Synthesis and testing of the first azobenzene mannobioside as photoswitchable ligand for the bacterial lectin FimH

Article abstract of DOI:10.3762/bjoc.9.26

In order to allow spatial and temporal control of carbohydrate-specific bacterial adhesion, it has become our goal to synthesise azobenzene mannosides as photoswitchable inhibitors of type 1 fimbriae-mediated adhesion of E. coli. An azobenzene mannobioside 2 was prepared and its photochromic properties were investigated. The E→Z isomerisation was found to be highly effective, yielding a long-lived (Z)-isomer. Both isomers, E and Z, show excellent water solubility and were tested as inhibitors of mannosidespecific bacterial adhesion in solution. Their inhibitory potency was found to be equal and almost two orders of magnitude higher than that of the standard inhibitor methyl mannoside. These findings could be rationalised on the basis of computer-aided docking studies. The properties of the new azobenzene mannobioside have qualified this glycoside to be eventually employed on solid support, in order to fabricate photoswitchable adhesive surfaces.

Full text of DOI:10.3762/bjoc.9.26

Synthesis and testing of the first azobenzene
mannobioside as photoswitchable ligand
for the bacterial lectin FimH
Vijayanand Chandrasekaran, Katharina Kolbe, Femke Beiroth
and Thisbe K. Lindhorst*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 223–233.
Christiana Albertina University of Kiel, Otto Diels Institute of Organic
Chemistry, Otto-Hahn-Platz 3/4, D-24098 Kiel, Germany, Fax: +49
431 8807410
doi:10.3762/bjoc.9.26
Received: 08 November 2012
Accepted: 18 January 2013
Published: 01 February 2013
Email:
Thisbe K. Lindhorst* - tklind@oc.uni-kiel.de
This article is part of the Thematic Series "Molecular switches and cages".
Guest Editor: D. Trauner
* Corresponding author
Keywords:
azobenzene glycosides; bacterial adhesion; E/Z photoisomerisation;
FimH antagonists; mannobiosides; molecular switches; sweet
switches
© 2013 Chandrasekaran et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
In order to allow spatial and temporal control of carbohydrate-specific bacterial adhesion, it has become our goal to synthesise
azobenzene mannosides as photoswitchable inhibitors of type 1 fimbriae-mediated adhesion of E. coli. An azobenzene mannobio-
side 2 was prepared and its photochromic properties were investigated. The E→Z isomerisation was found to be highly effective,
yielding a long-lived (Z)-isomer. Both isomers, E and Z, show excellent water solubility and were tested as inhibitors of mannoside-
specific bacterial adhesion in solution. Their inhibitory potency was found to be equal and almost two orders of magnitude higher
than that of the standard inhibitor methyl mannoside. These findings could be rationalised on the basis of computer-aided docking
studies. The properties of the new azobenzene mannobioside have qualified this glycoside to be eventually employed on solid
support, in order to fabricate photoswitchable adhesive surfaces.
Introduction
Adhesion of bacteria to surfaces can be a severe problem both action of adhesive organelles called fimbriae. They project from
in vivo and in vitro. Hence, inhibition of bacterial adhesion by the surface of bacteria and contain lectin domains to attach to
powerful antagonists is highly desirable, however, ideally on certain carbohydrate ligands of a glycosylated surface such as
demand, that is, in a specific and spatially as well as temporally the glycocalyx of eukaryotic target cells (Figure 1A) [1-4]. This
resolved way. Often bacterial adhesion depends on the inter- offers the possibility to inhibit bacterial adhesion by designed
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Figure 1: The α-(1→3)-linked mannobioside α-D-Man-(1→3)-D-Man 1 (B) is a potent disaccharide ligand for the bacterial lectin FimH and can thus
inhibit type 1 fimbriae-mediated bacterial adhesion to glycosylated surfaces (A). Introduction of an azobenzene aglycone moiety turns glycoside 1 into
a putative photoswitchable antagonist 2 of mannose-specific bacterial adhesion, displaying an (E)-, as well as a (Z)-form (C). In a future perspective
azobenzene glycosides such as 2 can be further functionalised to be attached to oligofunctional core molecules or immobilised on surfaces (D).
antagonists of the respective carbohydrate-specific bacterial [13,14]. In the case that the E→Z isomerisation process is high-
lectins [5]. In order to expand the scope of carbohydrate-based yielding and the lifetime of the (Z)-form of the azobenzene
antiadhesives, it has become our goal to make photoswitchable glycoside is long enough, it can be employed in bacterial adhe-
ligands of bacterial lectins to allow blocking of bacterial adhe- sion assays independently from the more stable (E)-isomer.
sion in a photocontrolled manner.
Eventually, this type of azobenzene mannobioside can be
further functionalised to be attached to various supports such as
One of the best-known fimbriae are the type 1 fimbriae of oligofunctional core molecules [15] or surfaces, to achieve
uropathogenic E. coli (UPEC), which comprise the α-D- switchable adhesive surfaces in continuation of our work on
mannosyl-specific lectin FimH at the tip of the fimbrial shaft. glycoarrays [16-18] (Figure 1D).
FimH antagonists are currently considered as new therapeutics
for the treatment of urinary tract infections [6]. The carbo- In this account, we describe the synthesis of the azobenzene
hydrate specificity of FimH has been investigated in great detail mannobioside 2 as well as of mannoside 6, investigation of their
[7] and its structure is well-known from several X-ray studies photochromic properties, and testing of mannobioside 2 as an
[8-11]. It has turned out that the 1,3-linked mannobioside α-D- inhibitor of type 1 fimbriae-mediated bacterial adhesion. Inter-
Man-(1→3)-D-Man (1, Figure 1B) is an ideal disaccharide pretation of the test results was supported by computer-aided
ligand for FimH [3,12]. All other isomeric mannobiosides do docking studies.
not bind favourably to FimH. Therefore, we have designed the
Results
respective azobenzene mannobioside 2 (Figure 1C) in order to
make a photoswitchable FimH antagonist available. Photoirra- Synthesis of azobenzene mannobioside 2
diation of azobenzene glycosides at ~365 nm effects E→Z For the preparation of azobenzene mannobioside 2, the azoben-
isomerisation of the N=N double bond, and thermal relaxation zene mannoside 6 was prepared first. Thus, mannosylation of
or irradiation at ~450 nm leads to Z→E back isomerisation the hydroxy-functionalised azobenzene 4 by using the mannosyl
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trichlororacetimidate 3 [19] led to the respective azobenzene orthoester 8, which, without intermediate purification steps,
α-mannosides 5 in 81% yield (Scheme 1). Treatment of 5 under could be carried on in a sequence of silyl ether-deprotection
Zemplén conditions [20] furnished the deprotected mannoside 6 leading to the intermediate 9, acetylation of the 4- and
in a basically quantitative reaction. Then, a standard protecting- 6-hydroxy groups, and then acid-mediated regioselective ring
group strategy was employed to allow the synthesis of the 3-OH opening of the 2,3-orthoester in the same pot to yield the free
unprotected mannoside 10, which is a key intermediate serving 3-OH azobenzene mannoside 10 in an overall yield of 43%.
as the glycosyl acceptor in the following disaccharide synthesis. Thus, the required protecting group pattern was obtained in a
First, regioselective protection of the primary 6-hydroxy group highly efficient way, based on the regioselective opening of
in 6 was accomplished by using TBDMS chloride in pyridine to orthoacetates to yield a vicinal arrangement of equatorial OH
yield 7. Then, triethylorthoacetate was employed to make the and axial O-acetyl groups [21,22]. The acetylation pattern was
Scheme 1: Synthesis of azobenzene mannoside 6 and azobenzene mannobioside 2 by glycosylation.
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clearly confirmed by 1H NMR spectroscopy showing the isomerisation, and the life time of the resulting (Z)-isomer is
expected downfield shift for the H-3 signal resonating at long enough to test this isomer independently from the more
4.32 ppm (H-2: 5.30 ppm, H-4: 5.17 ppm).
stable (E)-form.
Biological testing of azobenzene mannobio-
Next, glycosylation of the key intermediate 10 by using the
mannosyl donor 3 gave the desired mannobioside 11 in 76% side 2
yield. Finally, removal of the O-acetyl groups according to As a test system for mannose-specific bacterial adhesion, fluo-
Zemplén led to the unprotected 1,3-linked target mannobioside rescent GFP-transfected E. coli bacteria (pPKL1162) [23] were
α-D-Man-(1→3)-D-Man (2).
employed and tested on a mannan-coated polystyrene microtiter
plate surface. In this setup the amount of bacterial adhesion
With the two azobenzene glycosides 6 and 2 at hand, their solu- correlates with fluorescence intensity and can be quantified by
bility and photochromic properties were then investigated and using a standard microtiter plate reader. For inhibition of
compared. Mannoside 6 showed only poor solubility in most bacterial adhesion, two sets of serially diluted solutions of 2
organic solvents, except for DMSO. Unfortunately, it was also were prepared to inhibit adhesion of fluorescing E. coli to the
not soluble in water, or in water/DMSO mixtures, which would mannan surface. In one case, a stock solution of (E)-2 was seri-
allow biological testing. Mannobioside 2, on the other hand, ally diluted, in the second case, this stock solution of (E)-2 was
showed good solubility in polar organic solvents as well as in irradiated for 15 minutes to obtain the pure (Z)-2 isomer for
pure water. Thus, it was amenable to biological testing in subsequent serial dilution. The effect of both isomers as inhibi-
aqueous buffer.
tors of mannose-specific bacterial adhesion was then measured
in a concentration-dependent way. From the testing results
E→Z photoisomerisation of azobenzene mannoside 6 was sigmoidal inhibition curves were obtained (Supporting Informa-
studied in DMSO, while isomerisation of azobenzene manno- tion File 1) from which IC50 values for every individual inhib-
bioside 2 was performed in water. Photoirradiation was carried itor were deduced. The IC50 value reflects the concentration at
out in the dark at room temperature by employing a 365 nm which a compound inhibits 50% of bacterial adhesion to a
LED. Photostationary states (PSS) were reached after mannan-coated surface. The determined IC50 values were refer-
10 minutes of irradiation for both compounds. E→Z isomerisa- enced to the inhibitory potency of methyl α-D-mannoside
tion was observed by both 1H NMR and UV–vis spectroscopy. (MeMan) and p-nitrophenyl α-D-mannoside (pNPMan), res-
The E/Z ratios of the ground state (GS) as well as of the pectively, each tested on the same plate. Thus, relative inhib-
photostationary state were determined on the basis of the itory potencies (RIP values) were obtained, which allow com-
integration of the anomeric H-1 protons in the 1H NMR spec- parison of inhibitory potencies of different inhibitors, even
trum. Half-life were determined by UV–vis spectroscopic when they were not tested in the same experiment. The testing
observation of the thermal Z→E relaxation process (Supporting results collected in Table 2 show that the inhibitory power of
Information File 1). The respective data are collected in mannobioside 2 is roughly the same, regardless of whether its
Table 1.
(E)- or (Z)-form was employed. Inspection of their relative
inhibitory potencies reveals that both isomers of 2 are equally
Fortunately, the mannobioside 2 is ideally suited for biological potent inhibitors of type 1 fimbriae-mediated bacterial
testing as it is soluble in water and aqueous buffer, respectively. adhesion, similar to the power of the well-known mannoside
Photoirradiation of the (E)-isomer leads to almost quantitative pNPMan.
Table 1: Characterisation of the (E)- and (Z)-isomers of azobenzene glycosides 6 and 2.
UV–vis
azobenzene
glycoside
E/Za
(GS)
E/Za
(PSS)
H-1 (ppm)
(E)-isomer
H-1 (ppm)
(Z)-isomer
absorption
maxima (nm)
λmax(E), λmax(Z)
half-life, τ1/2 (h)
6
2
99:1
95:5
3:97
4:96
5.54b
5.65d
5.34b
5.52d
347, 440c
339, 429e
89
178.5
aaccording to the integration ratio of H-1(E) and H-1(Z) in the 1H NMR spectrum;
b10 mM concentration in DMSO-d6;
c50 µM concentration in DMSO;
d8 mM concentration in D2O;
e65 µM concentration in H2O.
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Table 2: Inhibition of adhesion of E. coli to a mannan-coated surface. The inhibitory potencies of (E)- and (Z)-2 are compared to the standard inhibi-
tors MeMan and pNPMan. a
MeMan
pNPMan
(E)-2
(Z)-2
5.205 ± 0.416
0.064 ± 0.018
0.078 ± 0.006
0.073 ± 0.001
0.084 ± 0.002
IC50 ± SD (mM)
0.073 ± 0.003
RIP (MeMan) ± SD
IP ≡ 1
81 ± 25
71 ± 1
RIP (pNPMan) ± SD
IP ≡ 1
0.94 ± 0.07
0.87 ± 0.02
aAverage values from duplicate results; SD: standard deviation (from one assay); RIP: relative inhibitory potency referenced to either MeMan or
pNPMan, each tested on the same microtiter plate.
In order to support the interpretation of the obtained test results,
Table 3: FlexX scoring values for the (E)- and the (Z)-isomer of 2
binding of (E)-2 and (Z)-2 to the bacterial lectin FimH was
investigated by computer-aided docking studies to get an idea
of their interactions with the carbohydrate-recognition domain
(CRD) of the lectin.
based on two different crystal structures in comparison to MeMan and
pNPMan.
Ligand
“open-gate” structure “closed-gate” structure
[9]
[10]
MeMan
pNPMan
(E)-2
−22.5
−24.9
−28.8
−28.7
−23.3
−27.4
−20.4
−21.6
Docking of azobenzene mannobioside 2 into
the carbohydrate binding site of FimH
To visualise complexation of the (E)- and (Z)-isomers of
azobenzene mannobioside 2 within the CRD of FimH FlexX
[24-26], flexible docking and consensus scoring [27,28], as
implemented in Sybyl 6.9 [29], was employed. Docking was
based on two different X-ray structures of FimH. They differ in
the conformation of the so-called tyrosine gate at the entrance
of the CRD, formed by the side chains of Y48 and Y137. One
structure is crystallised in an “open-gate” conformation [9],
another in the “closed-gate” conformation [10]. Affinity of any
FimH ligand is improved when it exerts favourable interactions
with the tyrosine gate of FimH. Thus, this substructure is an
important feature of the rim of the carbohydrate binding site of
this lectin.
(Z)-2
gate structure of FimH are almost equal (−28.8 and −28.7), and
also the scoring values obtained with the closed-gate structure
do not differ significantly (−20.4 and −21.6). Interestingly, the
predictions for pNPMan and also MeMan are the opposite,
suggesting better binding to the closed-gate conformation of
FimH, as described earlier [31]. Representative snapshots as
depicted in Figure 2 show that both isomers have the terminal
mannoside complexed within the CRD of the lectin, as
expected, and furthermore, that in both cases the azobenzene
moiety exerts effective interactions with the tyrosine gate
involving both benzene rings.
Before minimisation of the ligands, the bond angle of the N=N
double bond of the azobenzene moiety was manually set as Regardless of whether the (E)- or the (Z)-form of 2 is
180° for (E)-2 and as 90° for (Z)-2 [30]. Then docking was complexed with FimH, favourable π–π interactions can be
performed holding the FimH CRD fixed whereas the ligands formed between the azobenzene moiety and the tyrosine gate at
were allowed to change their conformations under the influ- the entrance of the CRD, though in different ways. The only
ence of the force field. A FlexX scoring value has been attri- difference that is seen is that, apparently, the interactions of
buted to each of the 30 obtained conformations (Table 3). This mannobioside 2 with the open-gate conformation of FimH are
value correlates with the binding affinity of the ligand for the advantageous over those with the closed-gate form. From the
FimH CRD, more negative values suggesting higher binding bioassay in solution phase it can certainly not be decided, which
affinity than less negative ones.
conformation the bacterial lectin adopts to interact with com-
pound 2; however, our test results confirm that both isomers of
Docking gave very similar results for both isomers of manno- the azobenzene mannobioside 2 have the same power as inhibi-
bioside 2, (E)-2 and (Z)-2. Scoring values based on the open- tors of FimH-mediated bacterial adhesion.
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Figure 2: Connolly [32,33] descriptions of the FimH CRD with the docked azobenzene mannobioside 2. Top: (E)-isomer (A, closed-gate; B, open-gate
conformation). Bottom: (Z)-isomer (C, closed-gate; D, open-gate conformation).
Discussion
mannose ring acts as a spacer moiety, sticking out straight from
The azobenzene mannobioside 2 was selected as a photoswitch- the CRD and placing the azobenzene portion in an orientation
able inhibitor of the bacterial lectin FimH based on earlier find- that allows flexible interactions with the tyrosine gate at the
ings about the inhibitory potency of several mannobiosides entrance of the carbohydrate binding site of the lectin.
[34,35]. Its synthesis was straightforward and high-yielding. It
has very convenient photochromic properties as the E→Z Apparently, the affinity of 2 to the open-gate form of FimH is
isomerisation is almost quantitative and the resulting (Z)-isomer higher than to the closed-gate conformation, a finding that
is especially long-lived. Both isomers are very well water- differs from many other docked FimH ligands. Here, the higher
soluble and could be independently tested as inhibitors of affinity for the open-gate FimH can be explained by strong π–π
mannose-specific bacterial adhesion and showed an equal and stacking of the first aromatic ring of the azobenzene unit with
high inhibitory potency in the range of the well-known high- the tyrosine gate.
affinity inhibitor p-nitrophenyl α-D-mannoside (pNPMan). This
result can be rationalised by computer docking, showing that As both isomers of 2 interact equally well with FimH, they
regardless of the configuration of the N=N double bond of the can’t be used to switch type 1 fimbriae-mediated bacterial adhe-
azobenzene moiety in 2, favourable interactions can be formed sion in solution. On the other hand, the obtained results support
with the tyrosine gate of the FimH CRD. While the terminal the idea to immobilise the azobenzene mannobioside on a solid
mannoside portion is complexed in the carbohydrate binding support to photocontrol the adhesive properties of the resulting
site, the first mannoside does not add significantly to the surface. In this approach the azobenzene N=N double bond can
affinity and this is in accordance with other studies on the com- be used as a hinge region to bend down the terminal mannose
plexation of oligosaccharides by FimH [11]. However, this moiety of the compound, which is critical for specific bacterial
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adhesion. Thus, upon E→Z isomerisation, the ligand will no For NMR assignments the following numbering was used:
longer be available for the interaction with the FimH-termi-
nated type 1 fimbriae that mediate adhesion. In this approach,
the second mannose moiety of the mannobioside is important
both to mediate hydrophilicity and to intensify the steric effect
that photoswitching has on the exposition of the terminal
mannoside.
Conclusion
The azobenzene mannosides presented herein resemble a struc-
ture quite similar to biaryl mannosides, which have been intro-
duced lately and shown to be of medical relevance as FimH (E)-p-(Phenylazo)phenyl 2,3,4,6-tetra-O-acetyl-α-D-
antagonists [6]. Thus, our novel “sweet switches” [15] appear to mannopyranoside (5). To a solution of the mannosyl donor 3
be highly promising FimH ligands, with the additional feature (5.00 g, 10.2 mmol) and p-hydroxyazobenzene (4, 2.01 g,
of a photoswitchable moiety. The biomedicinal potential of 10.2 mmol) in dry CH2Cl2 (100 mL) BF3·etherate (1.88 mL,
azobenzene glycosides seems even higher when their favour- 15.2 mmol) was added at 0 °C under N2 atmosphere, and the
able physiological properties are considered, such as low reaction mixture was stirred at this temperature for 15 min.
toxicity [36] and receptor specificity of the azobenzene aglycon Then, stirring was continued at rt for about 6 h, and then the
[37]. It will be our next goal to employ derivatives of azoben- reaction was quenched by the addition of satd. aq. NaHCO3
zene mannobioside 2 for immobilisation to test the solution (50 mL). The phases were separated, the aqueous phase
photoswitching of adhesion on surfaces.
was extracted with CH2Cl2 (2 × 150 mL), and the combined
organic phases were dried over MgSO4. This was filtered, and
the filtrate was concentrated under reduced pressure. Purifica-
tion of the crude product by column chromatography (cyclo-
Experimental
Materials and general methods
p-Hydroxyazobenzene was purchased from Sigma Aldrich and hexane/ethyl acetate, 3:1) gave the title glycoside 5 as an orange
used without further purification. Moisture-sensitive reactions crystalline solid (4.33 g, 8.19 mmol, 81%). Mp 53–55 °C; Rf
were carried out under nitrogen in dry glassware. Thin-layer 0.35 (cyclohexane/ethyl acetate 2:1); [α]20D +0.86 (c 0.9,
chromatography was performed on silica-gel plates (GF 254, DMSO); 1H NMR (500 MHz, CDCl3) δ 7.92 (d, J = 9.0 Hz,
Merck). Detection was effected by UV and/or charring with 2H, H-9, H-11), 7.89 (d, J = 8.5 Hz, 2H, H-14, H-18),
10% sulfuric acid in EtOH followed by heat treatment at 7.53–7.45 (m, 3H, H-15, H-16, H-17), 7.23 (d, J = 9.0 Hz, 2H,
~180 °C. Flash chromatography was performed on silica gel 60 H-8, H-12), 5.62 (d, J1,2 = 1.8 Hz, 1H, H-1), 5.58 (dd, J2,3 =
(Merck, 230–400 mesh, particle size 0.040–0.063 mm) by using 3.6 Hz, J3,4 = 10.1, 1H, H-3), 5.49 (dd, J1,2 = 1.8 Hz, J2,3 =
distilled solvents. Optical rotations were measured with a 3.6 Hz, 1H, H-2), 5.39 (dd~t, J3,4 = J4,5 = 10.0 Hz, 1H, H-4),
Perkin-Elmer 241 polarimeter (sodium D-line: 589 nm, length 4.30 (dd, J5,6a = 5.4 Hz, J6a,6b = 12.0 Hz, 1H, H-6a), 4.14–4.07
of cell: 1 dm) in the solvents indicated. 1H and 13C NMR (m, 2H, H-5, H-6b), 2.21, 2.06, 2.05, 2.03 (each s, each 3H, 4
spectra were recorded on Bruker DRX-500 and AV-600 spec- OAc); 13C NMR (125 MHz, CDCl3) δ 170.5, 169.9, 169.9,
trometers at 300 K. Chemical shifts are reported relative to 169.7 (4 C=O), 157.6 (C-7), 152.6 (C-13), 148.4 (C-10), 130.8
internal tetramethylsilane (δ = 0.00 ppm) or D2O (δ = (C-16), 128.9 (C-15, C-17), 124.6 (C-9, C-11), 123.1 (C-14,
4.76 ppm). Full assignment of the peaks was achieved with the 18), 116.8 (C-8, C-12), 95.7 (C-1), 69.4 (C-5), 69.3 (C-2), 68.8
aid of 2D NMR techniques (1H/1H COSY and 1H/13C HSQC). (C-3), 65.9 (C-4), 62.1 (C-6), 20.9, 20.7, 20.7, 20.6 (4 COCH3);
IR spectra were measured with a Perkin Elmer FT-IR Paragon IR (ATR) : 2929, 1743, 1598, 1496, 1366, 1209, 1029 cm−1;
1000 (ATR) spectrometer. ESI mass spectra were recorded on ESIMS (m/z): [M + Na]+ calcd for C26H28N2O10, 551.5; found,
an Esquire-LC instrument from Bruker Daltonics. MALDI-TOF 551.1.
mass spectra were recorded on a Bruker Biflex III instrument
with 19 kV acceleration voltage, and 2,5-dihydroxybenzoic acid (E)-p-(Phenylazo)phenyl α-D-mannopyranoside (6). To a
(DHB) was used as the matrix. UV–vis absorption spectra were solution of the acetyl-protected glycoside 5 (600 mg,
performed on Perkin-Elmer Lambda-241 or Varian Cary-5000 1.14 mmol) in dry MeOH (6 mL), a catalytic amount of solid
at a temperature of 18 ± 1 °C. Photoirradiaton was carried out NaOMe was added under N2 atmosphere, and the reaction mix-
by using a LED (emitting 365 nm light) from the Nichia ture was stirred for 5 h at rt. Then it was neutralized with
Corporation (NC4U133A) with a FWHM of 9 nm and an Amberlite IR 120 ion-exchange resin and filtered. The filtrate
optical power output (Po) ~ 1 W.
was evaporated under reduced pressure to yield the deprotected
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Beilstein J. Org. Chem. 2013, 9, 223–233.
mannoside 6 as a pale yellow solid (393 mg, 1.09 mmol, 96%). J1,2 = 1.7 Hz, 1H, H-1), 4.08 (dd, J1,2 = 1.8 Hz, J2,3 = 3.4 Hz,
Mp 183–185 °C; Rf 0.34 (ethyl acetate/MeOH 4:1); [α]20D 1H, H-2), 3.98 (dd, J5,6a = 1.8 Hz, J6a,6b = 11.2 Hz, 1H, H-6a),
+1.40 (c 1.0, DMSO); 1H NMR (500 MHz, DMSO-d6) δ 7.86 3.94 (dd, J2,3 = 3.5 Hz, J3,4 = 9.1 Hz, 1H, H-3), 3.82 (dd, J5,6b
(d, J = 8.7 Hz, 2H, H-9, H-11), 7.82 (d, J = 7.7 Hz, 2H, H-14, = 6.5 Hz, J6a,6b = 11.3 Hz, 1H, H-6b), 3.71 (t, J = 9.4 Hz, 1H,
H-18), 7.53–7.45 (m, 3H, H-15, H-16, H-17), 7.26 (d, J = H-4), 3.65 (mc, 1H, H-5), 0.83 (s, 9H, tert-butyl), 0.04, 0.05
8.7 Hz, 2H, H-8, H-12), 5.53 (bs, 1H, H-1), 3.89 (dd~bs, 1H, (each s, each 3H, 2 Si-CH3) ppm; 13C NMR (125 MHz, MeOH-
H-2), 3.73 (dd, J3,4 = 9.2 Hz, J3,2 = 3.0 Hz, 1H, H-3), 3.58–3.47 d4) δ 160.4 (C-7), 154.1 (C-13), 149.18 (C-10), 131.8 (C-16),
(m, 3H, H-6a, H-4, H-6b), 3.38 (mc, 1H, H-5); 13C NMR 130.2 (C-15), 125.5 (C-17), 123.6 (C-9), 118.2 (C-11), 100.0
(125 MHz, DMSO-d6) δ 158.9 (C-7), 155.5 (C-13), 151.9 (C-1), 76.2 (C-5), 72.3 (C-2), 71.7 (C-3), 68.6 (C-4), 64.4 (C-6),
(C-10), 130.9 (C-16), 129.4 (C-15, C-17), 124.3 (C-9, C-11), 26.4 (C(CH3)3), 19.1 (C(CH3)3), −5.13 (2 Si-CH3) ppm; IR
122.3 (C-14, C-18), 117.1 (C-8, C-12), 98.7 (C-1), 75.2 (C-5), (ATR) : 3337, 2928, 1599, 1498, 1229, 1006, 685 cm−1;
70.6 (C-3), 69.9 (C-2), 66.6 (C-4), 60.9 (C-6); UV, λmax: ESIMS (m/z): [M + Na]+ calcd for C24H34N2O6Si, 497.1;
347 nm; ε = 25907 ± 529 L × mol−1 × cm−1; IR (ATR) : 3337, found, 497.2;.
2920, 1599, 1584, 1496, 1227 cm−1; MALDI-TOFMS (m/z):
[M + H]+ calcd for 361.36; found, 361.21; anal. calcd for (E)-p-(Phenylazo)phenyl 6-O-tert-butyldimethylsilyl-2,3-O-
C18H20N2O6: C, 59.99; H, 5.59; N, 7.77; found: C, 61.07; H, (ethylorthoacetyl)-α-D-mannopyranoside (8). To a solution
5.80; N, 8.07.
of mannoside 7 (500 mg, 1.05 mmol) in toluene (8.0 mL),
triethylorthoacetate (773 µL, 4.22 mmol) and a catalytic amount
NMR spectroscopic data for (Z)-6. 1H NMR (500 MHz, of p-toluenesulfonic acid were added at rt, and the reaction mix-
DMSO-d6) δ 7.32 (t, J = 7.8 Hz, 2H, H-15, H-17), 7.18 (t, J = ture was stirred for 3.5 h, after which TLC showed complete
7.4 Hz, 1H, H-16), 6.97 (d, J = 8.9 Hz, 2H, H-9, H-11), 6.82 consumption of the starting material. Then, it was neutralised
(dd, J = 8.2 Hz, J = 6.7 Hz, 4H, H-8, H-12, H-14, H-18), 5.32 with triethylamine (100 µL), and the solution was diluted with
(d, J1,2 = 1.6 Hz, 1H, H-1), 3.77 (dd, J2,3 = 3.1 Hz, J1,2 = water (10 mL). It was extracted with toluene (2 × 20 mL), and
1.9 Hz, 1H, H-2), 3.62 (dd, J3,4 = 9.3 Hz, J3,2 = 3.3 Hz, 1H, the extract was concentrated under reduced pressure to get
H-3), 3.52 (dd, J5,6a = 2.1 Hz, J6a,6b= 11.8 Hz, 1H, H-6a), crude 8 (600 mg) as a red viscous syrup, which was used in the
3.48–3.36 (m, 2H, H-4, H-6b), 3.31 (ddd, J4,5 = 9.4 Hz, J5,6a = next reaction step without further purification.
5.8 Hz, J5,6b = 2.1 Hz, 1H, H-5); 13C NMR (125 MHz, DMSO-
d6) δ 155.4 (C-7), 153.8 (C-13), 147.3 (C-10), 129.1 (C-15, (E)-p-(Phenylazo)phenyl 2,3-O-(ethylorthoacetyl)-α-D-
C-17), 127.0 (C-16), 122.5 (C-8, C-12), 119.4 (C-14, C-18), mannopyranoside (9). The crude intermediate 8 (600 mg) was
116.7 (C-9, C-11), 98.7 (C-1), 74.8 (C-5), 70.3 (C-3), 69.7 dissolved in CH2Cl2 (6.0 mL), tetrabutylammonium fluoride
(C-2), 66.3 (C-4), 60.7 (C-6); UV, λmax: 440 nm, ε = 2635 ± 76 (1 M solution in THF, 1.68 mL) was added, and the reaction
L × mol−1 × cm−1.
mixture was stirred at rt for 4 h, after which TLC showed
complete consumption of the starting material. Then, it was
(E)-p-(Phenylazo)phenyl 6-O-tert-butyldimethylsilyl-α-D- concentrated under reduced pressure to obtain crude 9 as a dark
mannopyranoside (7). To a solution of the azobenzene red viscous syrup (594 mg), which was used in the next reac-
mannoside 6 (3.00 g, 8.33 mmol) in pyridine (30.0 mL) tert- tion step without further purification.
butyldimethylchlorosilane (1.38 g, 9.17 mmol) was added and
the reaction mixture was stirred at rt for 18 h, after which TLC (E)-p-(Phenylazo)phenyl 2,4,6-tri-O-acetyl-α-D-
showed complete consumption of the starting material. The mannopyranoside (10). The crude orthoester-protected
reaction was quenched with MeOH (2.0 mL) and further diluted mannoside 9 (594 mg) was dissolved in pyridine (2.5 mL), and
with ethyl acetate (150 mL). Then it was washed with satd. aq. acetic anhydride (1.26 mL) was added for O-acetylation. The
NaHCO3 solution (30 mL) and the aqueous phase extracted reaction mixture was stirred at rt for 3 h. Then, pyridine was
with ethyl acetate (2 × 50 mL). The combined organic phases removed under reduced pressure, and the residue was dissolved
were dried over MgSO4 and filtered, and the filtrate concen- in ethyl acetate (20 mL) and washed with satd. aq. NaHCO3
trated under reduced pressure to obtain the crude product. Puri- solution (10 mL). The aqueous phase was extracted with ethyl
fication by flash column chromatography (CH2Cl2/MeOH 3:7) acetate (2 × 25 mL), the combined organic phases were dried
gave the title compound as a dark orange solid (3.16 g, over Na2SO4 and filtered, and the filtrate concentrated was
6.66 mmol, 80%). Mp 74 °C; Rf 0.59 (CH2Cl2/MeOH 7:1); under reduced pressure to obtain a syrupy intermediate. It was
[α]20D +73 (c 0.97, MeOH); 1H NMR (500 MHz, MeOH-d4) δ dissolved in 80% acetic acid (2.5 mL), and the mixture was
7.94–7.88 (m, 4H, H-9, H-11, H-14, H-18), 7.57–7.49 (m, 3H, stirred at rt for 1.5 h to effect regioselective cleavage of the
H-15, H-16, H-17), 7.30 (d, J = 9.0 Hz, 2H, H-8, H-12), 5.62 (d, orthoester. Then, ethyl acetate (50 mL) was added and the
230

Beilstein J. Org. Chem. 2013, 9, 223–233.
organic layer was washed with water (5 mL) and dried over 1.94 (each s, each 3H, 7 OAc) ppm; 13C NMR (125 MHz,
MgSO4. It was filtered, and the filtrate was evaporated to obtain CDCl3) δ 170.6, 170.5, 170.4, 170.0, 169.9, 169.8, 169.6 (7
the crude product, which purified by column chromatography COCH3), 157.4 (C-7), 152.6 (C-13), 148.4 (C-10), 130.8
(cyclohexane/ethyl acetate 2:1) to yield the free 3-OH title (C-16), 129.1 (C-15, C-17), 124.6 (C-9, C-11), 122.71 (C-14,
mannoside 10 as a bright orange solid (220 mg, 0.453 mmol, C-18) 116.7 (C-8, C-12), 99.1 (C-1′), 95.6 (C-1), 74.8 (C-3),
43% over three steps). Mp 144–146 °C; Rf 0.21 (cyclohexane/ 70.8 (C-2), 69.9 (C-2′), 69.9, 69.6 (C-5, C-5′), 68.3 (C-4), 67.4
ethyl acetate); [α]20D +70 (c 0.96, CH2Cl2); 1H NMR (C-4′), 65.9 (C-3′), 62.5, 62.7 (C-6, C-6′), 20.9, 20.8, 20.7, 20.7,
(500 MHz, CDCl3) δ 7.92 (d, J = 8.9 Hz, 2H, H-9, H-11), 7.89 20.7, 20.6, 20.6 (7 COCH3) ppm; IR (ATR) : 1743, 1213,
(d, J = 7.9 Hz, 2H, H-14, H-18), 7.53–7.44 (m, 3H, H-15, H-16, 1032, 838 cm−1; ESIMS (m/z): [M + Na]+ calcd for
H-17), 7.19 (d, J = 8.9 Hz, 2H, H-8, H-12), 5.69 (d, J1,2 = C38H44N2O18, 839.3; found, 839.2.
1.4 Hz, 1H, H-1), 5.30 (dd, J1,2 = 1.7 Hz, J2,3 = 3.8 Hz, 1H,
H-2), 5.17 (t, J = 10.0 Hz, 1H, H-4), 4.32 (m, 2H, H-3, H-6a), (E)-p-(Phenylazo)phenyl 3-O-(α-D-mannopyranosyl)-α-D-
4.11 (dd, J5,6b = 2.2 Hz, J6a,6b = 12.4 Hz, 1H, H-6b), 4.05 (mc, mannopyranoside (2). The acetyl-protected disaccharide 11
1H, H-5), 2.23, 2.15, 2.03 (each s, each 3H, 3 OAc), 1.62 (bs, (50 mg, 61.2 µmol) was dissolved in dry MeOH (2 mL) and a
OH) ppm; 13C NMR (150 MHz, CDCl3) δ 171.3, 170.6, 170.4 catalytic amount of solid NaOMe was added under N2 atmos-
(3 COCH3), 157.7 (C-7), 152.6 (C-13), 148.3 (C-10), 130.8 phere. The reaction mixture was stirred for 5 h at rt, and then it
(C-16), 129.1 (C-15, C-17), 124.6 (C-9, C-11), 122.7 (C-14, was neutralized with Amberlite IR 120 ion-exchange resin. It
C-18), 116.7 (C-8, C-12), 95.42 (C-1), 71.99 (C-2), 69.14 (C-5), was then filtered and thoroughly washed with MeOH (2 ×
69.04 (C-4), 68.47 (C-3), 62.15 (C-6), 20.97, 20.91, 20.69 (3 20 mL), and the filtrate was evaporated to obtain the crude pro-
COCH3) ppm; IR (ATR) : 3453, 2961, 1737, 1228, 1023, duct, which after purification by flash column chromatography
798 cm−1; ESIMS (m/z): [M + H]+ calcd for C24H26N2O9, (CH2Cl2/methanol 9:1) gave the final mannobioside 2 as a pale
509.1; found, 509.2.
yellow solid (29.3 mg, 56.1 µmol, 92%). Mp 107–109 °C; Rf
0.08 (CH2Cl2/MeOH 9:1); [α]20D +18.4 (c 0.48, MeOH);
(E)-p-(Phenylazo)phenyl 3-O-(2,3,4,6-tetra-O-acetyl-α-D- 1H NMR (500 MHz, D2O) δ 7.81 (d, J = 8.2 Hz, 2H, H-9,
mannopyranosyl)-2,4,6-tri-O-acetyl-α-D-mannopyranoside H-11), 7.75 (d, J = 7.4 Hz, 2H, H-14, H-18), 7.53–7.49 (m, 3H,
(11). The 3-OH unprotected mannoside 10 (50 mg, 103 µmol) H-15, H-16, H-17), 7.24 (d, J = 8.3 Hz, 2H, H-8, H-12), 5.65 (s,
and the mannosyl donor 3 (101 mg, 206 µmol) were dissolved 1H, H-1), 5.16 (s, 1H, H-1′), 4.29 (mc, 1H, H-2), 4.14 (dd, J2,3
in dry CH2Cl2 (10 mL), and the mixture was cooled to −10 °C = 3.1 Hz, J3,4 = 10.3 Hz, 1H, H-3′), 4.06 (mc, 1H, H-2′),
under N2 atmosphere. To this ice-cooled solution BF3·etherate 3.89–3.81 (m, 3H, H-3, H-4, H-4′), 3.79–3.62 (m, 6H, H-6a,
(13 µL, 108 µmol) was added and the mixture was stirred at H-6b, H-5, H-5′, H-6a′, H-6b′) ppm; 13C NMR (125 MHz,
0 °C for about 30 min. Then, the reaction mixture was allowed D2O) δ 158.2 (C-7), 151.3 (C-13), 148.2 (C-10), 131.4 (C-16),
to warm to rt and stirred for another 4 h. The reaction mixture 129.6 (C-15, C-17), 124.4 (C-9, C-11), 122.2 (C-14, C-18),
was then quenched by the addition of a catalytic amount of 117.3 (C-8, C-12), 102.4 (C-1′), 97.8 (C-1), 77.9 (C-3′), 73.8
solid NaHCO3 and concentrated under reduced pressure to (C-5), 73.5 (C-3), 70.5 (C-4′), 70.1 (C-2′), 69.5 (C-2), 66.9
obtain the crude product as a dark reddish-brown syrup. Purific- (C-5′), 65.9 (C-4), 61.1 (C-6), 60.6 (C-6′) ppm; IR (ATR)
:
ation by column chromatography (CH2Cl2/ethyl acetate 8:2) 3318, 2927, 1599, 1231, 1007, 685 cm−1; MALDI-TOFMS
gave the acetyl-protected mannobioside 11 as a pale yellow (m/z): [M + Na]+ calcd for C24H30N2O11, 545.18; found,
solid (64 mg, 78 µmol, 76%). Mp 84–85 °C; Rf 0.57 (CH2Cl2/ 545.17; UV, λmax: 339 nm, ε = 14776 ± 729 L × mol−1 × cm−1;
ethyl acetate 8:2); [α]20D +103 (c 0.86, CH2Cl2); 1H NMR anal. calcd for C24H30N2O11 × 1.1 H2O: C, 52.11; H, 6.09; N,
(500 MHz, CDCl3) δ 7.92 (d, J = 9.0 Hz, 2H, H-9, H-11), 7.89 5.07; found: C, 52.04; H, 5.79; N, 5.06.
(d, J = 7.1 Hz, 2H, H-14, H-18), 7.46–7.38 (m, 3H, H-15, H-16,
H-17), 7.18 (d, J = 9.0 Hz, 2H, H-8, H-12), 5.65 (d, J1,2 = NMR-spectroscopic data for (Z)-2. 1H NMR (500 MHz, D2O)
1.7 Hz, 1H, H-1), 5.46 (dd, J1,2 = 1.8 Hz, J2,3 = 3.5 Hz, 1H, δ 7.32 (t, J = 7.1 Hz, 2H, H-15, H-17), 7.24 (t, 1H, H-16), 7.00
H-2), 5.40 (t, J = 10.1 Hz, 1H, H-4′), 5.30 (mc, 1H, H-3′), 5.26 (dd, J = 1.9 Hz, J = 8.9 Hz, 2H, H-9, H-11), 6.95 (dd, J =
(mc, 1H, H-4), 5.09 (d, J1,2 = 1.7 Hz 1H, H-1′), 5.06 (dd, J1,2 = 1.9 Hz, J = 8.9 Hz, 2H, H-8, H-12), 6.91 (dd, J = 1.3 Hz, J =
1.9 Hz, J2,3 = 2.9 Hz, 1H, H-2′), 4.41 (dd, J2,3 = 3.5 Hz, J3,4 = 7.8 Hz, 2H, H-14, H-18), 5.52 (s, 1H, H-1), 5.12 (s, 1H, H-1′),
9.9 Hz, 1H, H-3), 4.30 (dd, J5,6b = 6.3 Hz, J6a,6b = 12.7 Hz, 1H, 4.22 (mc, 1H, H-2), 4.07 (mc, 1H, H-3′), 4.03 (dd, J1,2 = 1.7 Hz,
H-6a), 4.24 (dd, J5′,6b′ = 5.8 Hz, J6a′,6b′ = 12.3 Hz, 1H, H-6a′), J2,3 = 3.2 Hz, 1H, H-2′), 3.85–3.82 (m, 2H, H-3, H-4),
4.14–4.10 (m, H-5′, H-6b), 4.08 (dd, J5,6a = 2.4 Hz, J6a,6b = 3.79–3.59 (m, 7H, H-4′, H-6a, H-6b, H-5, H-5′, H-6a′, H-6b′)
12.3 Hz, 1H, H-6b), 3.99 (ddd, J4,5 = 10.2 Hz, J5,6a = 2.3 Hz, ppm; 13C NMR (125 MHz, D2O) δ 155.3 (C-7), 153.5 (C-13),
J6a,6b = 5.8 Hz, 1H, H-5), 2.20, 2.09, 2.08, 2.03, 2.00, 1.97, 146.9 (C-10), 129.2 (C-15, C-17), 128.2 (C-16), 123.4 (C-8,
231

Beilstein J. Org. Chem. 2013, 9, 223–233.
14.Kramer, R. H.; Fortin, D. L.; Trauner, D. Curr. Opin. Neurobiol. 2009,
19, 544–552. doi:10.1016/j.conb.2009.09.004
C-12), 120.3 (C-14, C-18), 116.9 (C-9, C-11), 102.4 (C-1′),
97.8 (C-1), 77.8 (C-3′), 73.7 (C-5), 73.4 (C-3), 70.4 (C-4′), 70.1
(C-2′), 69.4 (C-2), 66.8 (C-5′), 65.9 (C-4), 61.0 (C-6), 60.6
(C-6′) ppm; UV, λmax: 429 nm, ε = 1699 ± 68 L × mol−1 ×
cm−1.
15.Chandrasekaran, V.; Lindhorst, T. K. Chem. Commun. 2012, 48,
7519–7521. doi:10.1039/c2cc33542e
16.Weissenborn, M. J.; Castangia, R.; Wehner, J. W.; Šardzík, R.;
Lindhorst, T. K.; Flitsch, S. Chem. Commun. 2012, 4444–4446.
doi:10.1039/c2cc30844d
17.Grabosch, C.; Kolbe, K.; Lindhorst, T. K. ChemBioChem 2012, 13,
1874–1879. doi:10.1002/cbic.201200365
Supporting Information
18.Wehner, J. W.; Weissenborn, M. J.; Hartmann, M.; Gray, C. J.;
Šardzík, R.; Eyers, C. E.; Flitsch, S. L.; Lindhorst, T. K.
Org. Biomol. Chem. 2012, 10, 8919–8926. doi:10.1039/c2ob26118a
19.Jung, K.-H.; Hoch, M.; Schmidt, R. R. Liebigs Ann. Chem. 1989,
1099–1106. doi:10.1002/jlac.198919890276
Supporting Information File 1
Photoisomerization studies, UV–vis spectra, NMR spectra,
bioassay and docking results.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-26-S1.pdf]
20.Zemplén, G.; Pacsu, E. Ber. Dtsch. Chem. Ges. B 1929, 62,
1613–1614. doi:10.1002/cber.19290620640
21.Lindhorst, T. K.; Bruegge, K.; Fuchs, A.; Sperling, O.
Beilstein J. Org. Chem. 2010, 6, 801–809. doi:10.3762/bjoc.6.90
22.Oscarson, S.; Tidén, A.-K. Carbohydr. Res. 1993, 247, 323–328.
doi:10.1016/0008-6215(93)84266-9
Acknowledgements
Financial support by the DFG (collaborative network SFB677)
and FCI (Fonds der Chemischen Industrie) is gratefully
acknowledged. We thank Max Britz for technical assistance.
23.Hartmann, M.; Horst, A. K.; Klemm, P.; Lindhorst, T. K.
Chem. Commun. 2010, 46, 330–332. doi:10.1039/b922525k
24.Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol. Biol. 1996, 261,
470–489. doi:10.1006/jmbi.1996.0477
References
25.Rarey, M.; Kramer, B.; Lengauer, T. J. Comput.-Aided Mol. Des. 1997,
11, 369–384. doi:10.1023/A:1007913026166
1. Ohlsen, K.; Oelschlaeger, T. A.; Hacker, J.; Khan, A. S.
Top. Curr. Chem. 2009, 288, 17–65. doi:10.1007/128_2008_10
2. Klemm, P.; Schembri, M. A. Int. J. Med. Microbiol. 2000, 290, 27–35.
doi:10.1016/S1438-4221(00)80102-2
26.Kramer, B.; Rarey, M.; Lengauer, T. Proteins: Struct., Funct., Genet.
1999, 37, 228–241.
doi:10.1002/(SICI)1097-0134(19991101)37:2<228::AID-PROT8>3.0.C
O;2-8
3. Mulvey, M. A. Cell. Microbiol. 2002, 4, 257–271.
doi:10.1046/j.1462-5822.2002.00193.x
27.Clark, R. D.; Strizhev, A.; Leonard, J. M.; Blake, J. F.; Matthew, J. B.
J. Mol. Graphics Modell. 2002, 20, 281–295.
4. Kau, A. L.; Hunstad, D. A.; Hultgren, S. J. Curr. Opin. Microbiol. 2005,
8, 54–59. doi:10.1016/j.mib.2004.12.001
doi:10.1016/S1093-3263(01)00125-5
5. Hartmann, M.; Lindhorst, T. K. Eur. J. Org. Chem. 2011, 3583–3609.
doi:10.1002/ejoc.201100407
28.Charifson, P. S.; Corkery, J. J.; Murcko, M. A.; Walters, W. P.
J. Med. Chem. 1999, 42, 5100–5109. doi:10.1021/jm990352k
29.SYBYL, Version 6.9; Tripos, Inc.: St. Louis, MO.
30.Dokić, J.; Gothe, M.; Wirth, J.; Peters, M. V.; Schwarz, J.; Hecht, S.;
Saalfrank, P. J. Phys. Chem. A 2009, 113, 6763–6773.
doi:10.1021/jp9021344
See for a review.
6. Scharenberg, M.; Schwardt, O.; Rabbani, S.; Ernst, B. J. Med. Chem.
2012, 55, 9810–9816. doi:10.1021/jm3010338
7. Knight, S. D.; Bouckaert, J. Top. Curr. Chem. 2009, 288, 67–107.
doi:10.1007/128_2008_13
31.Sperling, O.; Fuchs, A.; Lindhorst, T. K. Org. Biomol. Chem. 2006, 4,
3913–3922. doi:10.1039/b610745a
8. Choudhury, D.; Thompson, A.; Stojanoff, V.; Langerman, S.;
Pinkner, J.; Hultgren, S. J.; Knight, S. D. Science 1999, 285,
1061–1066. doi:10.1126/science.285.5430.1061
32.Connolly, M. L. Science 1983, 221, 709–713.
doi:10.1126/science.6879170
9. Hung, C.-S.; Bouckaert, J.; Hung, D.; Pinkner, J.; Widberg, C.;
Defusco, A.; Auguste, C. G.; Strouse, R.; Langermann, S.;
Waksman, G.; Hultgren, S. J. Mol. Microbiol. 2002, 44, 903–918.
doi:10.1046/j.1365-2958.2002.02915.x
33.Connolly, M. L. J. Appl. Crystallogr. 1983, 16, 548.
doi:10.1107/S0021889883010985
34.Sharon, N. FEBS Lett. 1987, 217, 145–157.
doi:10.1016/0014-5793(87)80654-3
10.Bouckaert, J.; Berglund, J.; Schembri, M.; Genst, E. D.; Cools, L.;
Wuhrer, M.; Hung, C.-S.; Pinkner, J.; Slättegård, R.; Zavialov, A.;
Choudhury, D.; Langermann, S.; Hultgren, S. J.; Wyns, L.; Klemm, P.;
Oscarson, S.; Knight, S. D.; Greve, H. D. Mol. Microbiol. 2005, 55,
441–455. doi:10.1111/j.1365-2958.2004.04415.x
35.Lindhorst, T. K. Ligands for FimH. In Synthesis and Biological
Applications of Glycoconjugates; Renaudet, O.; Spinelli, N., Eds.;
Bentham Science e-Books, 2011; pp 12–35.
36.Hartmann, M.; Papavlassopoulos, H.; Chandrasekaran, V.;
Grabosch, C.; Beiroth, F.; Lindhorst, T. K.; Röhl, C. FEBS Lett. 2012,
586, 1459–1465. doi:10.1016/j.febslet.2012.03.059
37.Garcia-Amorós, J.; Díaz-Lobo, M.; Nonell, S.; Velasco, D.
Angew. Chem., Int. Ed. 2012, 51, 12820–12823.
doi:10.1002/anie.201207602
11.Wellens, A.; Garofalo, C.; Nguyen, H.; Van Gerven, N.; Slättegård, R.;
Hernalsteens, J. P.; Wyns, L.; Oscarson, S.; De Greve, H.;
Hultgren, S.; Bouckaert, J. PLoS One 2008, 3, e2040.
doi:10.1371/journal.pone.0002040
12.Dubber, M.; Sperling, O.; Lindhorst, T. K. Org. Biomol. Chem. 2006, 4,
3901–3912. doi:10.1039/b610741a
13.Russew, M.-M.; Hecht, S. Adv. Mater. 2010, 22, 3348–3360.
doi:10.1002/adma.200904102
232

Beilstein J. Org. Chem. 2013, 9, 223–233.
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