Click inspired synthesis of hexa and octadecavalent peripheral galactosylated glycodendrimers and their possible therapeutic applications

Article abstract of DOI:10.1039/c9nj02564b

A Cu(i)-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction (CuAAC) has been utilized for the synthesis of novel glycodendrimers containing a rigid hexapropargyloxy benzene centered core with 6- and 18-peripheral β-d-galactopyranosidic units. Struct

Full text of DOI:10.1039/c9nj02564b

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Volume 42
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March 2018
Pages 3963-4776
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DOI: 10.1039/C9NJ02564B
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Click inspired Synthesis of Hexa and Octadecavalent Peripheral
Galactosylated Glycodendrimer and their Possible Therapeutic
Applications
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
a
a
b
a
a
Anand K. Agrahari , Anoop S. Singh , Ashish Kumar Singh , Nidhi Mishra , Mala Singh ,
b*
a*
Pradyot Prakash and Vinod K. Tiwari
transduction processes, for instance, surface sensing and adhesion
by the bacteria and virus, drug effectors modalities, immunological
Cu(I)-Catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction
CuAAC) has been utilized for the synthesis of novel
(
glycodendrimers containing rigid hexapropargyloxy benzene cascades, cellular interactions, cell cycle regulation and
centered core with 6- and 18-peripheral β-D-galactopyranosidic differentiation, and cancer cell aggregation as well as its metastatic
units. Structures of novel glycodendrimers and intermediates
are well elucidated by NMR spectroscopy, MALDI-TOF MS, IR
and SEC analysis. The therapeutic evaluations of developed
glycodendrimers were investigated and found potentially good
as anti-bacterial, anti-biofilm, and anti-tumour agent.
6
spread. Therefore, over the last few years, investigators across the
globe have put their efforts to explore the potency of
carbohydrates in drug discovery and development and also exploit
the desired biological interactions by tuning the spatial
arrangements of sugar residues on dendritic scaffolds for better
mimics/decoys.2,7 The outstanding interaction profiles of large
Introduction
dendritic glycoconjugates, due to their avidity, pave the path to use
Dendrimers are well-known to possess in general high
monodispersity, good biocompatibility, multivalency, and high
pharmacokinetics. Moreover, the synthesis of the exactly
controllable size of dendrimers by only deciding the number of
generation, charge, and dimension of the molecule as biological
them as drugs per se in different therapeutic fields.8-10 The
development of newer moieties with potential therapeutic activity
against drug-resistant pathogens with minimal toxicity was the
main intent of the study. The most alluring solution seems to be the
inhibition of initial bacterial attachment to target cells/ surfaces
using anti-adhesive molecules. Bacterial biofilms are the
1
system can be possible with various easy synthetic methods. These
properties of dendrimers make them interesting for the further
detailed investigation.1-3 Carbohydrates are fundamental entities to
sustain and nurture the living system. Since long back,
carbohydrates and their derivatives have been widely explored in a
different area of science ranging from chemical biology to catalysis
and medicinal chemistry to material science.4 Dendritic sugar
architectures modulate a wide array of biological phenomena,
which may be due to either specific recognition of functional
motif(s) directly by the glycodendrimers or they self-assemble on
noteworthy illustrations of adherence phenomena on inanimate as
well as animate surfaces.11 We are interested in the biocompatible
neutral motifs of galactose because It has been reported that
galactosylated or mannosylated dendrimers are more
therapeutically active compared to other sugars.12 Cu(I)-catalyzed
azide-alkyne cycloaddition (Click chemistry)13 nowadays is widely
exploited in the field of carbohydrate chemistry for fostering regio-,
chemoselective with a quantitative yield of glycoconjugates, such as
glycopeptides, glycopolymers, polysaccharides, glycodendrimers,
glycol-arrays, glyco-macrocylces, etc.2a,14-16 Regioselective triazole
generated from azide-alkyne stitching is the bioisostere of the
amide functional group. Amide shows natural connector in the
5
the molecular scaffolds as and when needed. This is evidenced by
the fact that multivalent carbohydrate-protein interactions are
pivotal in the majority of biological recognition and dissemination/
biological system and being the bioisostere of amide, triazole is also
an important pharmacophore17a and therefore Click inspired
triazoles has been widely used as a linker to adhere two distinct
scaffolds.17
Considering the importance of glycodendrimer, herein we wish to
report the Click inspired synthesis of novel glycodendrimers which
were evaluated for their biological activities against multi-drug
resistant bacterial isolates and human colorectal carcinoma cells
(HCT116), and also their biocompatibility profiling.
a.Department of Chemistry, Institute of Science, Banaras Hindu University,
Varanasi-221005, India
E-mail: Tiwari_chem@yahoo.co.in; Vinod.Tiwari@bhu.ac.in
b.Department of Microbiology, Institute of Medical Sciences, Banaras Hindu
University, Varanasi-221005, India. E-mail: pradyot_micro@bhu.ac.in
1
†
Electronic Supplementary Information (ESI) available: Copies of H and
13C NMR of developed glycodendrimers and their precursors (1-10) has
been provided. MaldI-TOF spectra, IR, SEC and DLS spectrum has also been
given. See DOI: 10.1039/x0xx00000x
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Results and discussion
Our overall strategy for the formation of G(0) and G(1) type
glycodendrimer involves the Cu(I)-catalyzed azide-alkyne 1,3-
View Article Online
DOI: 10.1039/C9NJ02564B
dipolar
cycloaddition
reaction
(CuAAC)
of
hexakis(propargyloxymethyl) benzene core unit with galactosylated
azide as well as multi functionalized dendritic azide 8 (Scheme S1).
We followed the convenient convergent method for the
construction of glycodendrimers 9 and 10 and thus our strategy
began with the synthesis of six arm hexakis(propargyloxymethyl)
benzene core unit (1) which was obtained from commercially
available hexakis (bromomethyl) benzene. When hexakis (bromom
ethyl)benzene reacted with propargyl alcohol in presence of NaH in
anhydrous THF at room temperature, propargyl group displaced the
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Scheme 2. Synthesis of galactosylated dendritic architecture: (a)
di-tert-butyl dicarbonate, t-BuOH/MeOH, 18h, r.t.; (b) propargyl
bromide, KOH, r.t.; (c) TFA, Dry DCM, 0˚C-rt; (d)
Chloroacetylchloride, DIPEA, Dry DCM, 0˚C-rt; (e) Acetyl
bromide and afforded the desired core 1 with a 60% yield (Scheme
). The synthesized core was fully characterized by H and 13C NMR,
1
1
MS and FT-IR spectrometry. The formation of the core was further galactosylated azide 2, CuI, DIPEA, anhydrous dichloromethane; (f)
confirmed by single crystal XRD, which exhibited the unequivocal
formation of our desired core moiety 1.
3
NaN , DMF, 24h, r.t.
Scheme 1. Synthesis of hexakis((prop-2-yn-1-yloxy)methyl)benzene
ortep diagram of Crystal
&
After the successful synthesis of the core, we focused to
synthesize other parts of glycodendrimers i.e. galactose azide
2
and Dendron 8. For this purpose, we followed the well
reported work for the synthesis of galactose azide i.e. acetyl
2
1
8
protection of D-galactose by acetic anhydride and I2. followed
by treatment with HBr in acetic acid affording selective
bromination at the C-1 position and then azidation to produce
desired galactose azide. Whereas, for the synthesis of
galactosylated dendritic wedge , we started with N-BOC
protection of tris-hydroxyaminomethane followed by
15
Scheme 3. Synthesis of glycoconjugate cluster 9a and 9b
8
1
propargylation using propargyl bromide in presence of KOH to Complete vanishing of the alkenyl-H peak of core 1 in H NMR and
furnish
aminomethane
characterized by NMR. Deprotection of amine group of
N-(tert-butyloxycarbonyl)tris[(propargyloxy)methyl] azide peak of compound 2 in IR spectrum demonstrated the purity
5
with 64% yield. The product was well
of the desired product. The appearance of triazolyl peak at 7.87 and
145.3 ppm, in H and 13C NMR, respectively showed the formation
of the triazole ring. Moreover, the ratio of the anomeric and
triazolyl proton is 1:1, which confirms the formation of symmetric
glycodendrimer with one triazole linker for each sugar moiety.
1
9
5
by
1
reaction with trifluoroacetic acid followed by treatment with
chloroacetyl chloride in presence of DIPEA, produced 2-
chloroacetamide-tris[(propargyloxy)methyl]aminomethane
61%) Scheme ). At the end of the sequence, 2-
chloroacetamide-tris[(propargyloxy)
AB monomers) was clicked with galactosylated azide (end moieties through dipolar cycloaddition reaction of alkyne tethered
groups) to afford the multivalent galactosylated dendritic
compound . The Appearance of triazolyl peak at 7.83 ppm
6
(
(
2
methyl]aminomethane Glycodendrimer 10a was synthesized by amalgamating two
(
3
core 1 with the azide functionalized dendritic wedge 8 in the
presence of CuI and DIPEA which resulted in the final 18 peripheral
galactosylated glycodendrimer 10a. Compound 10a was purified by
column chromatography (SiO ) and characterized by its NMR, IR,
2
and MALDI-TOF MS. Absence of the azide peak in FT-IR of
7
and disappearance of the alkenyl-H peak in NMR clearly
confirmed the formation of compound 7. Further, azide
functionalized dendritic architecture
reacting with NaN in DMF at r.t. for 24 h (88%) (Scheme 2).
Furthermore, six armed core unit
8 was prepared by
7
3
1
was clicked with glycodendrimer 10a showed the formation of first-generation
galactosylated azide (end group) to afford multivalent
glycodendrimer 9a with 78% yield after purification by flash
column chromatography. Compound 9a was characterized by
glycodendrimer 10a. Furthermore, SEC analysis shows the size
progression from 9a to 10a and the low dispersity index (Ð) (1.03 to
.01) reveals the monodispersity of the developed
1
1
13
H and C NMR, IR, MALDI-TOF MS and SEC analysis (Scheme
glycodendrimer.20
3
).
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biofilm biomass reduction in a dose dependent VmiewanAnrtieclre Ownliitnhe
increasing concentration of dendrimers 9a aDndOI1: 010a.1(0F3ig9u/Cre9N2J)0. 2564B
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Figure 2: Comparative evaluation of cellular viability of HCT116 cell
lines upon exposure to dendrimers 9a, 10a and the drug 5-Fluoro
Uracil.
Cell viability upon treatment with dendrimer 10a decreased to
10.33, 6 and 1.667 log10 CFU/mL at concentrations 8, 16, and 32
µg/mL, respectively, whereas bacterial count reduced to zero when
the concentration was escalated to 128 µg/mL compared to control
where log10 CFU/mL was found to be 19.67. The minimum biofilm
inhibitory concentration (MBIC) of 10a was found to be 2 and 8
μg/mL against drug-resistant S. aureus and E. coli biofilms,
respectively. The MBIC data is in consonance with the results of the
biofilm disruption test. At the concentration 4 µg/mL, dendrimer
0a was found to inhibit the biofilm formation by 54% but, no
sooner, the concentration was increased to 64 µg/mL then,
significant inhibition of 83.5%, was observed against MRSA
1028/2018), while at the same concentration complete inhibition
was noted in Staphylococcus aureus (ATCC 25923). Interestingly,
st
Scheme 4. Synthesis of 1 generation glycodendrimer 10a and 10b
1
(
Figure 1: SEC chromatogram of glycodendriners 9a and 10a
To make the glycodendrimers water-soluble, compound 9a and 10a dendrimer 9a was less effective against the biofilms of tested
were quantitatively de-O-acetylated using NaOMe in MeOH by isolates at the said concentration where merely 12% and 68.8 %
applying Zemplén reaction method to afford the water soluble inhibition was realized (Figure 3). Remarkably, the inhibition was
dendrimers 9b and 10b (Scheme
glycodendrimers 9b and 10b were characterized by using standard
spectroscopic techniques such as, NMR, and IR analysis.
The biological data clearly reveal the potential antibacterial activity
of the synthesized glycodendrimers 9a and 10a against both gram-
negative and gram-positive pathogens irrespective of their
susceptibility profile with other conventional drugs. The MIC ranged
from 2 to 32 µg/mL (Table 1). For comparison, MIC indices of
vancomycin (for gram-positive isolates) and meropenem (for gram-
negative) were also evaluated. Of note dendrimer 9a (relatively
smaller dendrimer) showed mediocre activity against the tested
isolates.
3 and 4). Both the less pronounced in the case of gram-negative isolate.
Figure 3: (I) Plot depicting reduction in colony forming units (CFUs) of
Biofilm, an important adaptation in bacteria wherein they remain Staphylococcus aureus MRSA (1028/2018) with escalating concentration
enmeshed within self-produced exopolysaccharides, bestows them of glycodendrimer 10a. (II) Tissue culture plate assay for biofilm
1
1,21-
protection against antimicrobials and host immune responses.
quantification exhibiting dose-dependent reduction in biomass in
presence of dendrimers 9a and 10a. Well A, B, C, and D depict the
effective concentration of 8, 16, 32, and 64 µg/mL respectively.
23
We investigated bacterial viability in biofilms post treatment. A
sharp reduction in CFUs was noted. Besides, we also observed
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View Article Online
DOI: 10.1039/C9NJ02564B
Table 1: Minimum inhibitory concentration and minimum biofilm inhibitory concentration of glycodendrimers 9a and 10a
Drugs
MIC/MBIC (µg/ml)
Staphylococcus
aureus, MRSA(1672/2018) aureus ATCC 29213
MIC/MBIC (µg/ml)
Staphylococcus
MIC/MBIC (µg/ml)
Staphylococcus
aureus USA300
MIC/MBIC
(µg/ml)
Escherichia coli,
(2147/2018)
MIC/MBIC
(µg/ml)
Escherichia coli
ATCC 25922
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Vancomycin/
Meropenem
4/< 0.1953
> 0.25 / 0.114
2 / 0.125
1/ 0.03125
0.25 /0.125
9a
32/ 16
16/ 4
4/ 4
4/ 2
8/ 4
2/ 1
32/ 16
32/ 8
8/ 8
8/ 4
10a
Freshly grown bacteria were then challenged with the MIC investigated the percentage of growth inhibition in identical cell
concentration of the dendrimer 10a (16 and 32 µg/mL for MRSA cultures treated with 5 to 200 µg/mL of 9a and 10a. The results
and E. coli respectively, data shown in micrograph) and 9a (32 and (Figure 5) indicated that the concentration of 20 µg/mL, 9a
3
2 µg/mL for MRSA and E. coli respectively, not shown in inhibited the cell proliferation by ~88%, whereas 10a resulted in
micrograph) and the image has been taken after staining with the virtually complete inhibition of proliferation that is, glycodendrimer
red fluorescent dye propidium iodide. After 6 hrs of drug exposure, 10a was dramatically more effective than 9a. At the 150 µg/mL
the said bacteria produced an intense PI-staining, indicating the cell concentrations of 10a and 200 µg/mL of 9a, were both highly anti-
death (Figure 4). Fluorescent micrographs showed very intense proliferative against HCT116, and therefore, no differences were
uptake of PI post 10a treatment, indicating compromised cell observed in the effectiveness of 9a and 10a at these
membrane permeability. Red signals veiled complete cellular concentrations.
obliteration. Thus, the above results indicated that dendrimers 9a
and 10a fostered bacterial killing by disrupting their cell
membranes.
Figure 4: Fluorescent Micrographs of (A) Staphylococcus aureus and (B)
Escherichia colifor the evaluation of anti-bacterial potential of
compound 10a. PI staining of S. aureus and E. coli after dendrimer 10a
treatment reveals the substantial bacterial deaths by the presence of
abundant red emission wavelength. At MIC concentration of dendrimer
Figure 5: Dose dependent anti-tumour assay of glycodendrimer 10a
1
0a, the clear uptake of PI symbolizes membrane perturbations.
against HCT116. (1) Untreated, (Negative control), (2) Treated HCT116
cells with 2 µg/mL 10a, (3) Treated HCT116 cells with 16 µg/mL 10a, (4)
Treated HCT116 cells with 64 µg/mL 10a, (5) Treated HCT116 cells with
To ensure the anti-tumour potential of dendrimers 9a and 10a, the
in vitro cellular viability was evaluated by SRB assay against HCT116
cells.24 Incubation of HCT116 with compound 9a did not influence
cell viability in the tested range, which can easily be conjectured (Positive control)
from the fact that upon exposure to 2 µg/mL of compound 9a, cell
2
56 µg/mL 10a; (6) Treated with 256 µg/mL with 5-fluoro uracil
The phase contrast microscopy was used to evaluate the
viability was around 97% which remained 77% when exposed to
56 µg/mL (Figure 5). Unlike compound 9a, the exposure to the
morphological alterations if any. We noted the alterations in
cellular morphology and adherences, which eventually resulted in
the cellular deaths upon treatment with compound 10a even at the
concentration of 2 µg/mL compared to the control in the dose
dependent manner. Thus, the outcome is in agreement with the
results obtained after SRB assay.
Damage to the cell membrane is one of the hallmarks of the drug-
toxicity.23 To ensure whether the glycodendrimers 9a and 10a
foster any cell membrane damage, we investigated the treated and
2
compound 10a had pronounced hostile consequences over the cell
viability in a concentration-dependent manner. For instance, at 2
µg/mL concentration, around 96% cell viability was noted but as the
concentration was escalated to 256 µg/mL; cell viability reduced to
53%. After exposure for 48 hrs, the GI50 and LC50 values for
compounds 9a and 10a were estimated and for 10a were 15.8 and
159.6 µg/mL, respectively, whereas for 9a, were 10.7 and 123.2
µg/mL, respectively.24,25 On consideration of the GI50 and LC50, we
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untreated cells using lactate dehydrogenase assay. LDH is a 20.4 and 20.1 ppm. IR (KBr): νmax 3395.50, 3145V.i8ew0,Art2ic9le2O8n.3lin3e,
cytosolic enzyme and its presence in the cell soup indicates the 2111.58, 1755.66, 1674.53, 1529.27, 1459.9D4,O1I:41302.1.02329,/aCn9dN1J0327516.47B8
membrane damage. Result demonstrates that the LDH activity (in cm .
terms of mean absorbance) in percentage after 48 hrs exposures of
the SiHa cells to the glycodendrimers 9a and 10a (see, supporting
Table S1). At any given time, SiHa cells incubated with dendrimers
-
1
Physical data of glycodendrimer 9a:
,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl azide (184 mg, 0.493
mmol) was dissolved in dry DCM, synthesized core compound 1 (30
mg, 6.17x10 mmol) was added to the solution, CuI (0.21 mg, 0.111
mmol) and DIPEA (32.0µL, 0.185 mmol) were added to the reaction
mixture and stirred under argon atmosphere for 12h. When the
reaction shows the complete disappearance (monitored by TLC) of
the compound 1, the mixture was passed through cellite to remove
the metal, 20 mL of DCM was added to the obtained the filtrate and
taken in separating funnel, the organic layer was washed with water
2
9a and 10a at various concentrations (32, 64, 128, 256, 512, 1024
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µg/mL) showed insignificant LDH activity than the group inoculated
with amphotericin B in the same concentration range, signifying the
biocompatibility of dendrimers 9a and 10a. At the concentration of
1
024 µg/mL, around 67% cytotoxicity was noted in amphotericin B
treated cells while on the same concentration, no significant
leakage was noted in 9a and 10a treated cells. This indicates
biocompatibility of compounds 9a and 10a. In addition, as evident
from the differential light scattering experiment, the dendrimers
exfoliate in the aqueous phase owing to its potential to open up in
aqueous environments; we found its potential applicability in using
as anti-adhesive. The two distant peaks depict agglomeration
however; in the exploitation of therapeutic capacity this minimal
aggregation exhibits minimal contrivances. The larger extent is
showing the exfoliation which is prerequisite for being the anti-
biofilm agent. The compounds are more soluble in DMSO than in
aqueous phase therefore when this binary system is used for the
DLS experiment two different peaks appeared depicting two
different particle size distribution (See, Supporting information,
(
2 4
2 x 20 mL). Organics were dried over anhydrous Na SO and
evaporated to afford crude product. Purification of the compound
was done by flash column chromatography in (2% methanol/DCM)
to obtain the compound 9a as yellow solid. Yield (131 mg, 78%); R
f
=
1
0
.4 (5% methanol/DCM); H NMR (500 MHz, CDCl
3
): δ 7.87 (s, 6H,
), 5.56-5.44 (m, 12H, H , H ),
), 4.56-4.48 (m, 24H, H6a, H6b, OCH CH=CH),
), 4.04-3.99 (m, 12H, OCH Ar), 2.11 (s, 18H, 6 x
), 1.93-1.90 (m, 36H, 12 x COCH ), 1.74 (s, 18H, 6 x COCH );
13C NMR (125 MHz, CDCl
): δ 170.3, 170.1, 169.8, 168.9, 145.3,
37.9, 122.2, 85.9, 73.7, 70.9, 67.9, 66.9, 63.7, 61.0, 60.3, 54.7,
42.9, 21.0, 20.6, 20.5 and 20.2 ppm. IR (KBr): νmax 3479.91, 3146.72,
Triazolyl-H), 5.85 (d, J = 9.5 Hz, 6H, H
.22-5.19 (m, 6H, H
.28-4.2 0 (m, 6H, H
4
2
3
5
4
1
2
5
2
COCH
3
3
3
3
1
Figure S30). The smaller size shows exfoliation in the aqueous
phase while the larger particle size shows aggregation.26-28
-1
2934.72, 1755.54, 1639.17, 1434.53, 1371.90 cm , MALDI-TOF MS:
+
m/z C114
H
144
N
18
O
60Na , calculated = 2748.8696; found = 2748.9348
In conclusion, we successfully explored Click chemistry for the
development of targeted glycodendrimers. The in vitro results (M+Na)+.
obtained in the current study indicate its possible therapeutic
potential with regard to antibacterial, anti-biofilm and anti-tumor
activities.
Synthesis of first generation glycodendrimer 10a:
-2
Compound 1 (12 mg, 2.47x10 mmol) and compound 8 (0.266 mg,
2
0
.185 mmol) were dissolved in DCM. CuI (9 mg, 4.73 x 10 mmol)
and DIPEA (13 µL, 7.74 x 10-2 mmol) both were added to the
solution and stirred at r.t. for 12 h, the disappearance of the core
i.e. compound 1 (monitored by TLC) inferred the completion of the
reaction. The reaction mixture was passed through cellite and
obtained filtrates were taken in separating funnel, DCM (20 mL)
was mixed and followed by washing with water (2x 30 mL). The
organic layer was collected and dried over anhydrous sodium
sulphate and evaporated to give residue 10a, which was purified by
flash column chromatography (2-9%, M-D). The product 10a was
Experimental
Synthesis of azide functionalized first generation dendron (8):
Compound 7 (0.3 g, 0.21 mmol) was dissolved in Dry DMF (3.0 mL)
in an R.B., then NaN (40 mg, 0.625 mmol) was added to the
3
reaction mixture and stirred for 12 h at r.t., after completion of the
reaction (monitored by TLC) solvent was evaporated in continuation
to that ethyl acetate (30 mL) was added to the mixture and taken
up in a separating funnel, washed with water (3 x 15 mL) followed
by brine solution. Further, the organic layer was collected and dried
over anhydrous sodium sulphate and reduced under high vacuum
to obtain the crude compound which was further subjected to
column chromatography to afford compound 8 as yellow solid. Yield
obtained as off white solid. Yield : (157 mg, 70%); R
f
= 0.50 (10%
): δ 7.90 (br s, 18H,
Peripheral triazolyl-H), 7.87 (br s, 6H, Inner triazolyl-H), 7.03 (br s,
H, NH), 5.97-5.95 (m, 18H, H ), 5.59-5.55 (m, 18H, H ), 5.50 (br s,
18H, H ), 5.30-5.25 (m, 18H, H ), 4.55 (br s, 72H, H6a, H6b,
OCH CH=CH), 4.29 (br s, 18H, H ), 4.15-4.07 (m, 36H, CH ), 3.74-
.69 (m, 36H, CH CON, OCH Ar, OCH CH=CH), 2.15-2.12 (m, 54H, 18
x COCH ), 1.96-1.95 (m, 108 H, 36 x COCH ), 1.74 (s, 54H, 18 x
COCH ): δ 170.4, 170.1, 169.9, 169.1,
); 13C NMR (125 MHz, CDCl
65.3, 145.1, 139.3, 138.0, 125.7, 122.2, 114.0, 85.8, 73.7, 70.8,
1
methanol/DCM); H NMR (500 MHz, CDCl
3
6
4
2
1
3
1
(
0.265 g, 88%); R
f
= 0.5 (5% Methanol/DCM); H NMR (500 MHz,
): δ 7.87 (s, 3H, Triazolyl-H), 6.62 (s, 1H, NH), 5.91 (d, J = 9.5
), 5.61 (d, J = 9.5 Hz, 3H, H ), 5.56 (d, J = 3.0 Hz, 3H, H ),
.30-5.27 (m, 3H, H ), 4.67-4.61 (m, 6H, OCH CH=CH), 4.29-4.27 (m,
H, H6a), 4.19 (d, J = 7.0 Hz, 6H, CqCH O), 3.87-3.86 (m, 3H, H6b),
.84 (s, 2H, ClCH ), 3.77 (d, J = 9.5 Hz, 3H, H ), 2.22 (s, 9H, COCH ),
.05 (s, 9H, COCH ), 2.01 (s, 9H, COCH ), 1.83 (s, 9H, COCH
): δ 170.3, 170.0, 169.8, 169.0, 166.7, 145.3,
CDCl
Hz, 3H, H
5
3
3
2
3
2
5
2 q
C
3
2
2
2
4
2
3
3
3
1
2
3
3
2
1
2
5
3
); 13C
3
68.7, 68.0, 67.0, 64.4, 61.1, 50.8, 40.5, 20.7, 20.6, 20.2 and 19.6
3
3
ppm. IR (KBr): νmax 3454.46, 3144.32, 2925.33, 2854.91, 1755.21,
NMR (125 MHz, CDCl
3
-1
1
636.08, 1552.67, 1463.37, 1432.7, 1371.64 cm ; MALDI-TOF MS:
121.5, 86.0, 73.8, 70.7, 68.8, 67.8, 66.8, 64.5, 61.0, 59.8, 52.6, 20.6,
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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Journal Name
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2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
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5
5
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6
+
m/z C372
(
H
484
N
78
O
192Na
3
calculated = 9187.0296; found = 9187.0693
Ertapenem (10 µg), Gentamicin (120 µg), ImipenVieewmAr(ti1cl0e Oµnlgin)e,
DOI: 10.1039/C9NJ02564B
Levofloxacin (5 µg), Meropenem (10 µg), and Piperacillin &
M+3Na+4H)+
.
tazobactum (100/10 µg).
“De-O-acetylation” (Zemplèn reaction) procedure for the synthesis
Minimum Inhibitory Concentration (MIC) determination
MIC of the dendrimers 9a and 10a was determined by the
broth micro dilution method as described earlier with minor
modifications.21,23 Methicillin resistant Staphylococcus aureus
(MRSA, USA300, Lab code 1028/2018), Methicillin sensitive
Staphylococcus aureus (MSSA, ATCC 25923), Escherichia coli
(ATCC 25922, Lab code: 2764/2018) were used in the current
study and bacteria were cultured in Brain Heart Infusion Broth
(BHI) media (HiMedia laboratories, Mumbai). Initially the
bacteria were streaked from −80°C glycerol stock onto Brain
Heart Infusion Agar (BHIA) plate and single colony was
inoculated into BHI broth (50% brain heart with 4% glucose)
and incubated at 37°C for 24 hrs. From there, 106 CFU/mL
bacterial cell suspensions were taken for all subsequent
experiments. The freshly prepared stock solution of 9a and 10a
of glycodendrimer 9b and 10b:
Glycoconjugate cluster 9a or 10a was dissolved in a mixture of
anhydrous methanol: anhydrous THF: anhydrous DCM in the ratio
-3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
of 3:0.5:0.5 by fixing the cluster molarity 2.5 x 10 M. A freshly
prepared solution of NaOMe (1M, 30-40µL approx.) was added until
the solution pH became 9-10. The reaction was stirred for 48 h at
room temperature. After that, Milli-Q water was poured to
solubilize the whole mixture and neutralized by ion exchange resin
+
(
Amberlite 120 H ) till pH reaches in between 6-7, followed by
filtration, and the solvent was evaporated under reduced pressure
to afford the deprotected glycodendrimers 9b and 10b. The
developed compound further characterized by the NMR, and
MALDI-TOF MS and IR spectroscopy.
Physical data of glycodendrimer 9b:
1
White solid, yield (26 mg, 88%); H NMR (500 MHz, D
H, Triazolyl-H), 5.57 (d, J = 8.5 Hz, 6H, H ), 4.40 (br s, 12H,
OCH CH=CH), 4.30 (br s, 12H, H , H ), 4.12-4.08 (m, 6H, H ), 3.925-
.920 (m, 6H, H6a), 3.83-3.80 (m, 6H, H6b), 3.73-3.70 (m, 6H, H ),
Ar); 13C NMR (125 MHz, D
3.59-3.51(m, 12H, OCH O): δ 146.7,
2
O): δ 8.21 (s,
(
20 mg/mL in DMSO) were used for the study. The stock was
6
4
diluted in a series of two-fold dilutions ranging from 0.5 to 64
µg/mL in sterile BHI broths in microtiter wells. Each well of 96-
well microtiter plate was then inoculated with 200 µl of
standardized cell suspension (106 CFU/mL) and incubated at
2
2
3
1
3
5
2
2
143.9, 137.7, 130.3, 125.7, 124.8, 88.1, 78.2, 73.0, 69.7, 68.5, 64.7,
62.7 and 60.7 ppm. IR (KBr): νmax 3425.79, 2925.43, 2851.8, 1633.62,
1457.3, 1404.34, 1093.77 cm ; MALDI-TOF MS: for C66H N O
100 18 37
Calculated = 1736.6492; found = 1736.1666 (M+H
3
7°C for next 24 hrs along with the test compounds. The MIC
-1
+
of compounds 9a and 10a against the said bacterial isolates
were delineated as their minimum concentration at which no
perceivable bacterial growth was manifested as outlined by
CLSI. Positive controls were devoid of compounds 9a and 10a
while the sterile broth was used as negative control.
Experiments were performed in triplicate.
O+2H)+.
2
Physical data of glycodendrimer 10b: White solid, yield (28 mg,
4%); 1H NMR (500 MHz, D
O): δ 8.06 (br s, 18H, Peripheral
triazolyl-H), 7.88 (br s, 6H, Inner triazolyl-H), 5.51-5.49 (m, 18H, H ),
), 3.90 (br s,
8H, H6a), 3.80 (br s, 18H, H6b), 3.69-3.67 (m, 24H, OCH CH=CH,
); C NMR (125
O): δ 170.3, 147.6, 145.1, 133.2, 131.2, 130.1, 126.7, 126.6,
25.1, 109.5, 89.0, 79.2, 73.9, 70.6, 69.5, 68.5, 64.4, 61.7 and 61.2
ppm. IR (KBr): νmax 3419.51, 2925.22, 1686.02, 1642.3, 1401.87,
8
2
4
5
2 1 2 3
.02 (br s, 12H, CH CON), 4.45-4.36 (m, 54H, H , H , H
1
2
13
2 5 2 2 q
OCH Ar), 3.62-3.50 (m, 90H, H , OCH CH=CH, CH C
Antibiofilm Activity Determination
MHz, D
2
Tissue Culture Plate Assay (TCP)
1
The antibiofilm assay was performed in 96-well tissue culture
plate as described previously with minor modifications.4,5
Briefly, the overnight cultures of MRSA (USA 300, Lab code:
-
1
1
195.52, 1095.55 cm .
1
028/2018) and E. coli (ATCC 25922, Lab code: 2764/2018)
Biological investigations of glycodendrimers:
were grown in Brain Heart Infusion broth. A volume of 180 µl
of each diluted bacterial suspension (0.5 McFarland’s, 108
CFU/mL) was dispensed into flat-bottom polystyrene 96-well
tissue culture plate along with 20 µl of dendrimers 9a and 10a
Growth inhibition assays of glycodendrimers 9a and 10a
We explored the effect(s) of glycodendrimers 9a and 10a over
multi-drug resistant clinical isolates of Escherichia coli (Lab
code: 2764/2018) and Methicillin resistant Staphylococcus
aureus (MRSA, lab code: 1028/2018) as described earlier.21,23,26
(
50 µg/mL) at 37°C without shaking for 24 hrs. Wells without
the said compounds were set as controls. As a positive control,
we used Staphylococcus epidermidis (ATCC 35984), a known
high biofilm former. After the respective incubations, biofilm
was quantitated by crystal violet (CV) assay as described
earlier.22 The assays were performed in triplicate, and the
results were expressed as mean OD570 ± the standard
deviation of the mean (SD).
Reduction = (Mean absorbance of the control-Mean
absorbance of the test sample)/(Mean absorbance of the
control) × 100
Further, effect against select control bacteria namely
Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC
2
5922) was also investigated. In this study, multi-drug
resistance was defined as resistance against at least 5 different
classes of drugs. Antibiotic susceptibility testing was
performed by modified Kirby–Bauer method in accordance
with the Clinical and Laboratory Standards Institute guidelines
%
2
018 using the following antibioticsm e.g. Ampicillin (10 µg),
Amikacin (30 µg), Amoxicillin/clavulanate (20/10 µg),
Ciprofloxacin (5 µg), Co-trimoxazole (23.75/1.25 µg),
6
| J. Name., 2012, 00, 1-3
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COMMUNICATION
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Fluorescent Microscopy for determination of anti-bacterial Instruments, Inc., Winooski, VT, USA). Each expVeierwimAreticnletOwnlianes
properties: run in triplicates. GI50 (i.e., the drug conc
64to
For fluorescent analysis of the effects of the said compounds, inhibit cell proliferation by 50%) and LC50 (i.e., the drug
we grew MRSA isolate and E. coli clinical isolates in chambered concentration required to kill 50% of the cultured cells) values
slides (Nunc, Denmark). Briefly, the overnight grown isolates were calculated as the mean of the three independent
were diluted 1:100 in fresh BHI broth to adjust its absorbance experiments.
to 0.2 at λmax 600nm. Fifty-microliters of its diluted suspension
was then dispensed into flat-bottom chambered slide
containing 480 µl of BHI broth and incubated for 3 hrs. This
was followed by the treatment with MIC dose of dendrimers
9a and 10a for next 3 hrs. Prior to staining,the residual broth
was aspirated and washed thrice by phosphate buffer (pH 7.5).
The 4% (v/v) paraformaldehyde was used for bacterial fixation
for 30 mins. The PI stock solution (1mg/mL) was prepared in
DMSO and stored frozen in aliquots of 100 µl. For use, stock
solutions were diluted with PBS to the concentration of 10
µg/mL. Fifteen-microliters of these staining solutions were
applied directly to the top of the biofilms. The Nikon Eclipse
microscope was used to detect the red fluorescence from the and 13C NMR of all the developed glycodendrimers and their precursors
stains. Propidium iodide was excited with the HeNe2 530nm (1-10b) has been provided. MaldI-TOF spectra, an IR, SEC and DLS
laser and emission fluorescence was collected with the 620 nm spectrum has also been given.
D
e
O
n
I:
t
1
r
0
a
.
t
1
i
0
o
3
n
9/
r
C
e
9
q
N
u
J
i
0
r
2
e
5
d
B
Acknowledgment
0
1
2
3
4
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6
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8
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1
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4
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0
Authors sincerely thank Science and Engineering Research Board
(SERB), New Delhi for the funding (Grant No.: EMR/2016/001123), CISC-
BHU for providing spectroscopic studies and Prof. B. Ray for SEC
analysis. PP and AKS acknowledge DST-PURSE grant sanctioned to
Microbiol. Deptt., IMS, BHU. AKA and AKS thanks CSIR and UGC for SRF.
We acknowledge CSIR-National Chemical Laboratory, Pune for providing the
facility for MALDI-TOF-MS and thankful to Dr. Shaziya Khanam (CSIR-Nehru
PDF), NCL Pune, for her help in MALDI-TOF-MS analysis of developed
glycodendrimers.
†
1
Electronic Supplementary Information (ESI) available: Copies of H
filter.
References
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.5 mL of the fresh medium containing various concentrations
of dendrimers 9a and 10a. A 2 mg/mL stock solution of said
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New Journal of Chemistry
View Article Online
DOI: 10.1039/C9NJ02564B
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Click inspired synthesis of hexa- and octadecavalent peripheral galactosylated
Glycodendrimer and its possible therapeutic Applications
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Anand K. Agrahari , Anoop S. Singh , Ashish Kumar Singh , Nidhi Mishra , Mala Singh , Pradyot Prakash and
Vinod K. Tiwaria*
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Click inspired glycodendrimers comprising of rigid hexapropargyloxy benzene core peripheral β-D-
galactopyranosidic units were developed and evaluated for their therapeutic potential.