryl H; 4.79,
d, J1,2 1.7 Hz, H1; 3.88–3.64, m, H3, H2, H4, H6, H6´, and
OCH2CH2CHHOman; 3.58–3.45, m, H5, OCH2CH2CHHOman,
OCH2CH2CH2aryl, OCH2CH2CHHOman; 3.49, s, CCH2O; 3.46–3.40,
m, OCH2CH2CH2aryl; 2.72, mc, OCH2CH2CH2aryl; 1.84, mc,
OCH2CH2CHHOman, OCH2CH2CH2aryl. 13C NMR (CDCl3, 125.47
MHz) δ 143.31 129.63, 129.61, 126.77, aryl C; 101.59, C1; 74.53, C5;
72.61, C3; 72.21, C2; 70.88, CH2CH2CH2aryl; 70.71, 70.56, CCH2O;
69.21, OCH2CH2CHHOman; 68.47, C4; 65.52, C6; 62.82,
OCH2CH2CHHOman; 49.28, CCH2O; 33.29, CH2CH2CH2aryl; 32.49,
CH2CH2CH2aryl; 30.77, OCH2CH2CHHOman. MALDI-TOF m/z
937.6 [M + Na]+ (Calc. for C37H62O21: 914.44).
Tris[3-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyloxy)propyloxy-
methyl](5-phenyl-2-oxapentyl)methane (21)
The triol (18) (98 mg, 0.25 mmol) and the mannosyl donor (4.31 g,
5.82 mmol) were dissolved in dry CH2Cl2 (6 mL) and treated with
TMSOTf solution (0.1 mL) according to the general mannosylation
procedure to yield the benzoylated cluster mannoside as a slightly
yellow foam (491 mg, 0.23 mmol, 91%). 1H NMR (CDCl3, 500 MHz)
δ 8.10, 8.04, 7.94, 7.83, mc, o-aryl H; 7.58–7.31, m, m- and p-aryl H;
6.14, dd, H4; 5.93, dd, J3,4 10.1, J2,3 3.3 Hz, H3; 5.71, dd, J1,2 1.8 Hz,
H2; 5.09, d, H1; 4.7, dd, J6,6´ 12.1, J5,6 2.5 Hz, H6; 4.48, dd, J5,6´ 4.1 Hz,
H6´; 4.44, ddd, J4,5 8.5 Hz, H5; 3.91, mc, OCH2CH2CHHOman; 3.68,
mc, OCH2CH2CHHOman; 3.56, dd, OCH2CH2CHHOman; 3.48, s,
CCH2O; 3.4, t, OCH2CH2CH2aryl; 2.65, dd, OCH2CH2CH2aryl; 1.98,
dddd, OCH2CH2CHHOman; 1.85, dddd, OCH2CH2CH2aryl. 13C NMR
(CDCl3, 125.76 MHz) δ 166.12, 165.45, 165.33, C=O; 142.15, 133.48,
133.11, 133.03, 129.86, 129.82, 129.78, 129.71, 129.34, 129.09,
128.95, 128.54, 128.47, 128.43, 128.39, 128.27, 128.22, 125.61, arylC;
97.7, C1; 70.52, C2; 70.45, OCH2CH2CH2aryl; 69.89, CCH2O; 68.75,
C5, 67.92, OCH2CH2CH2Oman; 66.86, C4; 65.86, OCH2CH2-
CH2Oman; 62.77, C6, 45.45, CCH2O; 32.28, OCH2CH2CH2aryl;
31.22, OCH2CH2CH2aryl; 29.71, OCH2CH2CH2Oman. MALDI-TOF
m/z 2186 [M + Na]+ (Calc. for C125H118O34: 2162.28).
Benzyltris[3-(α-D-mannopyranosyloxy)propyloxymethyl]methane (3)
The benzoyl-protected cluster mannoside (22) (287 mg, 0.13 mmol)
was dissolved in dry MeOH (100 mL), sodium methanolate (1 mL) was
added and the reaction mixture was stirred for 56 h at room
temperature. The deprotected product (110 mg, 0.13 mmol, 86%) was
1
obtained according to the general procedure to yield a white foam. H
NMR (500 MHz, CDCl3) δ 7.27–7.13, m, aryl H; 4.75, d, J1,2 1.7 Hz,
H1; 3.86–3.68, m, H3, H2, H4, H6, H6´, OCH2CH2CHHOman; 3.63, t,
OCH2CH2CHHOman; 3.58–3.45, m, H5, OCH2CH2CHHOman,
OCH2CH2CHHOman; 3.34, s, CCH2O; 2.65, s, CCH2aryl; 1.87, m,
OCH2CH2CHHOman. 13C NMR (CDCl3, 125.47 MHz) δ 138.88,
131.67, 128.92, 127.36, 127.09, aryl C; 101.61, C1; 74.58, C5; 72.63,
C3; 72.24, C2; 70.72, CCH2O; 68.96, OCH2CH2CHHOman; 68.62,
C4; 65.56, C6; 62.85, OCH2CH2CHHOman; 49.85, CCH2O; 36.4,
CCH2aryl; 30.94, OCH2CH2CHHOman. MALDI-TOF m/z 879.6
[M + Na]+ (Calc. for C38H64O21: 856.39).
Benzyltris[3-(2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyloxy)-
propyloxymethyl]methane (22)
The triol (19) (101 mg, 0.29 mmol) and the mannosyl donor (5.37 g,
7.25 mmol) were dissolved in dry CH2Cl2 (6 mL) and treated with
TMSOTf solution (0.1 mL) according to the general mannosylation
procedure to yield the benzoylated cluster mannoside as a slightly
yellow foam (539 mg, 0.256 mmol, 89%). 1H NMR (500 MHz, CDCl3)
δ 8.09, 8.04, 7.93, 7.83, m, o-aryl H; 7.59–7.23, m, m- and p-aryl H;
6.13, dd, H4; 5.94, dd, J3,4 10.1, J2,3 3.3 Hz, H3; 5.72, dd, J1,2 1.9 Hz,
H2; 5.11, d, H1; 4.70, dd, J6,6´ 9.6, J5,6´ 2.6 Hz, H6´; 4.47, dd, J5,6 4.1
Hz, H6; 4.44, mc, H5; 3.96, mc, OCH2CH2CHHOman; 3.73, mc,
OCH2CH2CHHOman; 3.26, mc, CCH2O; 2.72, mc, CCH2aryl; 2.01,
mc, OCH2CH2CHHOman. 13C NMR (CDCl3, 125,47 MHz) δ 166.13,
165.46, 165.36, C=O; 133.51, 133.4, 133.13, 130.11, 129.83, 129.78,
129.72, 129.34, 129.09, 128.94, 128.55, 128.45, 128.42, 128.42,
128.26, arylC; 97.75, C1; 70.53, C2; 70.32, CCH2aryl; 70.19, C3;
68.79, C5; 67.66, OCH2CH2CH2Oman; 66.87, C4; 65.90,
OCH2CH2CH2Oman; 62.81, C6; 44.54, CCH2O; 29.65,
OCH2CH2CH2Oman. MALDI-TOF m/z 2127.7 [M + Na]+ (Calc. for
C122H112O33: 2104.71).
Phenyltris[3-(α-D-mannopyranosyloxy)propyloxymethyl]methane (4)
The benzoyl-protected cluster mannoside (23) (287 mg, 0.13 mmol)
was dissolved in dry MeOH (100 mL), sodium methanolate (1 mL) was
added and the reaction mixture was stirred for 56 h at room
temperature. The deprotected product (109 mg, 0.13 mmol, 99%) was
1
obtained according to the general procedure to yield a white foam. H
NMR (CDCl3, 500 MHz) δ 7.47, 7.33, 7.23, mc, arylH; 4.75, d, J1,2 1.8
Hz, H1; 3.86–3.71, m, H3, H2, H4, H6, H6´, OCH2CH2CHHOman;
3.66, t, OCH2CH2CHHOman; 3.56–3.45, m, H5, OCH2CH2-
CHHOman, OCH2CH2CHHOman; 3.4, s, CCH2O; 1.84, m,
OCH2CH2CHHOman. 13C NMR (CDCl3, 125.47 MHz) δ 143.42,
128.91, 128.36, 127.28, aryl C; 101.59, C1; 74.52, C5; 73.81, CCH2O;

Trivalent Cluster Mannosides with Aromatic Partial Structure
93
72.61, C3; 72.21, C2; 69.16, OCH2CH2CHHOman; 68.49, C4; 65.49,
C6; 62.82, OCH2CH2CHHOman; 48.03 CCH2O; 30.77,
OCH2CH2CHHOman. MALDI-TOF m/z 865 [M + Na]+ (Calc. for
C38H64O21: 842.88).
[6] D. Choudhury, A. Thomson, V. Stojanoff, S. Langermann, J.
Pinkner, S. J. Hultgren, S. D. Knight, Science 1999, 285, 1061.
[7] F. G. Sauer, M. Barnhart, D. Choudhury, S. D. Knight, G.
Waksman, S. Hultgren, Curr. Opin. Struct. Biol. 2000, 10, 548.
[8] Th. K. Lindhorst, M. Dubber, U. Krallmann-Wenzel, S. Ehlers,
Eur. J. Org. Chem. 2000, 2027.
Acknowledgments
[9] B. Padias, H. K. Hall, D. A. Tomalia, J. R. McConnell, J. Org.
Chem. 1987, 52, 5305.
[10] K. W. Hellmann, S. Friedrich, L. H. Gade, W. S. Li, M.
McPartlin, Chem. Ber. 1995, 128, 29.
This work was funded within the collaborative network SFB
470 (DFG) and the Fonds of the German Chemical Industry
(FCI).
[11] G. Zemplén, E. Pascu, Ber. Dtsch. Chem. Ges. 1929, 62, 1613.
[12] F. Bien, T. Ziegler, Tetrahedron: Asymmetry 1998, 9, 781.
[13] M. Dubber, Th. K. Lindhorst, J. Org. Chem. 2000, 65, 5275.
[14] P. Klemm, B. J. Jørgensen, I. van Die, H. de Ree, H. Bergmans,
Mol. Gen. Genet. 1985, 199, 410.
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