Fighting Shigella by Blocking Its Disease-Causing Toxin

Article abstract of DOI:10.1021/acs.jmedchem.1c00152

Shiga toxin is an AB5 toxin produced by Shigella species, while related toxins are produced by Shiga toxin-producing Escherichia coli (STEC). Infection by Shigella can lead to bloody diarrhea followed by the often fatal hemolytic uremic syndrome (HUS). In the present paper, we aimed for a simple and effective toxin inhibitor by comparing three classes of carbohydrate-based inhibitors: glycodendrimers, glycopolymers, and oligosaccharides. We observed a clear enhancement in potency for multivalent inhibitors, with the divalent and tetravalent compounds inhibiting in the millimolar and micromolar range, respectively. However, the polymeric inhibitor based on galabiose was the most potent in the series exhibiting nanomolar inhibition. Alginate and chitosan oligosaccharides also inhibit Shiga toxin and may be used as a prophylactic drug during shigella outbreaks.

Full text of DOI:10.1021/acs.jmedchem.1c00152

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Fighting Shigella by Blocking Its Disease-Causing Toxin
Diksha Haksar, Mostafa Asadpoor, Torben Heise, Jie Shi, Saskia Braber, Gert Folkerts, Lluis Ballell,
Janneth Rodrigues, and Roland J. Pieters*
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ABSTRACT: Shiga toxin is an AB5 toxin produced by Shigella species,
while related toxins are produced by Shiga toxin-producing Escherichia coli
(STEC). Infection by Shigella can lead to bloody diarrhea followed by the
often fatal hemolytic uremic syndrome (HUS). In the present paper, we
aimed for a simple and effective toxin inhibitor by comparing three classes
of carbohydrate-based inhibitors: glycodendrimers, glycopolymers, and
oligosaccharides. We observed a clear enhancement in potency for
multivalent inhibitors, with the divalent and tetravalent compounds
inhibiting in the millimolar and micromolar range, respectively. However,
the polymeric inhibitor based on galabiose was the most potent in the series
exhibiting nanomolar inhibition. Alginate and chitosan oligosaccharides also
inhibit Shiga toxin and may be used as a prophylactic drug during shigella
outbreaks.
flexneri and S. sonnei points toward increased future morbidity
and mortality.10−19 As an alternative to antibiotics, synthetic
molecules based on Gb3 have been explored as potential
prophylactic treatment for STEC.20 Synsorb Pk, silicon dioxide
coupled to synthetic Pk, showed promising results in the
trapping of toxins and preventing toxic effects on renal cells.21
However, a subsequent clinical trial was unsuccessful in
diminishing diarrhea-associated HUS possibly due to late
administration of the drug to the GI tract, while the toxin was
already active systemically.22 Recommendations were made for
intervention in the circulation. This approach was explored in
several cases with antibodies and nanobodies, as summarized
recently.23 Smaller dendritic molecules were also explored in
this respect. The soluble STARFISH inhibitor with a
decavalent display of the Gb3 trisaccharide, reported by
Bundle et al., exhibited subnanomolar inhibition of Stx1, with
large potency gains over the divalent analogue and the Pk
trisaccharide itself.24 A modification of the STARFISH named
DAISY was observed to be effective against both Stx1 and Stx2
with nanomolar inhibition and in vivo activity in EHEC orally
infected mice by subcutaneous injection 24 h after infection.25
Several related SUPERTWIG structures (based on Gb3
conjugated to carbosilane dendrimers), developed by Nishika-
wa et al., were also identified as effective neutralizers of Stx
INTRODUCTION
■
Bacterial dysentery or shigellosis has been identified as one of
the major causes of mortality in children under 5 years of age.1
Shigellosis is caused by gram-negative bacteria from the
following four species of Shigella: Shigella dysenteriae, Shigella
flexneri, Shigella boydii, and Shigella sonnei through the fecal−
oral route. The pathology can include bloody diarrhea
(hemorrhagic colitis) followed by the often fatal hemolytic
uremic syndrome (HUS). HUS can occur if the pathogen also
produces the Shiga toxin (Stx). The toxin is produced by S.
dysenteriae serotype 1, but closely related toxins Stx1 and Stx2
are produced by Shiga toxin-producing Escherichia coli (STEC)
or enterohemorrhagic E. coli (EHEC), where Stx2 has been
reported to cause more severe infections.2 STEC outbreaks are
mostly food-borne with the largest ever reported in Germany
(2011) linked to sprout consumption.3
The Shiga toxin is an AB5 toxin composed of the toxic A
subunit and a pentameric B subunit that is responsible for the
binding of the toxin to its cell surface receptor globotriao-
sylceramide (Gb3; Galα1-4Galβ1-4Glcβ1-ceramide, also
known as CD77 or the Pk blood group antigen).4 Each of
the five B subunits can bind three Gb3 molecules
simultaneously.5,6 After the initial bloody diarrhea, the toxin
enters the bloodstream by poorly understood mechanisms.7
The ample presence of Gb3 molecules in the kidney targets the
toxin to this location. Once endocytosed, the toxin induces
multiple signaling pathways leading to blockage of protein
synthesis and induction of apoptosis8 and HUS. STEC
infections are treated with antibiotics, although their use is
controversial with respect to their ability to increase the risk of
HUS.9 The recent emergence of toxin-producing strains of S.
Received: January 26, 2021
Published: April 28, 2021
© 2021 The Authors. Published by
American Chemical Society
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a
Scheme 1. Synthesis of Galabiose Azide
a
Reagents and conditions: (i) HSPh, BF3·Et2O, DCM, r.t., 16 h, 90%; NaOMe, MeOH, r.t. 90% (ii) tBu2Si(OTf)2, pyridine, DMF, −40°C, >90%
(iii) TBDMSOTf, DMAP, pyridine, r.t. 70% (iv) TMSN3, SnCl4, DCM, 95%; NaOMe, MeOH, r.t., 16 h, 100% (v) BzCl, pyridine, DCM, −80°C, 2
h, 50% (vi) Tf2O, Ph2SO, TTBP, DCM, −60°C, 1 h, 72% (vii) NaOMe, MeOH; HF, pyridine; Ac2O, pyridine, 63% (viii) NaOH, MeOH, 90%.
Figure 1. Di- and tetravalent dendrimers.
with a dependency on their valency and structure.26
hexavalent structure provided protection after intravenous
injections starting 3 days after oral infection.
In the present paper, three classes of carbohydrate-
containing structures were investigated: dendritic-synthesized
multivalent inhibitors, glycopolymers, and natural oligosac-
charides. As the ligand, we chose to explore the potential of the
disaccharide (Galα1-4Galβ; galabiose) as a possible mono-
valent alternative to Gb3-based inhibitors. The intention here
A
was to explore what the minimal structural requirements for
potent toxin inhibition would be by minimizing the ligand and
the multivalent scaffold. For the dendrimers, ease of
preparation was central to the selection of di- and tetravalent
dendrimers utilized. Polymeric scaffolds were selected for
potency comparison. For the polymer scaffold, hyperbranched
polyglycerol (hPG) was used for their easy synthesis, high
functionalization, biocompatibility, and low in vivo toxicity.27 A
polyalkyne and a polyazide variant of hPG were prepared for
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their conjugation by employing the copper-catalyzed alkyne
azide cycloaddition (CuAAC) conjugations. In addition to the
synthesized compounds, commercially available oligosacchar-
ides such as nontoxic food grade alginate, chitosan, and fructo-
and galacto-oligosaccharides [alginate oligosaccharide (AOS),
chitosan oligosaccharide (COS), galactose oligosaccharide
(GOS), fructose oligosaccharide (FOS)]28 were tested for
Stx inhibition. These could serve as an even more viable
practical alternative that could be a part of a preventative food-
based approach during outbreaks with a focus on the
gastrointestinal phase of the toxin-producing Shigella pathoge-
nicity.
followed by azide substitution using sodium azide.32 Mesyl
substitution of the hPG was calculated at 8% (ca. 10 mesyl end
groups per molecule) using proton NMR, and complete
substitution with azide groups was confirmed by the absence of
the mesyl protons (1H NMR) and the appearance of the azide
stretching in the infrared spectra (IR) at 2110 cm−1.
Propargylation of hPG was performed in a single step using
propargyl bromide in 72% yield.33 The polymer was calculated
to be 16% functionalized, which means ca. 20 propargyl end
groups per molecule based on proton NMR, and the IR spectra
further confirmed this via the 2110 cm−1 peak (see the
Supporting Information).
hPG azide was conjugated by CuAAC to globotriose-NAc-
propargyl (7, Scheme 2) to obtain 8 (Figure 3) in 80% yield.
Similarly, conjugation of 1d to hPG-propargyl following
deprotection yielded final compound 9 (Figure 3) in 75%
yield over two steps. Final polymers 8 and 9 were characterized
by H NMR and also by IR to check for the absence of the
azide and alkyne stretching peaks, respectively.
RESULTS
■
For the synthesis of the monovalent galabiose reagent,
galactose pentaacetate was used as the common precursor
for the synthesis of the glycosyl donor and acceptor (Scheme
1). Glycosyl donor 1a was synthesized in three steps by
thioglycoside preparation as the first step followed by silyl
protection of the sugar. Glycosyl donor 1b was synthesized by
azidation using trimethylsilyl azide and benzoyl protection.
Trifluoromethanesulfonic anhydride-mediated glycosylation
afforded the disaccharide 1c in moderate yields. Deprotection
was performed over two steps without purification followed by
acetylation to obtain 1d which was used for conjugation with
various dendrimers.
Building block 3,5-dihydroxybenzoic acid was used as the
starting material for the synthesis of all four dendrimers
(Figure 1). 2a and 2c were synthesized using previously
reported procedures.29,30 Divalent 2b was conveniently
prepared by coupling methyl 3,5-bis(2-aminoethoxy)benzoate
to propargyl chloroformate and was obtained in 88% yield.
Amide coupling of 2a to dodecane-1,12-diamine using BOP
gave tetravalent dendrimer 2d in 60% yield. Dendrimers (2a,
2b, 2c, and 2d) were conjugated to 1d by CuAAC and
deprotected to obtain final compounds 3, 4, 5, and 6 (Figure
3) in good yields.
1
Previously, inhibitors were tested for inhibition in ELISA
assays using immobilization of the B subunit of Stx1 (Stx1B).24
In contrast, we used an assay in which FSL-Gb3 was
immobilized instead of the toxin (Figure S2), as this was
deemed more realistic since in vivo the toxin is also free to
move. FSL-GB3 is comprised of a functional component (F)
which is GB3, conjugated via an O(CH2)3NH spacer (S) to an
activated adipate derivative of dioleoylphosphatidylethanol-
amine (L). Monovalent 1e was used as the reference in the
ELISA and, as expected, showed millimolar inhibition of the
toxin with an IC50 of approx. 5 mM. Divalent 3 and 4 also
inhibited the toxin in the millimolar range (1 and 1.2 mM
respectively) (Table 1). Clearly, the small variation in the
spacer length between dendrimer 2a and 2b did not cause any
significant variation in potency. It was anticipated that if the
divalent ligands bridge between sites 1 and 2 on a single toxin
subunit,6 this would be more easily possible with the longer
spacer of 4. A stronger enhancement of the inhibition was
observed with the tetravalent compounds 5 and 6 as both
showed micromolar inhibition (20 and 13 μM respectively).
Here again, the toxin did not discriminate between the
elongated and more flexible dendrimer 2d backbone with
respect to 2c (Figure 2).
We expected both compounds to bridge between the
strongest of the three binding sites per subunit, the so-called
site 2,34 of the same pentamer separated by ca. 30 Å.6 For the
decavalent hPG-Gb3 polymeric inhibitor 8, low micromolar
inhibition was seen (IC50 = 3 μM). Indeed, the compound was
more potent than the tetravalent 5 and 6 but not much and the
inherently stronger trisaccharide ligand it contains could easily
be responsible for this difference. Gratifyingly, the more highly
substituted hPG-galabiose conjugate 9 was much more potent
with an IC50 of 8 nM and a relative potency per sugar of ca.
30,000. These data make it the first nanomolar Stx inhibitor
based on the disaccharide galabiose to the best of our
knowledge.
A number of natural or synthesized oligosaccharides were
subsequently tested for activity at a maximal nontoxic
concentration of 2% (Figure 4).35,36 Chitosan oligosaccharide
(COS) is a cationic polymer obtained from crustaceans and
consists of glucosamine repeating units and has several
promising applications.37 COS (degree of acetylation: ≥95%)
showed a 71% inhibition of the Stx1B with inhibitory effects
seen as low as a 0.5% COS concentration (Figure 5). AOS,
Figure 2. Inhibition of STx1B (0.1 μg/mL) binding to a GB3 covered
surface by compounds from left to right, 9 (blue), 8 (red), 1e (black),
and 5 (white).
Glycidol, a reactive hydroxy-epoxide, was used as an AB2
monomer, and polymerization was initiated using tris-
(hydroxymethyl)propane (TMP). TMP was partially deproto-
nated and used as an initiator for the anionic polymerization
carried out by slow monomer addition and yielding hPG-OH
of ca. 9.4 kDa with 125 OH end groups, calculated using
inverse-gated carbon and proton NMR.31 Azidation of the hPG
was performed in two steps by first substituting the hydroxy
groups of the hPG with the more reactive mesyl groups
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Figure 3. Dendrimeric- and polymeric-galabiose conjugates.
a
Scheme 2. Synthesis of Hyperbranched Polymers
a
Reagents and conditions: (i) MsCl, TEA, DMF, 0 °C−r.t., 16 h, 88%; NaN3, DMF, 60 °C, quant. (ii) NaH, KI, propargyl bromide, DMF, 0 °C−
r.t., 72%.
another naturally occurring polyuronic saccharide, is composed
of β-D-mannuronic acid and α-L-guluronic acid.38 AOS have,
among others, antitumor, antioxidative, immunoregulatory,
and anti-inflammatory activity.39 AOS did show 51% inhibition
at a concentration of 0.5%. Curiously, higher AOS concen-
trations reduced inhibition. FOS and GOS did not inhibit the
toxin. Lactose was used as a negative control and did not show
any activity.
In order to evaluate the potential toxicity of the most
effective Stx inhibitor, polymer 9, toxicity tests were under-
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a
Table 1. Results of Inhibition in the Stx1B ELISA Assay
b
c
entry
Construct
ligand
valency (% functionalization of polymer)
IC50 (μM)
rel.pot.
4968
4.6
rel. pot. per sugar
1
2
3
4
5
6
7
1e
3
4
5
6
galabiose
galabiose
galabiose
galabiose
galabiose
globotriose
galabiose
1
2
2
4
4968 1232
1070 283
1245 169
19.9 2.4
13.5 2.6
2.8 0.2
1
2.3
2
4
250
367
62.5
92
187
29,928
4
8
9
10 (8%)
20 (16%)
1,774
598,554
0.0083 0.0006
a
b
Determined in an ELISA-like assay with Stx1B (0.1 μg/mL) and wells coated with Gb3. Relative to the potency of galabiose for 3, 4, 5, 6, and 9.
c
Relative potency divided by the valency.
Figure 4. Structures of natural and synthetic inhibitors of Stx.
taken. Different concentrations of glycopolymer 9 (1, 10, and
100 nM) did not impair T84 cell viability after 24 h exposure
as indicated by the MTT assay, while 10% ethanol (positive
control) significantly reduced the cell viability (Figure 6a).
Furthermore, as depicted in Figure 6b, 1, 10, and 100 nM of
glycopolymer 9 did not significantly alter the transepithelial
electrical resistance (TEER) values compared to untreated
cells after 24 h, whereas 10% ethanol strongly decreased the
TEER values. TEER values (transepithelial electrical resist-
ance) indicate the barrier integrity of epithelial cells.
Figure 5. Stx1B binding inhibition by COS.
Figure 6. Effect of glycopolymer 9 on intestinal cell viability and integrity. (a) T84 cells grown on 96-well plates were exposed to 1, 10, and 100 nM
glycopolymer 9 or 10% ethanol (positive control) for 24 h, and cell viability was measured by the MTT reduction assay. The MTT values were
presented as the percentage MTT released by nontreated T84 cells as mean SEM of three independent experiments each performed in triplicate.
(b) T84 cells grown on transwell inserts exposed to 1, 10, and 100 nM glycopolymer 9 or 10% ethanol (positive control) for 24 h, and TEER was
measured as described in the Experimental Section. The TEER values are presented as mean (Ω·cm2) SEM of three independent experiments
each performed in triplicate. ** = P < 0.001 compared to control. **** = P < 0.0001 compared to control).
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residues 21−89 of Stx1B (GenBank: AAA98348.1) with a C-terminal
6× His tag was first inserted into an entry plasmid pENTR1A through
restriction sites Dra I and Xho I. The resultant plasmid Stx1B-His-
pENTR1A together with a destination vector pDEST 14 was further
subjected to the Gateway LR Clonase cloning reaction to achieve the
final protein expression construct Stx1B-His-pDEST14 following the
manufacturer’s instructions.
DISCUSSION AND CONCLUSIONS
■
A growing number of Shigella infections contain the deadly
Shiga toxin, and the related STEC is also still a major threat
without a proper therapeutic approach. In this study, we aimed
for a simple and effective toxin inhibitor by comparing three
classes of carbohydrate-based inhibitors: glycodendrimers,
glycopolymers, and oligosaccharides. The glycodendrimers
needed at least a tetravalent ligand to reach significant
inhibition. One reason could be that it requires bridging40
between the two highest affinity sites (sites 2) of neighboring
toxin subunits for a significant inhibitory effect. The smaller
divalent compounds were too short to bridge ca. 30 Å. It is
likely that in addition to the chelation binding mode,
aggregation of the toxin also takes place, as previously
noted,41 and also for the related cholera toxin.42,43 Of the
two glycopolymers, it is striking that the more highly
functionalized 9 was much more potent than 8, despite having
the weaker galabiose ligand. Clearly, the high density of
binding sites helps in inhibition as seen for the related cholera
toxin inhibition with similar polymers;44,45 however, with three
binding sites per subunit, that is, 15 in total, the effects are
more dramatic than those for the cholera toxin with one
binding site per subunit. Prior works, both theoretical and
practical involving the Shiga-like toxin, have clearly indicated
that the avidity effects as seen here are caused by intrinsic
inter- and intramolecular recognition events, but on top of
that, there is an important combinatorial factor that describes
the probabilities of binding events. This factor is very
important and favorable and was shown to increase rapidly
for higher valency systems, provided that the geometry of the
multivalent ligand is appropriate for the target. In the case at
hand, the particle-like nature of the polymer is particularly
suitable for toxins in comparison with other polymers.44
Furthermore, the polymers were both ca. 10 kDa, but the
ligand density is vastly different (valencies of 10 vs 20 for 8 and
9). Clearly, the statistical possibilities for higher ligand density
9 are far greater and can overcome the lower intrinsic binding
potency of the disaccharide versus trisaccharide ligand.52
Shigella spp. are highly infective bacteria. 10−100 microbes
are enough to cause infection that could become fatal,
especially when it produces the toxin, which is also the case
for STEC. The initial diarrhea followed by the toxin moving
into circulation provides a challenge for therapy. It takes ca. 5−
9 days between the initial gastroenteritis until HUS occurs.46
In this time window, a GI-based agent, for example, a food-
grade polysaccharide, such as, COS, can be beneficial. This is
true also as a preventative, in case of an outbreak which can
happen with Shigella. In order to prevent systemic diseases,
that is, HUS, a soluble nontoxic multivalent glycan with
sufficient potency will likely be helpful. As such, a further
optimized dendrimer or the glycopolymer 9 based on hPG can
be used. hPGs can be prepared on a large scale in an
economical manner and have also been used in circulation.47
The utility of hPG is also well established in terms of safety
and biocompatibility. We have also previously used a hPG
backbone to target the cholera toxin and the flu virus with
good results.44,48
Protein expression was carried out using E. coli BL21 cells
transformed with the plasmid Stx1B-His-pDEST14. The above E. coli
cells were grown in LB broth media containing 100 mg/mL ampicillin
at 37 °C until OD450 reached 0.6, and then, IPTG was added to the
culture at a final concentration of 1 mM to induce the expression of
recombinant proteins at room temperature for 16 h. At the end of
IPTG induction, E. coli cells were immediately lysed in the culture
using a B-PER direct bacterial protein extraction kit (Thermo Fisher,
Spain) with the supplementation of the protease inhibitor (EDTA-
free), DNase I, and lysozyme following the manufacturer’s
instructions. After centrifugation for 10 min at 12,000 rpm at 4 °C,
the supernatant was collected for further protein purification.
Protein purification was performed using a home-made column
packed with HisPur Ni-NTA resin (Thermo Fisher, Spain) according
to the manufacturer’s instructions. After the column was washed with
an equilibration buffer (20 mM sodium phosphate, 300 mM sodium
chloride, 10 mM imidazole, pH 7.4), it was then loaded with the
above supernatant containing 6× His tagged Stx1B at 4 °C for 1 h.
Unbound proteins were removed from the column by using a washing
buffer (20 mM sodium phosphate, 300 mM sodium chloride, 20 mM
imidazole, pH 7.4). Finally, 6× His tagged Stx1B was eluted from the
column using an elution buffer (20 mM sodium phosphate, 500 mM
sodium chloride, and 300 mM imidazole, pH 7.4) and later confirmed
by SDS-PAGE (Figure S1). Before applying for the later binding
assay, imidazole residues in the eluted proteins were removed using
10 kDa molecular weight cut off protein concentrators (Thermo
Fisher, Spain) and a buffer containing 20 mM sodium phosphate and
500 mM sodium chloride.
Shiga Toxin Inhibition Assay. A (Stx1B ELISA) 96-well plate
(Nunc PolySorp) was coated with a solution of FSL-GB3 (50 μL, 2
μg/mL) in phosphate-buffered saline (PBS) for 3 h at room
temperature. Unattached GB3 was removed by washing with PBS
(0.2% BSA), and the remaining binding sites of the surface were
blocked with BSA (1%) for 1 h, followed by washing with PBS (0.2%
BSA). Samples of Stx1B (50 μL, 0.1 μg/mL) and inhibitors were
transferred to the GB3-coated plate and incubated at r.t. for 1 h
followed by washing with PBS (0.2% BSA). HisProbe-HRP (4 mg/
mL, 1:2000 dilution, 100 μL/well) was incubated for 0.5 h followed
by washing with PBS (0.2% BSA).49 The HRP activity was measured
using the 1-Step Ultra TMB-ELISA substrate solution (100 μL/well)
for a maximum of 10 min. After quenching with H2SO4, the
absorbance in each well was measured at 450 nm. Compounds 3, 4, 5,
6, 8, and 9 were tested at least twice in duplicate or triplicate, whereas
compound 1e was tested once. Inhibition data from the experiments
were averaged and fitted in GraphPad Prism 8.3.0 with a nonfixed
Hill-slope.
Cell Culture. Human colonic epithelial T84 cells (ATCC CCL-
248) were cultured in Dulbecco’s modified Eagle’s medium: nutrient
mixture F-12 (DMEM/F-12; Gibco, Invitrogen, Carlsbad, CA, USA)
(1:1) supplemented with 10% fetal calf serum (FCS; Gibco),
penicillin (100 U/mL), and streptomycin (100 g/mL) (Biocambrex)
and maintained at 37 °C in a humidified incubator with 5% CO2. Τ84
cells were grown on plastic culture flasks (75 cm2) at a density of 3 ×
106 cells/mL. After 7 days, T84 cells were seeded on 0.3 cm2 high
pore density polyethylene terephthalate membrane transwell inserts
with 0.4 μm pores (Falcon, BD Biosciences, Franklin Lakes, NJ, USA)
placed in a 24-well plate (density of 3 × 105 cells/insert) or in 96-well
microtiter plates (Costar 3614, Corning, NY, USA) at a density of 3 ×
104 cells/well. Cells were passaged by addition of trypsin ethyl-
enediaminetetraacetic acid (EDTA) at 100% confluency every week.
The experiments were performed at passage number 51−55 on fully
confluent monolayers with TEER values >1000 Ω·cm2.
EXPERIMENTAL SECTION
■
Plasmid Construction, Protein Expression, and Protein
Purification. The Stx1B expression plasmid was constructed by
using the Gateway recombinant cloning kit (Thermo Fisher, Spain).
Briefly, a synthetic DNA cassette (Invitrogen, Spain) that encodes
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Oligosaccharides. Fructo-oligosaccharides (FOS) isolated from
chicory were obtained from Orafti (Wijchen, The Netherlands)
(purity >97%). Galacto-oligosaccharides (GOS) (Vivinal GOS
Powder, purity >70%) produced from lactose were provided by
FrieslandCampina (Amersfoort, The Netherlands). AOS prepared by
degradation of algin (purity >85%) and chitosan oligosaccharides
(COS) derived from rich marine biological sources (shrimp & crab
shells) (purity >90%) were both purchased from BZ Oligo Biotech
Co., Ltd. (Qingdao, Shandong, China). All oligosaccharide solutions
were freshly prepared through dissolution in DMEM/F12, and their
pH was adjusted to pH = 7.2−7.4.
TEER Measurement. For evaluating the epithelial integrity of the
T84 monolayer, TEER values were measured using a Millicell-ERS
Volt-Ohm-meter (Millipore, Temecular, CA, USA). As described
above, T84 cells were seeded at a density of 3 × 105 cells/insert and
cultured for 3 weeks. The inserts were placed in a 24-well plate with
300 μL of medium at the apical compartment and 700 μL of medium
at the basolateral compartment. Different concentrations of the
Glycopolymer 9 (1, 10 and 100 nM) were added to the apical
compartment of the transwell inserts. Transwell inserts of T84 cells,
treated with medium, were considered as the control group. The
TEER values were measured before and 24 h after exposure to
different concentrations of Glycopolymer 9 incubated at 37 °C in 5%
CO2. The TEER was expressed as Ω·cm2.
a Waters XBridge BEH Prep Amide column (5 μm, 250 × 10 mm) at
a flow rate of 2.4 mL/min was used. Runs were performed using a
standard protocol: 95−50% gradient buffer B in 60 min. UV-
absorption was measured at 254 and 210 nm. For commercial
oligosaccharides, AOS (food grade) was purchased from Qingdao Bz
Oligo Biotech Co. Ltd. COS (9012-76-4) with a degree of
deacetylation ≥95%. GOS was purchased from Friesland Campina
(Vivinal GOS powder, 69%). Bis-alkyne 2a29 and Tetra-alkyne 2c30
were synthesized according to the reported procedures, with the
spectral data in agreement with the reported values.
General CuAAC Procedure for the Synthesis of Multivalent
Galabiose Compounds 3, 4, 5, and 6. All tested compounds were
>95% pure by HPLC. The alkyne (2a, 2b, 2c, 2d, and 1 equiv) was
dissolved in DMF followed by the addition of the ligand 1d (1.2
equiv). Copper sulfate pentahydrate (0.1 equiv) was dissolved in
water separately and added to the reaction mixture. Sodium ascorbate
(0.3 equiv) was also dissolved in water separately and added to the
reaction mixture. The reaction was performed at 80 °C in the
microwave for 1 h. The reaction mixture was extracted using EtOAc
and water, followed by column purification (6% MeOH in DCM) to
obtain the purified compound which was further subjected to
deacetylation as described below.
General Procedure for the Deacetylation Reaction. The
peracetylated compound was dissolved in anhydrous methanol,
followed by addition of a catalytic amount of an aqueous NaOH
solution (1 M) and stirred at room temperature. The reaction was
monitored by TLC. Upon completion of the reaction, the mixture was
neutralized by the addition of Dowex marathon resin. The solvent was
evaporated, and the crude mixture was purified by preparative HPLC
to obtain the pure product (1e, 3, 4, 5, and 6) in >80% yields.
Azide 1c. Compound 1a (2.00 g, 3.13 mmol, 1 equiv), diphenyl
sulfoxide (1.2 g, 6.0 mmol, 2.6 equiv), and 2,4,6-tris-tert-
butylpyrimidine (2.232 g, 9.0 mmol, 3.0 equiv) were dissolved in
anhydrous DCM (45 mL) under an atmosphere of argon. Activated
molecular sieves (3 Å) were added. The solution was then cooled to
−40 °C, and trifluoromethanesulfonic anhydride (500 μL, 3 mmol,
1.3 equiv) was added. The mixture was stirred for 10 min, and
galactose acceptor 1b (2.261 g, 4.373 mmol, 1.4 equiv) was added as a
solution in anhydrous DCM (40 mL). The reaction was stirred for ca.
1.5 h at −40 °C and then quenched by addition of triethylamine (5
mL, excess). The mixture was diluted with DCM (100 mL) and
washed with 1 M HCl and saturated aqueous sodium bicarbonate.
The organic layer was dried with NaSO4, filtered, and concentrated in
vacuo. Purification was done by column chromatography (0−20%
EtOAc in petroleum ether) to yield the product (1.655 g, 1.58 mmol,
50%). 1H NMR (600 MHz, CDCl3): δ 8.09 (dd, J = 8.1, 1.4 Hz, 2H,
2 × CH Bz ortho), 8.04 (dd, J = 8.3, 1.3 Hz, 2H, 2× CH Bz ortho),
7.93 (dd, J = 8.2, 1.4 Hz, 2H, 2× CH Bz ortho), 7.62−7.56 (m, 1H,
CH Bz para), 7.50 (dddd, J = 8.8, 6.1, 3.0, 1.3 Hz, 2H, 2× CH Bz
para), 7.46 (t, J = 7.8 Hz, 2H, 2× CH Bz meta), 7.36 (td, J = 7.8, 3.7
Hz, 4H, 4× CH Bz meta), 5.69 (dd, J = 10.5, 8.6 Hz, 1H, H-2), 5.46
(dd, J = 10.5, 2.4 Hz, 1H, H-3), 5.02 (d, J = 3.1 Hz, 1H, H′-1), 4.83−
4.76 (m, 2H, H-1, H-6a), 4.68 (dd, J = 12.1, 7.3 Hz, 1H, H-6b), 4.41
(d, J = 2.5 Hz, 1H, H-4), 4.38 (d, J = 2.4 Hz, 1H, H′-4), 4.24−4.16
(m, 3H, H′-2, H-5, H′-6a), 4.12−4.07 (m, 2H, H′-3, H′-5), 4.05 (dd,
J = 12.7, 1.9 Hz, 1H, H′-6b), 1.03 (s, 9H, SiC(CH3)3), 1.00 (s, 9H,
SiC(CH3)3), 0.97 (s, 9H, SiC(CH3)3), 0.86 (s, 9H, SiC(CH3)3), 0.19
(s, 3H, SiCH3), 0.17 (s, 3H, SiCH3), 0.08 (s, 3H, SiCH3), 0.04 (s, 3H,
SiCH3). 13C NMR (151 MHz, CDCl3): δ 166.15 (CO, Bz), 165.82
(CO, Bz), 165.12 (CO, Bz), 133.60 (CH, Bz para), 133.36 (CH,
Bz para), 133.21 (CH, Bz para), 130.01 (2× CH, Bz ortho), 129.83
(2× CH, Bz ortho), 129.80 (2× CH, Bz ortho), 129.78 (C Bz),
128.94 (C Bz), 128.67 (C Bz), 128.57 (2× CH, Bz meta), 128.43 (2×
CH, Bz meta), 128.38 (2× CH, Bz meta), 101.45 (C′-1), 88.38 (C-
1), 75.69 (C′-5), 75.05 (C′-4), 75.04 (C-4), 73.17 (C-3), 70.94 (C′-
3), 70.04 (C′-2), 69.17 (C′-5), 68.68 (C-2), 67.02 (C′-6), 64.12 (C-
6), 27.46 (SiC(CH3)3), 27.36 (SiC(CH3)3), 26.23 (SiC(CH3)3),
26.21 (SiC(CH3)3), 23.41 (2× SiC(CH3)3), 18.36 (SiC(CH3)3),
18.22 (SiC(CH3)3), −3.91 (SiCH3), −4.23 (SiCH3), −4.35 (SiCH3),
−4.66 (SiCH3).
ViabilityMTT Assay. Cell viability was measured by MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduc-
tion assay (Sigma-Aldrich, St. Louis, Mo, USA). T84 cells were
seeded on a flat-bottomed 96-well plate at a density of 3 × 104 cells/
well and grown for 7 days until they reached 100% confluency.
Thereafter, cells were exposed to three concentrations of glycopol-
ymer 9 (1, 10, and 100 nM), and 10% ethanol was used as positive
control. The wells with medium (no treatment) were considered as
the control group. After a 24 h incubation at 37 °C in 5% CO2, the
medium was removed, and 120 μL of MTT working solution [20 μL
MTT (5 mg/mL) and 100 μL medium] was added to each well and
incubated for 2 h under the same conditions. Finally, DMSO was
added to lyse the cells and dissolve the purple-blue sediment. After 5
min of mild shaking, the absorbance value of each well was measured
at 595 nm using a Glomax Discover microplate reader. The viability of
the T84 cells was calculated based on the following equation: (mean
absorbance of treatment cells/mean absorbance of control cells) ×
100.
Statistical Analysis. Data were reported as mean values SEM
of three independent experiments (n = 3) routinely performed in
triplicate (3 wells/condition). Results were analyzed using Prism 8.0
GraphPad software (GraphPad, San Diego, CA, USA). Statistical
significance was determined using one-way ANOVA followed by
Bonferroni post-hoc test. Differences were considered statistically
significant when P < 0.05.
Chemistry. Chemicals were obtained from commercial sources
and were used without further purification unless noted otherwise.
The solvents were obtained as synthesis grade and stored on
molecular sieves (4 Å). TLC was performed on Merck-precoated
silica plates. Spots were visualized by UV light and 10% H2SO4 in
MeOH. Microwave reactions were carried out in a Biotage microwave
initiator (300W, Uppsala, Sweden). The microwave power was
limited by temperature control once the desired temperature was
1
reached. Sealed vessels of 2−5 and 10−20 mL were used. H NMR,
HSQC, COSY (600 MHz), and 13C (151 MHz) were performed on a
Bruker 600 spectrometer. Infrared (IR) spectroscopy was performed
using a universal attenuated total reflectance (UATR) accessory of a
PerkinElmer Spectrum Two FT-IR spectrometer. High-resolution
mass spectrometry analysis was recorded using an Agilent 6560 Ion
Mobility Q-TOF LC/MS instrument. Analytical HPLC and
Preparative HPLC runs were performed on a Shimadzu 20A HPLC
system. Analytical HPLC was performed using a Dr Maisch GmBh
C18-AQ column (5 μm) at a flow rate of 0.5 mL/min. The used
buffers were H2O (buffer A) and CH3CN (buffer B). Runs were
performed using a standard protocol: 2−100% gradient buffer B in 35
min; UV-absorption was measured at 254 nm. For Preparative HPLC,
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Azide 1d. Compound 1c (1655 mg, 1.58 mmol 1 equiv) was solved
in MeOH (30 mL), and an excess of K2CO3 was added. The reaction
was stirred at r.t. for 16 h and then filtered and concentrated in vacuo.
The crude product was dissolved in EtOAc and extracted with
aqueous NaHCO3, and the organic layer was separated, dried with
NaSO4, filtered, and concentrated again in vacuo. The product was
purified using column chromatography (10−50% EtOAc in petroleum
ether) to yield the product (1034 mg, 1.41 mmol, 89%).The
debenzoylated product (332 mg, 0.452 mmol, 1 equiv) was dissolved
in DCM (5 mL), and HF-Pyridine (70%, 0.4 mL) was added
dropwise at r.t. under continuous argon flow. The reaction was stirred
for 2 h and quenched by addition of solid CaCl2 (99 mg, 0.904 mmol,
2 equiv), pyridine (10 mL, 124 mmol, 272 equiv), Ac2O (5 mL, 53
mmol, 117 equiv), and DMAP (3 mg, 0.02 mmol, 0.05 equiv). The
reaction was stirred for 16 h at r.t. and then diluted with EtOAc and
washed with saturated, aqueous K2CO3. The organic phase was dried
with NaSO4, filtered, and concentrated in vacuo. The product was
purified using silica gel flash chromatography using a gradient of 40−
100% EtOAc in petroleum ether (154 mg, 0.232 mmol, 51% (over 3
steps)). The spectral data was in accordance with published data.50
1H NMR (600 MHz, CDCl3): δ 5.53 (dd, J = 3.4, 1.3 Hz, 1H, H′-4),
5.33 (dd, J = 11.0, 3.4 Hz, 1H, H′-3), 5.16 (dd, J = 11.0, 3.7 Hz, 1H,
H′-2), 5.12 (dd, J = 10.7, 8.6 Hz, 1H, H-2), 4.99 (d, J = 3.7 Hz, 1H,
H′-1), 4.83 (dd, J = 10.7, 2.7 Hz, 1H, H-3), 4.63 (d, J = 8.6 Hz, 1H,
H-1), 4.45 (ddd, J = 7.6, 5.8, 1.3 Hz, 1H, H′-5), 4.40 (dd, J = 11.4, 6.9
Hz, 1H, H-6a), 4.12 (dd, J = 11.3, 6.1 Hz, 1H, H-6b), 4.10−4.04 (m,
3H, H′6ab; H-4), 3.87 (t, J = 6.5 Hz, 1H, H-5), 2.09 (s, 3H, CH3 Ac),
2.07 (s, 3H, CH3 Ac), 2.05 (s, 3H, CH3 Ac), 2.04 (s, 3H, CH3 Ac),
2.04 (s, 3H, CH3 Ac), 2.00 (s, 3H, CH3 Ac), 1.95 (s, 3H, CH3 Ac).
13C NMR (151 MHz, CDCl3): δ 170.45 (CO, Ac), 170.37 (CO,
Ac), 170.32 (CO, Ac), 170.27 (CO, Ac), 169.98 (CO, Ac),
169.66 (CO, Ac), 168.90 (CO, Ac), 98.97 (C′-1), 88.11 (C-1),
76.27 (C-4), 74.03 (C-5), 72.40 (C-3), 68.30 (C′-2), 67.88 (C-2),
67.67 (C′-4), 67.18 (C′-5), 67.07 (C′-3), 61.81 (C-6), 60.42 (C′-6),
20.76 (CH3, Ac), 20.62 (CH3, Ac), 20.58 (CH3, Ac), 20.54 (CH3,
Ac), 20.51 (CH3, Ac), 20.47 (CH3, Ac).
Compound 2d. To a solution of 2a (16.4 mg, 82 μmol) in DCM
(1 mL) was added (COCl)2 (31 μL, 246 μL, 3 equiv) and DMF (10
μL). After stirring at r.t. for 1.5 h, the mixture was concentrated. The
resulting residue was coevaporated with 10 mL of anhydrous toluene
and then redissolved in DCM (1 mL) and cooled to 0 °C. A solution
of pyridine (1 mL), DCM (1 mg), and dodecane-1,12-diamine (10
mg) was added slowly to the reaction flask. The resulting mixture was
stirred at r.t. overnight. Solvents were removed, and the residue was
partitioned between EtOAc and water. The organic layer was
separated, washed with brine (1×), dried with anhydrous MgSO4,
filtered, and concentrated. The residue was purified by silica gel flash
chromatography and yielded a product (20 mg, 80%). 1H NMR (600
MHz, acetone-d6): δ 7.57 (t, J = 5.9 Hz, 2H, NH), 7.02 (s, 4H, 4×
CH arom), 6.64 (s, 2H, 2× CH arom), 4.71−4.67 (m, 8H, (4×
OCH2), 3.24 (q, J = 6.7 Hz, 4H, 4× CH propargyl), 2.97 (s, 4H, 2×
NHCH2), 1.92 (s, 4H, 2× CH2), 1.46 (t, J = 7.1 Hz, 4H, 2× CH2),
1.28−1.02 (m, 12H, 6× CH2). 13C NMR (151 MHz, acetone-d6): δ
165.64 (2× CO), 158.77 (4× CO, arom), 137.57 (2× C-CONH),
106.68 (4× CH), 104.54 (2× CH), 78.54 (4× CCH, alkyne), 76.38
(4× CCH, alkyne), 55.70 (4× OCH2), 39.64 (2× N-CH2), 29.32
(CH2), 29.30 (CH2), 29.17 (CH2), 29.15 (CH2), 29.04 (CH2), 29.01
(CH2), 28.90 (CH2), 28.77(CH2), 28.64 (CH2). HR-ESI-TOF/MS
(m/z): [M + H]+ calcd for C38H44N2O6, 625.3277; found, 625.3302.
Compound 3. 1H NMR (600 MHz, Deuterium Oxide): δ 8.23 (s,
2H, triazole), 7.07 (dd, J = 2.3, 0.9 Hz, 2H, 2× CHarom-2,6)), 6.68
(td, J = 2.3, 0.8 Hz, 1H, 1× CHarom-4), 5.58 (dd, J = 9.0, 0.8 Hz, 2H,
2× H-1), 5.18 (s, 4H, 2× OCH2), 4.90 (d, J = 4.0 Hz, 2H, 2× H′-1),
4.29 (t, J = 6.4 Hz, 2H, 2× H-5), 4.20 (t, J = 9.5 Hz, 2H, 2× H-2),
4.07 (d, J = 3.2 Hz, 2H, 2× H′-5), 3.96−3.68 (m, 14H, 2× H′-4, 2×
H′-2, 2× H′-6, 2× H-4, 2× H′-3, 2× H-3), 3.64−3.55 (m, 4H, 2× H-
6).
13C NMR (151 MHz, deuterium oxide): δ 174.24 (-COOH),
158.33 (2× Carom-3), 143.27 (2× −OCH2-C), 139.24 (COOH−C),
125.28 (2× N−CH), 108.97 (2× CHarom-2,6)), 105.72 (1×
CHarom-4), 100.48 (2× C′-1), 88.10 (2× C-1), 78.28 (2× C′-5),
77.24 (2× C-4), 72.69 (2× C′-3), 70.90 (2× C-5), 69.61 (2× C-3),
69.05 (2× C′-2), 68.98 (2× C′-4), 68.63 (2× C-2), 61.39 (2×
OCH2), 60.54 (2× C′-6), 60.06 (2× C-6). HR-ESI-TOF/MS (m/z):
[M + Na]+ calcd for C37H52N6O24, 987.2930; found, 987.2934.
Azide 1e. Compound 1d was deprotected using the general
procedure described above to obtain the final compound 1e in 90%
yield. The spectral data was in accordance with published data.51 1H
NMR (600 MHz, MeOD): δ 4.94 (d, J = 3.7 Hz, 1H, H′-1), 4.52 (d, J
= 8.4 Hz, 1H, H-1), 4.18 (t, J = 6.2 Hz, 1H, H-5), 3.99 (d, J = 3.0 Hz,
1H, H-4), 3.88 (d, J = 3.2 Hz, 1H, H′-4), 3.85−3.71 (m, 6H, H′-2,
H′-3, H′-5, H′-6ab, H-6a), 3.65 (dd, J = 11.2, 5.1 Hz, 1H, H-6b), 3.52
(dd, J = 10.0, 2.9 Hz, 1H, H-3), 3.41 (dd, J = 10.3, 1.9 Hz, 1H, H-2).
13C NMR (151 MHz, MeOD): δ 101.43 (C′-1), 91.39 (C-1), 78.37
(C-5), 76.70 (C-4), 73.43 (C-3), 71.62 (C′-5), 71.02 (C-2), 69.83
(C′-4), 69.66 (C′-2), 69.20 (C′-3), 61.30 (C-6), 59.70 (C′-6).
Compound 2b. Methyl 3,5-bis(2-(boc-amino)ethoxy)benzoate
(110 mg, 0.25 mmol, 1 equiv) was prepared as reported30 and
dissolved in 1:1 TFA/DCM and stirred at r.t. for 2 h before
concentrating in vacuo. The residue was dissolved in DCM (10 mL),
and TEA (139 μL, 1 mmol, 4 equiv) was added, and the mixture was
left stirring for 5 min at r.t. before cooling to 0 °C. Propargyl
chloroformate (55 μL, 0.55 mmol, 2.2 equiv) was added dropwise,
and the reaction was allowed to slowly warm up to r.t. and was left
stirring for 16 h. The reaction was diluted with an excess of DCM and
1 M aq HCl, and the organic layer was collected, dried with NaSO4,
filtrated, and concentrated in vacuo. The compound was purified by
column chromatography using a gradient of 0−50% EtOAc in
petroleum ether and with 1% TFA yielding the free acid (89 mg, 0.22
1
Compound 4. H NMR (600 MHz, deuterium oxide): δ 8.23 (s,
2H, triazole), 7.07 (s, 2H, 2× CHarom-2,6)), 6.66 (s, 1H, 1×
CHarom-4), 5.66 (d, J = 9.0 Hz, 2H, 2× H-1), 5.20 (d, J = 2.4 Hz,
4H, 2× NH−CO−CH2), 5.03 (d, J = 4.0 Hz, 2H, 2× H′-1), 4.41 (t, J
= 6.5 Hz, 2H, 2× H-5), 4.31 (t, J = 9.6 Hz, 2H 2× H-2), 4.20 (d, J =
3.0 Hz, 2H, 2× H-3), 4.13 (t, J = 5.1 Hz, 4H, 2× CONH−CH2−
CH2), 4.07−3.84 (m, 14H, 2× H′-4, 2× H′-3, 2× H′-2, 2× H′-5, 2×
H-4, 2× H-6), 3.78−3.68 (m, 4H, 2× H′-6), 3.52 (t, J = 5.2 Hz, 4H,
2× CONH-CH2-CH2). 13C NMR (151 MHz, deuterium oxide): δ
157.89 (2× Carom3,5), 125.11 (2× N−CH), 108.29 (2× CHarom-
2,6)), 100.47 (2× C′-1), 88.08 (2× C-1), 78.23 (2× C′-5), 77.20 (2×
C-4), 72.68 (2× C′-3), 70.89 (2× C-5), 69.59 (2× C-3), 69.06 (2×
C′-2), 68.97 (2× C′-4), 68.62 (2× C-2), 67.18 (2× OCH2), 60.53
(2× C′-6), 60.00 (2× C-6), 57.63 (2× NHCO−O−CH2), 40.09 (2×
OCH2−CH2).
HR-ESI-TOF/MS (m/z): [M + Na]+ calcd for C43H62N8O28,
1161.3571; found, 1161.3574.
1
Compound 5. H NMR (600 MHz, deuterium oxide): δ 8.26 (s,
4H, triazole), 7.06 (s, J = 1.9 Hz, 2H, 2× CHarom-2,6), 6.70 (s, 1H,
1× CHarom-4), 6.62 (d, J = 2.2 Hz, 4H, 4× CHarom-2′,6′), 6.44 (s,
2H, 2× CHarom-4′), 5.64 (d, J = 9.0 Hz, 4H, 4× H-1), 5.00 (d, J =
4.0 Hz, 4H, 4× H′-1), 4.95 (s, 8H, 4× triazole-CH2−), 4.35 (t, J = 6.4
Hz, 4H, 4× H-5), 4.29 (t, J = 9.6 Hz, 4H, 4× H-2), 4.17 (d, J = 3.2
Hz, 8H, 4× H-4, 2× CONH−CH2−CH2−), 4.00 (t, J = 6.3 Hz, 4H,
4× H′-5), 3.97−3.88 (m, 12H, 4× H-3, 4× H′-3, 4× H′4), 3.88−3.78
(m, 12H, 4× H′-6, 4× H′-2), 3.68 (d, J = 6.4 Hz, 8H, 4× H-6), 3.64
(s, 4H, 2× CONH−CH2−CH2−). 13C NMR (151 MHz, deuterium
oxide, extracted from HSQC): δ 125.03 (triazole), 108.89 (2×
CHarom-2,6, 104.45 1× CHarom-4), 106.57 (4× CHarom-2′,6′),
105.32 (2× CHarom-4′), 88.11(C-1), 100.53 (C′-1), 61.07 (triazole-
CH2-), 70.93 (C-5), 69.65 (C-2), 77.35, 66.73, 78.13 (C′-5), 68.94,
1
mmol, 88%). H NMR (600 MHz, methanol-d4): δ 7.20 (d, J = 2.4
Hz, 2H, 2× CHarom-2,6), 6.79 (d, J = 2.5 Hz, 1× CHarom-4), 4.68
(d, J = 2.4 Hz, 4H, 2× CH2, propargyl), 4.07 (t, J = 5.5 Hz, 4H, 2×
OCH2), 3.41 (t, J = 5.5 Hz, 4H, 2× NCH2), 2.89 (t, J = 2.5 Hz, 2H,
2× CCH). 13C NMR (151 MHz, Methanol-d4): δ 169.61 (CO,
acid), 161.30 (CO, aromatic), 158.13 (2× CO, carbamate), 134.17
(Carom-COOH), 109.32 (2× CHarom-2,6), 107.26 (CHarom-4),
79.44 (2× CCH), 75.80 (2× CCH), 68.11 (2× OCH2), 53.20
(2× CH2, propargyl), 41.43 (2× NCH2). HR-ESI-TOF/MS (m/z):
[M + Na]+ calcd for C19H20N2O8, 427.1117; found, 427.1116.
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72.72, 59.97, 68.70, 60.52 (C-6), 39.84 (CONH−CH2−CH2−). HR-
ESI-TOF/MS (m/z): [M − H]− calcd for C85H116N14O50,
2132.6964; found, 2132.6869.
Authors
Diksha Haksar − Department of Chemical Biology & Drug
Discovery, Utrecht Institute for Pharmaceutical Sciences,
Utrecht University, 3584 CG Utrecht, The Netherlands
Mostafa Asadpoor − Division of Pharmacology, Utrecht
Institute for Pharmaceutical Sciences, Utrecht University,
3584 CG Utrecht, The Netherlands
Torben Heise − Department of Chemical Biology & Drug
Discovery, Utrecht Institute for Pharmaceutical Sciences,
Utrecht University, 3584 CG Utrecht, The Netherlands
Jie Shi − Diseases of the Developing World (DDW), Global
Health R&D, GlaxoSmithKline, 28760 Madrid, Spain
Saskia Braber − Division of Pharmacology, Utrecht Institute
for Pharmaceutical Sciences, Utrecht University, 3584 CG
Utrecht, The Netherlands
Gert Folkerts − Division of Pharmacology, Utrecht Institute
for Pharmaceutical Sciences, Utrecht University, 3584 CG
Utrecht, The Netherlands
Lluis Ballell − Diseases of the Developing World (DDW),
Global Health R&D, GlaxoSmithKline, 28760 Madrid,
Spain
1
Compound 6. H NMR (600 MHz, deuterium oxide): δ 8.18 (s,
4H, 4H, triazole), 6.92 (s, 4H, 4× CHarom-2′,6′), 6.56 (s, 2H, 2× C
CHarom-4′), 5.54 (s, 4H, 4× H-1), 4.98 (s, 6H, 4× H′-1), 4.76 (s,
8H, 4× triazole-CH2−), 4.32 (s, 4H, 4× H-5), 4.25 (s, 4H, 4× H-2),
4.12 (s, 4H, 4× H-4), 4.01−3.49 (m, 32H, 4× H′-2, 4× H′-3, 4× H′-
4, 4× H′-5, 4× H′-6, 4× H-3, 4× H-6), 3.19 (s, 4H, 2× CONH−
CH2−), 1.42 (s, 4H, 2× CONH−CH2−CH2−), 1.05 (s, 16H,
CONH-(CH2)12). HR-ESI-TOF/MS (m/z): [M − H]− calcd for
C86H128N14O46, 2092.8107; found, 2092.8028.
Compound 8. hPG-azide (2.5 mg, 0.002 mmol of azide groups)
was dissolved in water followed by the addition of ligand 7 (1.8 mg,
0.0032 mmol, 1.6 equiv). Copper sulfate pentahydrate (0.1 equiv) was
dissolved in water separately and added to the reaction mixture. 0.3
equiv of sodium ascorbate was also dissolved in water separately and
added to the reaction mixture. The reaction was carried out at 100 °C
in the microwave for 1 h. Cuprisorb resin was added to the reaction
mixture and stirred to adsorb excess copper. The solvent was
evaporated, and the crude reaction mixture was purified by dialysis
using a cellulose based dialysis cassette (MWCO: 2k) against
deionized water for 3−4 days and freeze dried to get 8 in 80%
yield as an off-white solid. The disappearance of the azide stretching
peak in the IR spectra of the final compound confirmed that all of the
azido polymer were consumed. 1H NMR (600 MHz, deuterium
oxide): δ 8.03 (s, triazole), 5.18−3.16 (m, CH2 and CH, hPG-OH
backbone; GB3), 2.31−2.18 (m, GB3; CH3), 0.85 (s, hPG core, CH2).
Compound 9. hPG-propargyl (5 mg, 0.0095 mmol of propargyl
groups) was dissolved in water followed by the addition of 1d (8.2
mg, 0.012 mmol, 1.3 equiv) which was dissolved in DMF. Copper
sulfate pentahydrate (0.1 equiv) was dissolved in water separately and
added to the reaction mixture. 0.3 equiv of sodium ascorbate was also
dissolved in water separately and added to the reaction mixture. The
reaction was carried out at 80 °C in the microwave for 60 min.
Cuprisorb resin was added to the reaction mixture and stirred to
adsorb excess copper. The crude mixture was extracted using ethyl
acetate and water. The protected polymer conjugate was then
subjected to deacetylation using the standard procedure described
above. The solvent was evaporated, and the crude reaction mixture
was purified by dialysis using a cellulose-based dialysis cassette
(MWCO: 2k) against deionized water for 3−4 days and freeze-dried.
The final product 9 was obtained in 75% yield as a white solid. The
disappearance of the CCH stretching peak in the IR spectra of the
Janneth Rodrigues − Diseases of the Developing World
(DDW), Global Health R&D, GlaxoSmithKline, 28760
Madrid, Spain
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00152
Author Contributions
The manuscript was written through contributions of all
authors. All authors approved of the final version of the
manuscript
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the Tres Cantos Open Lab Foundation for
funding this project (TC-232), as well as for the participation
of GSK and making the Open Lab available.
1
final compound confirmed that all the polymer was consumed. H
NMR (600 MHz, deuterium oxide): δ 8.30 (s, triazole), 5.69 (d, J =
9.2 Hz, galabiose; H-1), 5.00 (d, J = 3.9 Hz, galabiose; H′-1), 4.51−
3.30 (m, CH2 and CH, hPG-OH backbone; galabiose; H-2, H-3, H-4,
H-5, H-6, H′-2, H′-3, H′-4, H′-5, H′-6), 1.25 (s, CH2 core), 0.84 (s,
CH3 core).
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ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00152.
General information; Stx1B inhibition curves; synthesis;
NMR spectra; IR spectra; and analytical HPLC traces
(PDF)
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AUTHOR INFORMATION
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Corresponding Author
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̈
Roland J. Pieters − Department of Chemical Biology & Drug
Discovery, Utrecht Institute for Pharmaceutical Sciences,
Utrecht University, 3584 CG Utrecht, The Netherlands;
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microbiology. Appl. Environ. Microbiol. 2012, 78, 4065−4073.
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