Potential Dental Biofilm Inhibitors: Dynamic Combinatorial Chemistry Affords Sugar-Based Molecules that Target Bacterial Glucosyltransferase

Article abstract of DOI:10.1002/cmdc.202000222

We applied dynamic combinatorial chemistry (DCC) to find novel ligands of the bacterial virulence factor glucosyltransferase (GTF) 180. GTFs are the major producers of extracellular polysaccharides, which are important factors in the initiation and development of cariogenic dental biofilms. Following a structure-based strategy, we designed a series of 36 glucose- and maltose-based acylhydrazones as substrate mimics. Synthesis of the required mono- and disaccharide-based aldehydes set the stage for DCC experiments. Analysis of the dynamic combinatorial libraries (DCLs) by UPLC-MS revealed major amplification of four compounds in the presence of GTF180. Moreover, we found that derivatives of the glucose-acceptor maltose at the C1-hydroxy group act as glucose-donors and are cleaved by GTF180. The synthesized hits display medium to low binding affinity (K_D values of 0.4–10.0 mm) according to surface plasmon resonance. In addition, they were investigated for inhibitory activity in GTF-activity assays. The early-stage DCC study reveals that careful design of DCLs opens up easy access to a broad class of novel compounds that can be developed further as potential inhibitors.

Full text of DOI:10.1002/cmdc.202000222

Accepted Article
Title: Dynamic Combinatorial Chemistry Affords Sugar-Based
Molecules Targeting Bacterial Glucosyltranseferase as Potential
Dental Biofilm Inhibitors
Authors: Alwin M. Hartman, Varsha R. Jumde, Walid A. M. Elgaher,
Evelien M. te Poele, Lubbert Dijkhuizen, and Anna Katharina
Herta Hirsch
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000222
Link to VoR: https://doi.org/10.1002/cmdc.202000222
01/2020

10.1002/cmdc.202000222
ChemMedChem
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Dynamic Combinatorial Chemistry Affords Sugar-Based
Molecules Targeting Bacterial Glucosyltranseferase as Potential
Dental Biofilm Inhibitors
Alwin M. Hartman+,[a,b] Varsha R. Jumde+,[a] Walid A. M. Elgaher+,[a] Evelien M. te Poele,[c,d] Lubbert
Dijkhuizen,[c,d] and Anna K. H. Hirsch*[a,b]
[a]
Dr. A. M. Hartman, Dr. V. R. Jumde, Dr. W. A. M. Elgaher, Prof. Dr. A. K. H. Hirsch
Department of Drug Design and Optimization
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) – Helmholtz Centre for Infection Research (HZI)
Campus Building E8.1, 66123 Saarbrücken, Germany
E-mail: Anna.Hirsch@helmholtz-hips.de
[b]
[c]
Department of Pharmacy, Saarland University, Campus Building E8.1, 66123 Saarbrücken, Germany
Dr. E. M te Poele, Prof. Dr. L. Dijkhuizen
Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB)
University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands
CarbExplore Research BV, Zernikepark 12, 9747 AN Groningen, The Netherlands
These authors contributed equally
[d]
[+]
Supporting information for this article is given via a link at the end of the document.
Abstract: We applied dynamic combinatorial chemistry (DCC) to find
novel ligands of the bacterial virulence factor glucosyltransferase
(GTF) 180. GTFs are the major producers of extracellular
polysaccharides, which are important factors in the initiation and
development of cariogenic dental biofilms. Following a structure-
based strategy, we designed a series of 36 glucose- and maltose-
based acylhydrazones as substrate mimics. Synthesis of the required
mono- and disaccharide-based aldehydes set the stage for DCC
experiments. Analysis of the dynamic combinatorial libraries (DCLs)
via UPLC-MS revealed major amplification of four compounds in the
presence of GTF180. Moreover, we found that derivatives of the
glucose-acceptor maltose at the C1-hydroxyl group are acting as
glucose-donors and are cleaved by GTF180. The synthesized hits
display medium to low binding affinity (KD values of 0.4–10.0 mM)
according to surface plasmon resonance (SPR). In addition, they were
investigated for the inhibitory activity using the GTF-activity assays.
The early stage DCC study reveals that careful design of DCLs opens
up easy access to a broad class of novel compounds that can be
developed further as potential inhibitors.
of Lactobacillus reuteri 180 were previously reported and
provided evidence for an α-retaining double displacement
mechanism using one nucleophilic residue.[5] Briefly, the α(1→2)-
glycosidic linkage of a donor substrate (e.g., sucrose) is cleaved,
resulting in the release of fructose and formation of a β-glucosyl–
enzyme intermediate. Subsequently, an acceptor substrate
attacks the β-glucosyl–enzyme intermediate, and the glucosyl
moiety is transferred to the acceptor (e.g., a glucan chain, maltose
or water molecule) with restoration of the α-anomeric
configuration (Figure 1).
Introduction
Cariogenic dental biofilm, also known as dental plaque, is a
causative agent for dental caries. An important factor for the
initiation and development of this oral disease is the fermentation
of dietary carbohydrates, of which sucrose is considered the most
cariogenic. It acts as a substrate for the synthesis of extracellular
(EPS) and intracellular (IPS) polysaccharides, which are involved
in the formation of the biofilm, having α-glucan as one of the main
components. The biofilm hosts bacteria and can promote their
adhesion to the tooth enamel. Glucosyltransferases (GTFs) are
the major producers of EPS, and are secreted by different strains
of bacteria. These GTFs, also known as glucansucrases (GSs),
therefore are potential targets in order to inhibit biofilm formation
and therefore prevent dental caries.[1–3]
Figure 1. Reaction scheme of the proposed catalytic mechanism of GTF180 via
α-retaining double displacement, leading to retention of the α-configuration.[5]
Reportedly, glucansucrases can be inhibited by natural as well as
synthetic compounds. Natural inhibitors can for example be found
in culture broths of bacteria, as was the case for acarbose (Table
1). In 1977, researchers from Bayer discovered α-amylase
inhibitors from broths of Actinoplanes strains SE 50, SE 82 and
SB 18, of which BAY g 5421 (acarbose) was the most potent.
They postulated that acarbose could be a transition-state
analogue.[6] Since then, acarbose has been used as an
antidiabetic drug throughout the world, and was found to have
cardiovascular benefits.[7–9] Newbrun et al. showed that acarbose
also inhibits GSs,[10] and its mode of action was confirmed by
Glucansucrases are enzymes which are part of the glycoside
hydrolase family GH70, consisting of four catalytically essential
conserved sequences. To the superfamily of GH-H also belong
the glycoside hydrolase families 13 and 77.[4] Cocrystal structures,
containing the catalytic and C-terminal domains of glucansucrase
1
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Figure 2. Schematic illustration of target-directed dynamic combinatorial chemistry (tdDCC) using the acylhydrazone linkage, formed by the reversible reaction
between a glucose- or maltose-linked arylaldehyde and three representative hydrazides. Binding to the target protein causes a change in equilibrium, which, in turn,
leads to the amplification of the hit compounds.
cocrystal structure.[11] Compounds from plant sources such as
polyphenols in green tea extracts,[12] theaflavins in black tea
extracts,[13] curcumin[14] and oxyresveratrol[15] showed marked
inhibitory effects on the biofilm of the cariogenic pathogen
Streptococcus mutans, however, high concentrations and
sufficient time were required. Their antibiofilm activities were
mainly exerted through direct inhibition of the bacterial GTFs or
down-regulation of GTF expression.[12–15] On the other hand,
small molecule GTF inhibitors can be categorized into only two
groups, hydroxychalcones and compounds with a high number of
heteroatoms, especially nitrogen.[16] In view of this limited number
of inhibitors, we were interested in discovering a novel chemical
class that addresses the promising yet underexploited antibiofilm
target GTF180.
To identify new ligands of a protein, dynamic combinatorial
chemistry (DCC) has become an attractive strategy. DCC allows
a target protein to alter the equilibrium of a mixture of products,
also known as dynamic combinatorial library (DCL). Due to the
change in equilibrium in presence of a protein, good binders get
amplified and will therefore be selected as hits (Figure 2). The
conditions, reactions, protocols, analysis and applications of DCC
were reviewed before.[17–20] For example, Lehn et al identified
inhibitors of the plant lectin concanavalin A using a carbohydrate
DCL and dynamic deconvolution.[21] The group of Beau probed
DCC to discover binders for the glycosidase hen egg-white
lysozyme (HEWL).[22] Ernst and coworkers used DCC to identify
submicromolar binders of the bacterial adhesin FimH via
adjusting the ratio of building blocks and establishing a protein-
capturing protocol.[23] Furthermore, we demonstrated that DCC
can be applied to challenging targets involved in protein–protein
interactions by discovering stabilizers of 14-3-3(ζ)–synaptopodin
complex.[24] In this work, we exploited the power of DCC to identify
new inhibitors of the sugar-modifying enzyme GS using the well-
established acylhydrazone formation as a reversible reaction.
Mono- or disaccharide motifs as substrate mimics constitute the
building blocks of four DCLs.
such as sucrose binds at the catalytic site –1 with its glycosidic
linkage in near proximity to the catalytic triad Asp1025, Glu1063,
and Asp1136 (highlighted in orange in Figure 3A), and is cleaved
by GS. On the other hand, the acceptor substrate, which accepts
a glucose molecule such as maltose, binds at subsites +1 and +2
(Figure 3A).
Inspired by these substrates, we designed the basic scaffold of
acylhydrazone using glucose and maltose linked to benzaldehyde
at para position (A1 and A2, respectively, Scheme 1), which
would be elongated by a hydrophobic hydrazide moiety for full
occupation of the active site. We excluded sucrose (a glucose-
donor) due to its liability to cleavage by GTFs and opted for
glucose instead. On the other hand, p-hydroxybenzaldehyde was
chosen for glycosylation as it fits perfectly into subsite +2
permitting π–π interactions with Trp1065 (Figure 3A). Moreover,
the para position holds the optimum distance between the sugar
moiety and the hydrazide, mimicking the α(1→4) glycosidic
connection of α-glucans. Subsequently, we selected 18
chemically different and commercially available hydrazides (H1–
H18) that were divided into two groups (Hydrazides I and II,
Scheme 1). Each aldehyde (A1 or A2) was allowed to react
separately with both sets of hydrazides (I and II), resulting in four
series of substrate mimetics (DCLs 1–4, Scheme 1).
Docking of all possible 36 acylhydrazones into GTF180-ΔN active
site indicated a favorable binding with high-energy scores (–7.2 to
–10.4 kcal mol-1) similar to that of acarbose (Table S1).
Compound A2H2 for instance, is anchored to subsite –1 through
the maltose moiety, establishing six H-bonds with the catalytic
residues Asp 1025 and Glu 1063 as well as Arg 1023, His 1135,
Asp 1458, and Gln 1509. The aromatic ring of the aldehyde part
is located at subsite +2 and involved in a pi–pi interaction with Trp
1065. An additional hydrogen bond is formed between C=O of the
acylhydrazone linker as H-acceptor and NH2 of Arg 1088 as H-
donor. The piperidine and tert-butyl motifs of the hydrazide in part
occupy the distal region of the active site through hydrophobic
interactions with Met 1092 and Asn 1138 and are partially
exposed to the solvent (Figure 3). Worth mentioning, the maltose
moiety of the A2-derived acylhydrazones is forced into a different
binding site (subsites –1 and +1) and a new binding mode
different to that of free maltose. These modifications could affect
the binding energy and recognition by the enzyme as an acceptor
substrate.
Results and Discussion
Designing of Dynamic Combinatorial Library
We adopted structure-based design using the co-crystal
structures of Lactobacillus reuteri 180 GTF180-ΔN with two
disaccharides, namely sucrose as donor substrate (PDB: 3HZ3)
and maltose as an acceptor substrate (PDB: 3KLL).[5] The active
site of GS is relatively wide and it can be divided into subsites –1
(the catalytic pocket), +1, and +2 (Figure 3A). The donor substrate
Synthesis of Glucose and Maltose-Based Building Blocks
The aldehydes A1 and A2 were synthesized according to the
routes shown in Scheme 2. α-D-Glucose penta-acetate (1) was
reacted with a solution of HBr in acetic acid, resulting in 1-bromo
glucose tetra-acetate (2). The crude product 2 was coupled to p-
hydroxybenzaldehyde using silver (I) oxide, yielding compound
2
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Figure 3. (A) Putative binding mode of A2H2 (yellow) compared to maltose (cyan) in the GTF180-ΔN active site using Molecular Operating Environment (MOE):
hydrophobic surface (green), polar surface (magenta), exposed (red). The maltose moiety of A2H2 occupies the catalytic subsite –1 with a glyosidic bond accessible
to the catalytic residues Asp1025, Glu1063 and Asp1136 (orange); (B) 2D ligand interactions.
Scheme 1. Structures of mono- and disaccharide-based aldehydes (A1 and A2) and hydrazides (I and II) used in the DCC experiments. Each aldehyde was reacted
separately with the two hydrazide libraries resulting in the formation of four dynamic combinatorial libraries (DCLs 1–4).
3
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Table 1. Structures, amplification factors, binding affinities and maximum responses of the hits from DCLs 1–4, GTF substrate and cleavage compounds.
Structure
DCL
Compound
Amplification fold[a]
KD (mM)[b]
Rmax (RU)[c]
R1
H
R2
DCL1
A1H2[d]
1.8
1.6 ± 0.4
5 ± 1
DCL2
DCL3
A2H6
1.5
2.1
3.2
n.d.
n.d.
A1H12
A2H12
H
0.4 ± 0.1
10 ± 2
11 ± 2
DCL4
DCL2
140 ± 30
A2H2
(GTF substrate)
-
-
5 ± 1
8 ± 1
20 ± 2
34 ± 5
A1H8
(cleavage product
of A2H8)
DCL2
H
Acarbose (positive control)
-
0.18 ± 0.01
4 ± 1
[a] Calculated as (%P/%B), where %P and %B are the relative peak areas of the compound in the UV-chromatograms of the protein-templated reaction and blank
reaction, respectively; [b] KD: equilibrium dissociation constant determined by SPR; [c] Rmax: maximum analyte binding capacity; [d] Compound A1H2 was also
observed in DCL2 as a cleavage product of A2H2.
3.[25] Deprotection of the acetate groups by sodium methoxide
using the classical Zemplen deacetylation[26] gave aldehyde A1 in
a quantitative yield. The same route was used for the synthesis of
A2, however, the starting material β-maltose first had to be
acetylated[27] (Scheme 2).
DCL Formation
Each library consisted of one aldehyde (300 µM), one group of
hydrazides (300 µM each), aniline (10 mM) and DMSO (10% v/v)
in sodium acetate buffer (pH 5.2). Aniline enhances the rate at
which the acylhydrazone formation reaches equilibrium, as it
serves as nucleophilic catalyst to form Schiff bases with the
corresponding aldehydes.[28] The use of 10% DMSO as a
cosolvent was feasible thanks to the stability of GTFs at a DMSO
concentration of up to 20%.[29] It assures the solubility of building
blocks and products, preventing any undesired shift in equilibrium
due to precipitation. A desired shift in the equilibrium, also known
as the template effect, was achieved by the addition of the target
protein GTF180 (30 µM). The protein was added following a pre-
equilibrated approach, i.e., after an equilibrium was reached in the
blank library (3 h for these building blocks). A blank reaction (DCL
without protein) was prepared in parallel for monitoring the
amplification.
Scheme 2. Synthetic route towards aldehydes A1 and A2. Reagents and
conditions: (a) Ac2O, HBr 33% in AcOH, 0˚C – rt, 15 h, 70% (2) and 52% (6)
over two steps; (b) Ag2O, MeCN, rt, overnight 40% (3) and 62% (7); (c) NaOMe,
MeOH, Amberlite H+ resin, quantitative (A1) and 70% (A2); (d) Ac2O, HClO4,
AcOH, rt, 1 h.
4
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A2H6
A1H2
A1H8
A
B
Figure 4. Analysis of dynamic combinatorial library of aldehyde A2 with hydrazide library I (DCL2): A) UV-chromatogram at 290 nm of the blank reaction at 6 h; B)
UV-chromatogram at 290 nm of the protein-templated reaction at 6 h.
Monitoring the DCLs
Analysis of the NMR spectra of the prepared acylhydrazones
revealed two sets of signals. This can be ascribed to the presence
of cis/trans-amide CO-NH conformers of the energetically more
stable E imine C=N configuration as the Z isomer is usually
disfavored by steric repulsion.[33–35]
Owing to the reversible nature of the acylhydrazone linkage, we
investigated the chemical stability of the compounds under the
conditions of SPR and GTF180 activity assays. Encouragingly, all
six acylhydrazones showed marked stability in physiological pH
7.4 at room temperature for 24 h as well as in acidic pH 4.7 at
37 °C up to 3 h with less than 5% degradation as indicated by the
UPLC chromatograms (Figures S4–S15). These results are in
agreement with previous findings using UV/VIS spectroscopy and
1H NMR techniques.[34,36]
The DCLs were allowed to stir at room temperature and were
regularly monitored via UPLC-MS on hourly basis along with the
zero hour sample. Samples were prepared by taking 100 µL of
the corresponding library and raising pH to > 8 by the addition of
NaOH (2 M, 8 µL) to freeze the equilibrium, followed by
acetonitrile (100 µL) for protein denaturation and liberation of
protein-bound ligands. The mixture was centrifuged at 9,720 g for
2 min, and the supernatant was subjected to UPLC-MS analysis.
Samples of the blank reaction were treated in the same manner.
The formation of the acylhydrazones reached equilibrium within
three hours. It was at this time point that we added the GTF180-
∆N and continued the analysis via UPLC-MS. The distribution of
the products in the DCLs of the blank library versus the protein
library can be compared by the relative peak areas from the UV-
chromatograms (Figure 4, S1–S3 and Tables S2–S5). We
selected the most amplified ligation product of each library for
synthesis and biological evaluation (Table 1).
Since aniline could cause false-positive hits from the reaction with
the aldehydes in DCC,[30] we screened the LCMS chromatograms
for the possible imine products or hemiaminal intermediates and
no corresponding peaks were detected. Other factors could result
in false-positive or false-negative hits such as concentration and
purity of the protein template, buffer composition, and binding
interaction (aggregation) between the DCL members.[31] To avoid
such pitfalls, we used low concentration of purified GTF180 (0.1
equivalent to the building blocks) and selected the optimum buffer
and pH for DCC according to our systematic stability
monitoring.[20] Furthermore, we utilized cheminformatics to
exclude potential aggregators from the DCLs via the aggregator
advisor.[32]
Interestingly, analysis of DCL2, featuring the maltose-derived
aldehyde component, in the presence of GTF after 6 h revealed
the formation of two compounds that show molecular weights
corresponding to acylhydrazones of the glucose-based aldehyde
A1 (A1H2 and A1H8). These compounds can result from
cleavage of the α-glycosidic bond between the two sugar units of
maltose for the DCL2 members (A2H2 and A2H8) (Figure 4 and
Table S3). Both compounds possess a non-planar hydrophobic
alicyclic substituent on the hydrazide moiety. This finding
indicates that these compounds can indeed bind to the active site,
act as a substrate and get cleaved by the enzyme in agreement
with our docking study (Figure 3). Accordingly, we also
synthesized an example of the substrate molecules (A2H2,
featuring the hydrazide H2 that showed favorable amplification in
DCL1) and the hydrolysis products (A1H2 and A1H8) in order to
investigate their affinity and inhibitory activity toward GTF.
Binding Studies by Surface Plasmon Resonance (SPR)
We evaluated the binding affinities of the DCC hits, the substrate
compound A2H2 and the cleavage products using SPR. The
Lactobacillus reuteri GTF180-ΔN was immobilized covalently to a
carboxymethyldextran-coated sensor chip via amine coupling. In
order to ensure that the active site was accessible after
immobilization, we used acarbose as a positive control acting via
a
competitive inhibition mechanism.[10] Binding affinity
determination for acarbose showed a submillimolar dissociation
constant (KD) of 0.18 mM in line with the reported values for GTFs
from other bacterial strains.[10,37] Subsequently, we determined
the binding affinities of our hit compounds using 6–9 different
concentrations in the range of 0.0076–2.0 mM according to their
solubility. Except for compound A2H6, all compounds showed
concentration-dependent binding responses to GTF180-ΔN, yet
with KD values mainly about one order of magnitude higher than
acarbose (Table 1 and Figures S17–S23). Generally, the glucose-
bearing acylhydrazones exhibit higher affinity than the maltose
derivatives with compound A1H12 displaying the most promising
KD value of 0.4 mM. On the other hand, the sensorgrams of the
least amplified hit A2H6 showed low responses up to a
concentration of 1 mM and therefore its affinity could not be
determined (Figure S22). These unexpectedly weak affinities of
the new acylhydrazones may be attributed to the intrinsic weak
affinity and very weak inhibitory activity of their sugar moieties
especially maltose for GTF.[10] Moreover, the hydrophobic
hydrazide moieties of the compounds seem to be unfavorably
accommodated in the mainly hydrophilic active site of GTF180.
Furthermore, the rather rigid and planar acylhydrazone scaffold
possibly hinders a proper fit into the binding cavity.
GTF180 Activity Assay
The inhibitory effects of the compounds on GTF180 was
assessed via monitoring the hydrolysis of sucrose into glucose
5
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using the glucose oxidase/peroxidase (GOPOD) analysis.
Acarbose and the compounds at a concentration of 0.5 mM were
incubated with the enzyme for 30 min at 37 °C, then sucrose was
added, and the hydrolysis products were determined over time to
calculate the GTF activity. Consistent with our affinity results, only
acarbose showed about 70% inhibition of GTF180 activity at 0.5
mM, whereas none of the compounds exhibited significant
inhibition at the same concentration (Figure S24). Besides the
moderate to weak affinity of the compounds, the non-observed
residual inhibitory activity can be ascribed to the presence of a
high concentration of the substrate sucrose in the assay (20-fold
more than that of the compounds). Such large excess of the
substrate concentration can diminish or abolish the effects of
competitive inhibitors.[38] Altogether, these results suggest that the
new GTF ligands most probably act via competitive inhibition in
agreement with our design rationale.
parameters such as topological polar surface area, flexibility, and
water solubility would be required in order to improve the affinity
and inhibitory activity of this class. Alternatively, exploration of
non-carbohydrate chemical scaffolds should be envisaged.
Experimental Section
Materials and methods
Chemicals were purchased from commercial suppliers and used without
pretreatment. Solvents used for the experiments were reagent-grade and
dried, if necessary, according to standard procedures. The reactions were
performed under nitrogen atmosphere, unless otherwise stated. The yields
were calculated for the analytically pure compounds and were not
optimized. The purifications were performed using column
chromatography with Macherey-Nagel Silica 60 M 0.04–0.063 mm.
Preparative HPLC (Ultimate 3000 UHPLC+ focused, Thermo Scientific)
purification was performed on a reversed-phase column (C18 column, 5
μm, Macherey-Nagel, Germany). The solvents used for the
chromatography were water (0.1% formic acid) and MeCN (0.1% formic
acid), or EtOAc and DCM. NMR spectra were measured on a Bruker
Fourier 500 or Varian AMX400 spectrometers at (500 or 400 MHz for 1H)
and (126 or 101 MHz for 13C), respectively. The chemical shifts are
reported in parts per million (ppm) relative to the corresponding solvent
peak. The coupling constants of the splitting patterns are reported in Hz
and are indicated as broad (br) singlet (s), doublet (d), triplet (t) and
multiplet (m).
Conclusions
We described the first application of DCC to the bacterial
glucosyltransferase 180 belonging to the GS family, a potential
target for combating dental caries. We designed our compounds
to bear a glucose or maltose anchor targeting the active site,
resembling natural substrates, as a rational starting point. These
molecules were then expected to grow into the pocket by 18
different aromatic or aliphatic tails via DCC. A docking study of
the 36 DCC products into the active site of GTF180 supported our
rationale showing high binding energy scores. By separating the
complex library into four individual DCLs, we were able to analyze
the DCC experiments without overlap among the DCL
components. UPLC-MS analyses of the DCLs resulted in the
identification of four most amplified hit compounds, which we
synthesized and evaluated for their biophysical and biochemical
properties via SPR and a GTF-activity assay. Remarkably, we
discovered that the maltose-derived acylhydrazones A2H2 and
A2H8 with a lipophilic 5/6-membered alicyclic motif can be
cleaved by GTF180, acting as glucose-donor substrates, in
contrast to the parent maltose, which is known as an acceptor.
This indicates that modification of maltose at the C1-hydroxyl
group can alter its recognition by GTF from an acceptor substrate
to a donor. Acarbose showed moderate binding affinity for
GTF180 (KD 0.18 mM) and partial inhibition at 0.5 mM. In
comparison to acarbose, the hit compounds showed only
moderate to low affinities to GTF180. Results of the activity assay
are in line with the SPR measurements, showing no pronounced
inhibition at 0.5 mM, most probably due to weak affinity of the
sugar units, hydrophobicity of the hydrazide tails, and overall
rigidity of the scaffold due to the acylhydrazone linker.
Nevertheless, our endeavor for targeting the sugar-binding site of
GTF180 using DCC resulted in the identification of moderate to
weak binders that are indeed capable of binding to the active site
as indicated by the cleavage of the maltose derivatives A2H2 and
A2H8. This work demonstrates the utility of DCC for notoriously
challenging targets such as sugar-converting enzymes with
inherently weak ligand interactions and millimolar affinity.[22]
Besides saving time and resources, DCC can be particularly
advantageous in absence of structural information or a known
ligand to afford novel binders. We gave insight into some
challenges encountered by using a carbohydrate-based scaffold
for inhibiting GTFs. Optimization of the physicochemical
UPLC-MS analysis of DCC
UPLC-MS was carried out on a ThermoScientific Dionex Ultimate 3000
UHPLC System coupled to a ThermoScientific Q Exactive Focus with an
electrospray ion source. An Acquity Waters Column (BEH, C8 1.7 µm, 2.1
× 150 mm, Waters, Germany) equipped with a VanGuard Pre-Column
(BEH C8, 5 × 2.1 mm, 1.7 µm, Waters, Germany) was used for separation.
At a flow rate of 0.250 mL/min, the gradient of H2O (0.1% FA) and MeCN
(0.1% FA) was held at 5% MeCN for 1 min and then increased to 95% over
16 min. It was held there for 1.5 min before the gradient was decreased to
5% over 0.1 min where it was held for 1.9 min. The mass spectrum was
measured in positive mode in a range from 100–700 m/z.
HRMS analysis
High-resolution mass spectra were recorded with a ThermoScientific
system where a Dionex Ultimate 3000 RSLC was coupled to a Q Exactive
Focus mass spectrometer with an electrospray ion source. An Acquity
UPLC® BEH C8, 150 × 2.1 mm, 1.7 µm column equipped with
a
VanGuard Pre-Column BEH C8, 5 × 2.1 mm, 1.7 µm (Waters, Germany)
was used for separation. At a flow rate of 250 µL/min, the gradient of H2O
(0.1% FA) and MeCN (0.1% FA) was held at 10% B for 1 min and then
increased to 95% B over 4 min. It was held there for 1.2 min before the
gradient was decreased to 10% B over 0.3 min where it was held for 1 min.
The mass spectrum was measured in positive mode in a range from 120–
1000 m/z. UV spectrum was recorded at 254 nm.
General procedure for DCC experiments
The reaction mixture composition for each DCC library was obtained by
adding the hydrazides (each 3 μL, stock solutions 100 mM in DMSO) and
the aldehyde (3 μL, stock solutions 100 mM in DMSO) to a sodium acetate
buffer (590.5 µL, 0.1 M, pH 5.2). Aniline (5.55 µL, stock solution 1.8 M) was
added as well as DMSO, to reach a final concentration of DMSO in the
DCL of 10%. Protein (309.5 µL, stock solution 96.93 µM) was added
accordingly after 3 h of equilibration. Instead of protein, sodium acetate
buffer (309.5 µL) was added to another sample to serve as a blank reaction.
Final concentrations in the DCLs were: aniline (10 mM), aldehyde (300 µM),
hydrazides (300 µM each), protein (30 µM) and DMSO (10%). The DCLs
were left shaking at room temperature and were concurrently monitored at
regular intervals via UPLC-MS. After 6–7 h of shaking with protein, the
mixture was analyzed via UPLC-MS. For monitoring, 100 μL of the
corresponding library was mixed with 8 µL of NaOH (2 M) to raise pH > 8,
followed by adding 100 µL acetonitrile. The mixture was centrifuged at
9,720 g for 2 min, and the supernatant was analyzed via UPLC-MS.
6
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10.1002/cmdc.202000222
ChemMedChem
FULL PAPER
Chemistry
2-Cyclopentyl-N'-[(1E)-4-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl-
oxy)benzylidene]acetohydrazide (Acetylated A1H8)
Compounds 1 and 4 were purchased. Compounds 2,[39] 3,[40,41] 5,[27] 6,[42,43]
and 7[40] were prepared according to reported procedures.
The acylhydrazone was synthesized according to GP1 by using 2-
chlorophenoxyacetic acid hydrazide (18.9 mg, 0.13 mmol) in MeOH (1.8
mL) and p-(tetraacetyl-β-D-glucopyranosyl)benzaldehyde 3 (50.0 mg, 0.11
mmol). After purification, the acylhydrazone was obtained as a white solid
(59 mg, 93%). 1H-NMR (500 MHz, CD3OD) δ 8.05 (s, 1Htrans), 7.89 (s,
1Hcis), 7.75 (d, J = 8.8 Hz, 2Htrans), 7.63 (d, J = 8.8 Hz, 2Hcis), 7.07 (d,
J = 8.8 Hz, 2Htrans, 2Hcis), 5.46 – 5.35 (m, 2Htrans, 2Hcis), 5.22 – 5.07
(m, 2Htrans, 2Hcis), 4.39 – 4.24 (m, 1Htrans, 1Hcis), 4.21 – 4.01 (m,
2Htrans, 2Hcis), 2.74 (d, J = 7.4 Hz, 2Hcis), 2.40 – 2.12 (m, 3Htrans,
1Hcis), 2.10 – 1.93 (m, 12Htrans, 12Hcis), 1.91 – 1.78 (m, 2Htrans, 2Hcis),
1.77 – 1.53 (m, 4Htrans, 4Hcis), 1.38 – 1.16 (m, 2Htrans, 2Hcis); 13C-NMR
(126 MHz, CD3OD) δ 177.6, 172.4 (2C), 172.3 (2C), 171.6, 171.3 (2C),
171.1 (2C), 160.2, 159.9, 148.6, 144.8, 130.4 (2C), 130.3 (2C), 129.5 (2C),
117.9 (2C), 117.8 (2C), 99.2, 99.1, 74.1 (2C), 73.1 (2C), 72.7 (2C), 69.7
(2C), 63.1, 63.0, 41.6, 39.4, 38.6, 38.0, 33.5 (2C), 33.4 (2C), 25.9 (4C),
20.6 (4C), 20.5 (4C); HRMS (ESI) calcd for C28H37N2O11 [M+H]+: 577.2397,
found: 577.2360.
4-(β-D-Glucopyranosyloxy)benzaldehyde (A1)
Compound A1 was synthesized by a slight modification of the reported
method.[44] p-(Tetraacetyl-β-D-glucopyranosyl)benzaldehyde 3[40,41] (225
mg, 0.5 mmol) was dissolved in 3 mL dry MeOH in a flame-dried flask
under nitrogen. Next, a methanolic NaOMe solution (1.5 M, 0.5 mL) was
added dropwise, and the reaction was stirred for 4 h at room temperature.
The reaction was neutralized with DOWEX 50WX8 hydrogen-form ion
exchange resin, filtered and passed through a pad of charcoal to remove
any colored impurities and then evaporated to dryness in vacuo to obtain
pure compound A1 (137 mg, quantitative). NMR spectra show that A1 is
present as a hydrate form in the NMR solvent. 1H-NMR (400 MHz, CD3OD)
δ 7.34 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 8.7 Hz, 2H), 5.33 (s, 1H), 4.94 –
4.90 (m, 1H), 3.89 (dd, J = 12.0, 2.1 Hz, 1H), 3.69 (dd, J = 12.0, 5.4 Hz,
1H), 3.50 – 3.29 (m, 4H); 13C-NMR (101 MHz, CD3OD) δ 159.2, 133.5,
129.0 (2C), 117.3 (2C), 104.2, 102.2, 78.2, 78.0, 74.9, 71.4, 62.5; HRMS
(ESI) calcd for C13H15O7 [M–H]–: 283.0823, found: 283.0822.
4-(4-O-α-D-Glucopyranosyl-β-D-glucopyranosyloxy)benzaldehyde
(A2)
2-Cyclopentyl-N'-[(1E)-4-(β-D-glucopyranosyloxy)benzylidene]aceto-
hydrazide (A1H8)
p-(Heptaacetyl-β-D-maltosyl)benzaldehyde 7[40] (340.7 mg, 0.46 mmol)
was dissolved in 5 mL dry MeOH in a flame-dried flask under nitrogen.
Next, a methanolic NaOMe solution (1.5 M, 1 mL) was added dropwise,
and the reaction was stirred for 4 h at room temperature. The reaction was
neutralized with DOWEX 50WX8 hydrogen-form ion exchange resin,
filtered and passed through a pad of charcoal to remove any colored
impurities and then evaporated to dryness in vacuo to obtain pure
compound A2 (142 mg, 70%). 1H NMR (400 MHz, CD3OD) δ 9.84 (s, 1H),
7.87 (d, J = 8.7 Hz, 2H), 7.34 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 8.7 Hz, 2H),
7.08 (d, J = 8.7 Hz, 2H), 5.32 (s, 1H), 5.21 (t, J = 3.4 Hz, 2H), 5.09 (d, J =
7.8 Hz, 1H), 4.96 (d, J = 7.8 Hz, 1H), 3.96 – 3.43 (m, 22H), 3.32 – 3.25 (m,
2H); 13C NMR (101 MHz, CD3OD) δ 193.1, 163.8 (2C), 159.0 (2C), 133.4,
132.9, 132.4, 128.9, 117.8 (2C), 117.2 (2C), 104.3, 102.7 (2C), 101.9,
101.2, 80.7, 80.5, 77.6, 77.5, 76.8, 76.6, 75.0 (2C), 74.7 (2C), 74.4, 74.3,
74.0 (2C), 71.4 (2C), 62.6 (2C), 61.9 (2C); HRMS (ESI) calcd for C19H25O12
[M–H]–: 445.1346, found: 445.1353.
The acylhydrazone was synthesized according to GP2 by using compound
acetylated A1H8 (23.0 mg, 0.04 mmol) in MeOH (4 mL) and sodium
methoxide (0.32 mg, 0.006 mmol). After purification, the acylhydrazone
was obtained as a white solid (13 mg, 82%). 1H-NMR (500 MHz, CD3OD)
δ 8.05 (s, 1Htrans), 7.88 (s, 1Hcis), 7.73 (d, J = 8.8 Hz, 2Htrans), 7.61 (d,
J = 8.8 Hz, 2Hcis), 7.23 – 7.02 (m, 2Htrans, 2Hcis), 4.98 – 4.95 (m, 1Htrans,
1Hcis), 3.93 – 3.87 (m, 1Htrans, 1Hcis), 3.74 – 3.66 (m, 1Htrans, 1Hcis),
3.52 – 3.36 (m, 4Htrans, 4Hcis), 2.74 (d, J = 7.5 Hz, 2Hcis), 2.43 – 2.23
(m, 3Htrans, 1Hcis), 1.92 – 1.77 (m, 2Htrans, 2Hcis), 1.75 – 1.52 (m,
4Htrans, 4Hcis), 1.41 – 1.09 (m, 2Htrans, 2Hcis); 13C-NMR (126 MHz,
CD3OD) δ 177.5, 172.3, 160.9, 160.6, 149.0, 145.2, 130.2 (2C), 129.9 (2C),
129.6, 129.4, 117.9 (2C), 117.8 (2C), 101.9 (2C), 78.2 (2C), 78.0, 77.9,
74.9 (2C), 71.3 (2C), 62.5 (2C), 41.6, 39.4, 38.5, 38.0, 33.5 (2C), 33.4 (2C),
25.9 (4C); HRMS (ESI) calcd for C20H29N2O7 [M+H]+: 409.1975, found:
409.1964.
2-(2-Chlorophenoxy)-N'-[(1E)-4-(2,3,4,6-tetra-O-acetyl-β-D-gluco-
pyranosyloxy)benzylidene]acetohydrazide (Acetylated A1H12)
The acylhydrazone was synthesized according to GP1 by using 2-
chlorophenoxyacetic acid hydrazide (26.6 mg, 0.13 mmol) in MeOH (1.8
mL) and p-(tetraacetyl-β-D-glucopyranosyl)benzaldehyde 3 (50.0 mg, 0.11
mmol). After purification, the acylhydrazone was obtained as a white solid
(43 mg, 61%). 1H-NMR (500 MHz, CD3OD) δ 8.19 (s, 1Htrans), 7.93 (s,
1Hcis), 7.77 (d, J = 8.8 Hz, 2Htrans), 7.66 (d, J = 8.8 Hz, 2Hcis), 7.43 –
7.36 (m, 1Htrans, 1Hcis), 7.33 – 7.19 (m, 1Htrans, 1Hcis), 7.14 – 6.90 (m,
4Htrans, 4Hcis), 5.44 – 5.34 (m, 2Htrans, 2Hcis), 5.25 (s, 2Hcis), 5.21 –
5.15 (m, 1Htrans, 1Hcis), 5.15 – 5.09 (m, 1Htrans, 1Hcis), 4.76 (s, 2Htrans),
4.35 – 4.27 (m, 1Htrans, 1Hcis), 4.21 – 4.06 (m, 2Htrans, 2Hcis), 2.19 –
1.76 (m, 12Htrans, 12Hcis); 13C-NMR (126 MHz, CD3OD) δ 172.3 (2C),
171.6 (2C), 171.4, 171.3 (2C), 171.1 (2C), 167.1, 160.1, 159.8, 155.5,
155.0, 150.6 (2C), 146.1 (2C), 131.5, 131.4, 130.6, 130.3, 130.1, 129.8,
129.3, 128.9, 124.4, 124.1, 124.0, 123.1, 117.9, 117.8, 116.1, 115.3, 99.1
(2C), 74.1 (2C), 73.1 (2C), 72.6 (2C), 69.7, 69.6, 69.2 (2C), 67.3 (2C), 63.1,
63.0, 20.6 (4C), 20.5 (4C); HRMS (ESI) calcd for C29H32ClN2O12 [M+H]+:
635.1644, found: 635.1638.
General procedure for acylhydrazone formation (GP1):[36]
To the hydrazide (1 equiv) dissolved in MeOH, the corresponding aldehyde
(1.2 equiv) was added. The reaction mixture was stirred at room
temperature or refluxed until completion. After cooling to room temperature,
the reaction mixture was concentrated in vacuo. Purification of acetylated
products was performed by column chromatography and deprotected
sugars were purified by preparative HPLC, affording the corresponding
acylhydrazone in 60% to quantitative yields.
General procedure for the deprotection of the acetyl groups (GP2):[26]
The classical Zemplén deacetylation method of the O-acetyl protecting
groups with sodium methoxide in MeOH at room temperature was used.
The O-acetyl protected sugar was dissolved in MeOH (0.01 M), and a
catalytic amount of sodium methoxide (0.15 equiv) was added. The
reaction mixture was stirred at room temperature until complete
deprotection was achieved.
tert-Butyl 4-({(2E)-2-[4-(β-D-glucopyranosyloxy)benzylidene]hydra-
zino}carbonyl)piperidine-1-carboxylate (A1H2)
The acylhydrazone was synthesized according to GP1 by using 1-Boc-
isonipecotic acid hydrazide (26 mg, 0.1 mmol) in MeOH (1.0 mL) and p-
(β-D-glucopyranosyloxy)benzaldehyde A1 (14.5 mg, 0.05 mmol). After
purification, the acylhydrazone was obtained as a white solid (12 mg, 46%).
1H-NMR (500 MHz, CD3OD) δ 8.05 (s, 1Htrans), 7.88 (s, 1Hcis), 7.70 (d,
J = 8.6 Hz, 2Htrans), 7.60 (d, J = 8.6 Hz, 2Hcis), 7.15 – 7.06 (m, 2Htrans,
2Hcis), 4.98 – 4.92 (m, 1Htrans, 1Hcis), 4.12 (d, J = 13.3 Hz, 2Htrans,
2Hcis), 3.92 – 3.84 (m, 1Htrans, 1Hcis), 3.69 (dd, J = 12.1, 5.6 Hz, 1Htrans,
1Hcis), 3.51 – 3.34 (m, 6Htrans, 7Hcis), 2.45 (tt, J = 11.4, 3.6 Hz, 1Htrans),
1.89 – 1.52 (m, 4Htrans, 4Hcis), 1.45 (s, 9Htrans, 9Hcis); 13C-NMR (126
MHz, CD3OD) δ 178.6, 173.9, 161.0, 160.6, 156.5, 156.4, 149.3, 145.5,
130.2 (2C), 129.8 (2C), 129.5 (2C), 117.9 (2C), 117.8 (2C), 101.9 (2C),
81.2, 81.1, 78.2 (2C), 78.0, 77.9, 74.8 (2C), 71.3 (2C), 62.5 (2C), 49.8 (2C),
42.7 (2C), 39.5 (2C), 29.5 (2C), 28.9 (2C), 28.7 (6C); HRMS (ESI) calcd
for C24H36N3O9 [M+H]+: 510.2452, found: 510.2432.
2-(2-Chlorophenoxy)-N'-[(1E)-4-(β-D-glucopyranosyloxy)benzyl-
idene]acetohydrazide (A1H12)
The acylhydrazone was synthesized according to GP2 by using
acetylated A1H12 (26.1 mg, 0.04 mmol) in MeOH (4 mL) and sodium
methoxide (0.33 mg, 0.006 mmol). After purification, the acylhydrazone
1
was obtained as a white solid in quantitative yield (20 mg). H-NMR (500
MHz, DMSO-d6) δ 11.54 (br s, NH, 1Htrans, 1Hcis), 8.22 (s, 1Hcis), 7.96
(s, 1Htrans), 7.70 – 7.60 (m, 2Htrans, 2Hcis), 7.49 – 7.37 (m, 1Htrans,
1Hcis), 7.34 – 7.20 (m, 1Htrans, 1Hcis), 7.12 – 6.89 (m, 4Htrans, 4Hcis),
5.37 (br s, OH, 1Htrans, 1Hcis), 5.25 (s, 2Htrans), 5.16 (br s, OH, 1Htrans,
1Hcis), 5.08 (br s, OH, 1Htrans, 1Hcis), 4.96 – 4.88 (m, 1Htrans, 1Hcis),
4.74 (s, 2Hcis), 4.58 (br s, OH, 1Htrans, 1Hcis), 3.69 (d, J = 11.3 Hz,
1Htrans, 1Hcis), 3.53 – 3.41 (m, 1Htrans, 1Hcis), 3.39 – 3.11 (m, 4Htrans,
4Hcis);13C-NMR (126 MHz, DMSO-d6) δ 168.4, 158.7, 153.7, 153.5, 143.6
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000222
ChemMedChem
FULL PAPER
(2C), 130.1 (2C), 130.0 (2C), 128.6, 128.4, 128.3, 128.1, 127.8, 127.7,
122.1, 121.5, 121.4, 121.1, 116.4 (2C), 114.1 (2C), 113.8 (2C), 100.0 (2C),
77.1 (2C), 76.6 (2C), 73.2 (2C), 69.7 (2C), 69.6 (2C), 65.3 (2C), 60.6 (2C);
HRMS (ESI) calcd for C21H24ClN2O8 [M+H]+: 467.1221, found: 467.1205.
tert-Butyl 4-({(2E)-2-[4-(4-O-α-D-glucopyranosyl-β-D-glucopyranos-
yloxy)benzylidene]hydrazine}carbonyl)piperidine-1-carboxylate
(A2H2)
The acylhydrazone was synthesized according to GP1 by using 1-boc-
isonipecotic acid hydrazide (38 mg, 0.15 mmol) in MeOH (1.0 mL) and p-
(β-D-maltosyl)benzaldehyde A2 (22 mg, 0.05 mmol). After purification, the
acylhydrazone was obtained as a white solid (9.1 mg, 28%); 1H-NMR (500
MHz, CD3OD) δ 8.07 (s, 1Htrans), 7.90 (s, 1Hcis), 7.73 (d, J = 8.8 Hz,
2Htrans), 7.63 (d, J = 8.8 Hz, 2Hcis), 7.15 – 7.10 (m, 2Htrans, 2Hcis), 5.21
(d, J = 3.8 Hz, 1Htrans, 1Hcis), 5.01 – 4.99 (m, 1Htrans, 1Hcis), 4.18 –
4.09 (m, 2Htrans, 2Hcis), 3.96 – 3.41 (m, 12Htrans, 12Hcis), 3.30 – 3.24
(m, 2Htrans, 3Hcis), 2.47 (tt, J = 11.5, 3.8 Hz, 1H), 1.90 – 1.54 (m, 4Htrans,
4Hcis), 1.47 (s, 9Htrans, 9Hcis); 13C NMR (126 MHz, CD3OD) δ 178.6,
173.9, 160.9, 160.6, 156.5, 156.4, 149.3, 145.5, 130.3 (4C), 129.6, 129.5,
117.9 (2C), 117.8 (2C), 102.9 (2C), 101.7 (2C), 81.2 (2C), 81.1, 80.9 (2C),
80.8, 77.7 (2C), 76.8 (2C), 75.1 (2C), 74.9 (2C), 74.4 (2C), 74.2 (2C), 71.5
(2C), 62.8 (2C), 61.9 (2C), 61.9 (2C), 42.7 (2C), 39.5 (2C), 29.5 (2C), 28.7
(3C), 28.7 (3C); HRMS (ESI) calcd for C30H46N3O14 [M+H]+: 672.2980,
found: 672.2958.
7.07 (m, 3Htrans, 2Hcis), 7.06 – 6.90 (m, 1Htrans, 2Hcis), 5.26 (s, 2Hcis),
5.21 (d, J = 3.7 Hz, 1Htrans, 1Hcis), 5.05 – 4.97 (m, 1Htrans, 1Hcis), 4.76
(s, 2Htrans), 3.97 – 3.41 (m, 10Htrans, 10Hcis), 3.31 – 3.23 (m, 2Htrans,
2Hcis); 13C NMR (126 MHz, CD3OD) δ 171.4, 167.1, 161.1, 160.7, 155.6,
155.0, 151.0 (2C), 146.4 (2C), 131.5, 131.3, 130.5 (2C), 129.7 (2C), 129.6,
129.3 (2C), 128.9, 124.4, 124.1, 124.0, 123.1, 117.9, 117.8, 116.1, 115.2,
102.9 (2C), 101.7 (2C), 80.9, 80.8, 77.7 (2C), 76.8 (2C), 75.1 (2C), 74.9
(2C), 74.5 (2C), 74.2 (2C), 71.5 (2C), 69.2 (2C), 67.3 (2C), 62.8, 61.9;
HRMS (ESI) calcd for C27H34ClN2O13 [M+H]+: 629.1749, found: 629.1744.
Computational chemistry
All computational work was performed using Molecular Operating
Environment (MOE), version 2019.01, Chemical Computing Group ULC,
910–1010 Sherbrooke St. W. Montreal, Quebec, H3A 2R7, Canada.
Computational procedure was adopted from reported protocols[45,46] with a
slight modification as following.
Preparation of ligands and protein structure for docking
The 2D structures of 36 acylhydrazone products of DCL1–4 were sketched
using ChemDraw professional 17.0 and were pasted to the MOE window.
The compounds were subjected to an energy minimization up to a gradient
of 0.01 kcal mol−1 Å2 using the MMFF94x force field then they were saved
as mdb file. In the database viewer window, the acylhydrazone structures
were washed via compute | molecule | wash command. Deprotonation of
strong acids and protonation of strong bases were performed by choosing
the dominant protonation at pH 7 option in the wash panel. X-ray crystal
structure of the Lactobacillus reuteri 180 GTF180-ΔN in complex with
maltose (PDB code: 3KLL)[5] was used to perform the molecular docking
study. Potential was set up to Amber10:EHT as a force field and R-field for
solvation. Addition of hydrogen atoms, removal of water molecules farther
than 4.5 Å from ligand or receptor, correction of library errors, and tethered
energy minimization of binding site were performed via QuickPrep module.
Ligand–receptor docking
N'-[(1E)-4-(4-O-α-D-Glucopyranosyl-β-D-glucopyranosyloxy)benzyl-
idene]-2-(methylsulfonyl)acetohydrazide (A2H6)
The acylhydrazone was synthesized according to GP1 by using 2-
(methylsulfonyl)acetic acid hydrazide (12.8 mg, 0.08 mmol) in MeOH (0.8
mL) and p-(β-D-maltosyl)benzaldehyde A2 (30.2 mg, 0.07 mmol). After
purification, the acylhydrazone was obtained as a white solid (4.1 mg,
10%). 1H-NMR (500 MHz, CD3OD) δ 8.10 (s, 1Htrans), 7.95 (s, 1Hcis),
7.75 (d, J = 8.8 Hz, 2Htrans), 7.66 (d, J = 8.8 Hz, 2Hcis), 7.14 (d, J = 8.8
Hz, 2Htrans, 2Hcis), 5.21 (d, J = 3.8 Hz, 2Htrans), 5.02 – 5.01 (m, 1Htrans,
1Hcis), 4.68 (s, 2Hcis), 4.15 (s, 2Htrans), 3.96 – 3.38 (m, 12Htrans,
12Hcis), 3.19 (s, 3Hcis), 2.66 (s, 3H); 13C-NMR (126 MHz, CD3OD) δ 165.9,
161.2, 161.1, 160.8, 150.8, 146.8, 130.5 (2C), 129.8 (2C), 129.4, 129.1,
117.9 (4C), 102.9 (2C), 101.7 (2C), 80.8 (2C), 77.7 (2C), 76.8 (2C), 75.1
(2C), 74.8 (2C), 74.4 (2C), 74.2 (2C), 71.5 (2C), 62.8, 61.9, 60.0, 57.4,
42.6, 42.0, 40.4 (2C); HRMS (ESI) calcd for C22H33N2O14S [M+H]+:
581.1652, found: 581.1620.
The binding site was set to dummy atoms, which were calculated by the
site finder command, and the amino acid residues were chosen where
maltose binds in the GTF180-ΔN active site. Docking placement was
triangle matcher with an induced fit refinement option. The first scoring
function was alpha HB with 100 poses, followed by a refinement score
affinity dG with 10 poses.
Chemical stability determinations
Stability studies of the compounds in the SPR and GTF activity assays
were performed as previously reported[47] with slight modifications. Two
sets of samples were prepared according to the assay conditions. For SPR,
5 µL of 2 mM stock solution of the compounds in DMSO was added to 95
µL of HEPES buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005%
v/v tween 20, pH 7.4) and vortexed to attain final concentration of 100 µM
in a total volume (100 µL) containing 5% DMSO. The solutions of the
compounds were allowed to stir in a shaking mixer at 10 rpm at rt. Aliquots
of 5 µL were taken from the samples at time 0, 3, and 24 h, mixed with
MeOH (45 µL) to have a final concentration 10 µM, and samples were
submitted for UPLC-MS analysis. In the other set for GTF activity assay, 5
µL of 2 mM stock solution of the compounds in DMSO was added to a
mixture of 80 µL of sodium acetate buffer (100 mM, 8 mM CaCl2, pH 4.7)
and DMSO (15 µL) and vortexed to reach final concentration of 100 µM in
a total volume (100 µL) containing 20% DMSO. The solutions of the
compounds were incubated in a water bath at 37 °C. 5 µL Aliquots were
taken from the samples at time 0, 0.5, 1, and 3 h, mixed with MeOH (45
µL) and submitted for analysis. The samples were stored at –20 °C, if they
were not measured directly.
LCMS analyses were measured by Dionex UltiMate 3000 UHPLC+
focused/Thermo Scientific ISQ EC mass spectrometer system (Thermo
Fisher Scientific, Dreieich, Germany). The system consists of Dionex
UltiMate 3000 RS pump, RS autosampler, column compartment, diode
array detector, and single-quadrupole mass spectrometer, as well as the
standard software Chromeleon 7.2.9 for operation. RP Hypersil GOLD
C18, 1.9 µm (100 mm × 2.1 mm) column (Thermo Scientific, Dreieich,
Germany) was used as stationary phase, and a binary solvent system A
and B (A = water with 0.1% FA; B = MeCN with 0.1% FA) was used as
mobile phase. In a gradient run, the percentage of B was increased from
an initial concentration of 5% at 0 min to 100% at 4.2 min and kept at 100%
for 0.8 min. The injection volume was 5 µL and flow rate was set to 600
2-(2-Chlorophenoxy)-N'-{(1E)-4-[2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-
O-acetyl-α-D-glucopyranosyl)-β-D-glucopyranosyloxy]benzyl-
idene}acetohydrazide (Acetylated A2H12)
The acylhydrazone was synthesized according to GP1 by using 2-
chlorophenoxyacetic acid hydrazide (8.9 mg, 0.04 mmol) in MeOH (0.5
mL) and p-(heptaacetyl-β-D-maltosyl)benzaldehyde 7 (25.0 mg, 0.03
mmol). After purification, the acylhydrazone was obtained as a white solid
(30 mg, 48%). 1H NMR (500 MHz, CD3OD) δ 8.19 (s, 1Htrans), 7.92 (s,
1Hcis), 7.77 (d, J = 8.8 Hz, 2Htrans), 7.66 (d, J = 8.8 Hz, 2Hcis), 7.45 –
7.19 (m, 3Htrans, 2Hcis), 7.15 – 6.90 (m, 3Htrans, 4Hcis), 5.50 – 5.33 (m,
4Htrans, 4Hcis), 5.24 (s, 2Hcis), 5.11 – 5.00 (m, 2Htrans, 2Hcis), 4.93 –
4.85 (m, 1Htrans, 1Hcis), 4.76 (s, 2Htrans), 4.62 (s, 2Hcis), 4.60 – 4.50 (m,
2Htrans), 4.36 – 4.03 (m, 6Htrans, 6Hcis), 2.12 – 1.96 (m, 21Htrans,
21Hcis); 13C NMR (126 MHz, CD3OD) δ 172.3 (4C), 171.9 (2C), 171.8 (2C),
171.6 (2C), 171.3 (2C), 171.2 (2C), 167.1, 160.1, 159.7, 155.5, 154.9,
154.8, 150.6, 146.1, 131.5, 131.4 (2C), 130.6, 130.3, 130.0, 129.8, 129.3,
129.2, 128.9, 124.4, 124.1, 124.0, 123.9, 123.1, 117.9, 117.8, 116.1, 115.7,
115.3, 98.7 (2C), 97.3 (2C), 76.4 (2C), 75.0, 74.9, 73.6 (2C), 73.3 (2C),
71.7 (2C), 70.7, 69.9, 69.7 (2C), 69.2 (2C), 68.8, 67.3, 64.3 (2C), 63.1 (2C),
21.2 (2C), 20.8 (4C), 20.7 (2C), 20.6 (6C); HRMS (ESI) calcd for
C41H48ClN2O20 [M+H]+: 923.2489, found: 923.2446.
2-(2-Chlorophenoxy)-N'-[(1E)-4-(4-O-α-D-glucopyranosyl-β-D-gluco-
pyranosyloxy)benzylidene]acetohydrazide (A2H12)
The acylhydrazone was synthesized according to GP2 by using
acetylated A2H12 (27.0 mg, 0.03 mmol) in MeOH (3 mL) and sodium
methoxide (0.36 mg, 0.007 mmol). After purification, the acylhydrazone
was obtained as a white solid (17.2 mg, 93%). 1H NMR (500 MHz, CD3OD)
δ 8.19 (s, 1Htrans), 7.93 (s, 1Hcis), 7.76 (d, J = 8.8 Hz, 2Htrans), 7.65 (d,
J = 8.8 Hz, 2Hcis), 7.43 (dd, J = 7.9, 1.6 Hz, 1Htrans), 7.38 (dd, J = 7.9,
1.6 Hz, 1Hcis), 7.33 – 7.26 (m, 1Htrans), 7.26 – 7.20 (m, 1Hcis), 7.18 –
8
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10.1002/cmdc.202000222
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µL/min. For compound A2H6, the initial concentration of B was 1% and
the injection volume was 10 µL. Column temperature was 40 °C and UV
tracing was acquired at wavelength of 310 nm. MS (HESI) analysis was
carried out at a spray voltage of 3000 V (positive), and – 2000 V (negative),
and an ion transfer tube temperature of 300 °C. Spectra were acquired in
positive and negative modes from 100 to 1000 m/z.
A.K.H.H. gratefully acknowledges funding from the Netherlands
Organisation for Scientific Research (LIFT grant), the European
Research Council (ERC starting grant 757913) and the Helmholtz
Association’s Initiative and Networking Fund.
Binding studies by surface plasmon resonance (SPR)
Keywords: Dynamic combinatorial chemistry • Drug discovery •
Glucosyltransferase 180 • Glycosides • Synthesis
The SPR experiments were performed using a Reichert SR7500DC
surface plasmon resonance spectrometer (Reichert Technologies, Depew,
NY, USA), and medium density carboxymethyl dextran hydrogel
CMD500M sensor chips (XanTec Bioanalytics, Düsseldorf, Germany).
Double distilled (dd) water was used as the running buffer for
immobilization. HEPES buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA,
0.005% v/v tween 20, pH 7.4) containing 5% v/v DMSO was used as the
running buffer for binding study. All running buffers were filtered and
degassed prior to use. GTF180 (117 kDa) was immobilized in one of the
two flow cells by amine coupling according to reported procedure.[24,48] The
other flow cell was left blank to serve as a reference. The system was
initially primed with borate buffer 100 mM (pH 9.0), then the
carboxymethyldextran matrix was activated by a 1:1 mixture of N-ethyl-N′-
(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) 100 mM and N-
hydroxysuccinimide (NHS) 100 mM at a flow rate of 10 µL/min for 7 min.
GTF180 5 µM in 10 mM sodium acetate buffer (pH 4.0) was injected at a
flow rate of 10 µL/min for 7 min. Non-reacted surface was quenched by 1
M ethanolamine hydrochloride (pH 8.5) at a flow rate of 25 µL/min for 3 min.
A series of 10 buffer injections was run initially on both reference and
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Entry for the Table of Contents
Structure-based design in combination with acylhydrazone-based dynamic combinatorial chemistry (DCC) afforded inhibitors of
glucansucrase (GS), the main virulence factor responsible for dental caries. DCC offered a facile pathway for finding the first hits of GS
in the form of glucose- and maltose-based acylhydrazones, mimicking the GS substrate.
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