Multivalent assembly of a pyrene functionalized thio-N-acetylglucosamine: Synthesis, spectroscopic and WGA binding studies

Article abstract of DOI:10.1016/j.carres.2019.04.010

We introduce here a new fluorescent derivative of 1-thio-β-N-acetylglucosamine linked to a pyrene system through a triazolylpentyl spacer, designed to self-assemble into a multivalent glycocluster. The synthesis was achieved by efficient CuAAC click reaction between a pyrene functionalized with an azide group and a suitable alkynyl thiomonosaccharide. Spectroscopic studies by fluorometry indicated that the self-assembly in aqueous medium is modulated by concentration and pH changes, the latter due to the presence of the amino group close to the π system. Circular dichroism experiments revealed a moderate positive signal, suggesting that the pyrene-thioGlcNAc conjugate can aggregate into a chiral supramolecular assembly. The sugar moiety showed to specifically and reversibly interact with the wheat germ agglutinin, a fact that was demonstrated by turbidity assay. SEM microscopy of a lyophilized solution at pH 10 revealed a fibrillar morphology compatible with the presence of tubular micelles, whereas crystalline and amorphous solids are formed at lower pHs.

Full text of DOI:10.1016/j.carres.2019.04.010

CarbohydrateResearch479(2019)6–12
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Multivalent assembly of a pyrene functionalized thio-N-acetylglucosamine:
Synthesis, spectroscopic and WGA binding studies
Hugo O. Montenegroa,b, Pablo H. Di Chennaa,c, Carla C. Spagnuoloa,b,**, María Laura Uhriga,b,*
a Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica. Buenos Aires, Argentina
b Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-UBA. Centro de Investigación en Hidratos de Carbono (CIHIDECAR). Buenos Aires, Argentina
c Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-UBA, Unidad de Microanálisis y Métodos Físicos Aplicados a la Química Orgánica (UMYMFOR),
Buenos Aires, Argentina
A R T I C L E I N F O
A B S T R A C T
Keywords:
We introduce here a new fluorescent derivative of 1-thio-β-N-acetylglucosamine linked to a pyrene system
through a triazolylpentyl spacer, designed to self-assemble into a multivalent glycocluster. The synthesis was
achieved by efficient CuAAC click reaction between a pyrene functionalized with an azide group and a suitable
alkynyl thiomonosaccharide. Spectroscopic studies by fluorometry indicated that the self-assembly in aqueous
medium is modulated by concentration and pH changes, the latter due to the presence of the amino group close
to the π system. Circular dichroism experiments revealed a moderate positive signal, suggesting that the pyrene-
thioGlcNAc conjugate can aggregate into a chiral supramolecular assembly. The sugar moiety showed to spe-
cifically and reversibly interact with the wheat germ agglutinin, a fact that was demonstrated by turbidity assay.
SEM microscopy of a lyophilized solution at pH 10 revealed a fibrillar morphology compatible with the presence
of tubular micelles, whereas crystalline and amorphous solids are formed at lower pHs.
Thiosugars
Pyrene
Self-assembly
pH modulation
Fluorescence
WGA lectin
1. Introduction
between aromatic units provide additional properties to these materials
such as the possibility to correlate the dynamic process with an optical
Self-assembly of amphiphilic molecules has been exploited as a
useful approach to design supramolecular systems [1]. If a carbohy-
drate residue is chosen as the hydrophilic moiety, a supramolecular
multivalent ligand can be generated. Multivalent ligands have proved
to be excellent tools for the study of protein-carbohydrate interactions
and a wide variety of synthetic complex structures, have been reported
in literature [2]. On the other side, the achievement of multivalency by
supramolecular, reversible self-assembly, only requires the synthesis of
a rather simple and low molecular weight amphiphilic compound. If an
aromatic system is selected as the hydrophobic portion of the molecule,
this opens the possibility to make profit of the optical properties of such
systems. Giving the high sensibility of the fluorescence methods to
evaluate the behavior of these species, a responsive fluorophore as part
of the amphiphilic compound is highly desirable.
response. Pyrene and other dyes were previously used in combination
with saccharides to construct glycosidic self-assemblies [3,4] or to de-
velop molecular fluorescent probes including sugars to improve water
solubility and biocompatibility [5]. In this respect, some covalent
conjugates of pyrene and carbohydrates have been reported. For ex-
ample, a butylidene protected glucose residue has been attached to
pyrene and its optical response in micro-heterogeneous media has been
studied [6]. A α-linked mannose residue has been also attached to a
pyrene system through a hydrocarbon linker and the photophysical
properties have been studied [7]. Interestingly, a glucosamine N-pyr-
enyl-imino conjugate was synthesized by condensation of glucosamine
hydrochloride and pyrene carbaldehyde, and was shown to behave as a
ON-OFF receptor for Hg2+ [8]. Sialic acid was also conjugated to
pyrene and other aromatic scaffolds and binding assays with he-
magglutinin of Influenza A were performed [9]. Various sugar-pyrene
derivatives were reported as low molecular weight gelators able to
functionalize carbon nanotubes (SWCNTs) [10]. In another example,
bis-pyrenil sugar derivatives were found to be suitable fluorescent
sensors selective to Hg+2 [11].
Among the variety of aromatic systems available, it is well estab-
lished the ability of pyrene to generate well-defined supramolecular
assemblies by means of π-π stacking interactions. The properties of
aromatic systems have been the starting point for the development of
dynamic platforms as part of smart materials. The excitonic interactions
* Corresponding author. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica. Buenos Aires, Argentina.
** Corresponding author. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Orgánica. Buenos Aires, Argentina.
E-mail addresses: carlacs@qo.fcen.uba.ar (C.C. Spagnuolo), mluhrig@qo.fcen.uba.ar (M.L. Uhrig).
https://doi.org/10.1016/j.carres.2019.04.010
Received 27 February 2019; Received in revised form 12 April 2019; Accepted 29 April 2019
Availableonline30April2019
0008-6215/©2019ElsevierLtd.Allrightsreserved.

H.O. Montenegro, et al.
CarbohydrateResearch479(2019)6–12
Carbohydrate-lectin interactions are associated to a wide variety of
normal and abnormal processes [12–19]. Therefore, new synthetic li-
gands to identify lectin selectivity are very welcome not only to aid
glycomics but also as potential theranostic tools. Within the synthetic
strategy, multivalency of the ligand is mandatory in order to enhance
the binding affinity [18,20]. In recent years, our group focused on the
synthesis of multiglycosidic platforms with covalent nature [21–25]
and we have also explored self-assembled glycoclusters based on
pseudoglycolipid structures [26]. In this work, we designed a pyrene-
sugar derivative to construct a supramolecular multivalent platform
with J1,2 = 10.5 Hz, consistent with the trans-diaxial disposition of H-1
and H-2. No traces of the α-anomer (characterized by a J1,2 ≈ 5 Hz) was
detected. Click reaction of 3 and 5 in the presence of CuI and DIPEA
under microwave irradiation, led to 6, which, after purification by
column chromatography, was obtained in 72% yield (Scheme 2). The
1H-NMR spectrum of 6, showed protected multiplets for the aliphatic
methylenes of the hydrocarbon chain, the well-resolved signals corre-
sponding to the sugar protons, and the aromatic protons of the pyrene
ring at 8.0–8.4 ppm.
Finally, compound 7 was obtained by deacetylation of 6 with NH3/
MeOH/H2O and purified by reverse-phase column chromatography. It
is worthy to mention that purification of all pyrene-derived compounds
3, 4, 6 and 7, were not trivial, as π-π stacking association between
molecules made difficult the column chromatography procedures. This
fact accounted for the yields of the purified products. 1H-NMR spectrum
of compound 7 in CD3OD also showed well-resolved signals corre-
sponding to the aromatic, the sugar and the aliphatic protons.
The anomeric β-configuration of the sugar moiety in 6 and 7 were
also confirmed by 1H-NMR spectroscopy, as in both cases, the anomeric
protons appeared as doublets with J > 10 Hz. Moreover, the anomeric
signals shifted to 4.73 ppm for the case of 6, and 4.46 ppm for 7, ac-
counting for the presence of a sulfur atom in this position (see ESI).
On the other hand, the abilities of compounds 6 and 7 to behave as
gelators in a variety of solvents and mixtures of solvents were tested.
For instance, mixtures of chloroform/methanol were tested for 6 and
methanol/water for 7, but neither of them were able to form gels, in
contraposition to the pyrene-Glc conjugate reported by Nagarajan and
Das [10].
and its interaction with
a specific lectin was studied. N-Acet-
ylglucosamine is one of the most prevalent sugars in glycoconjugates,
being part of the structure of N- and O- linked glycoprotein glycans and
glycosaminoglycans. In addition, the –NHAc group, a known supra-
molecular synthon, would be able to participate in the self-assembly
processes through strong hydrogen bonding. Thus, in this study a β-
GlcNAc residue was chosen as the carbohydrate moiety. Moreover, as
we are interested in the development of glycomimetics with higher
stability to glycosidases than the natural O-glycosides, we introduced a
thio-glycosidic bond in its structure, whereas the linkage between the
aromatic system and the sugar was achieved by click chemistry.
Thus, we describe here an efficient synthesis of a novel amphiphilic
pyrene-thiosaccharide derivative based on click chemistry. We were
able to characterize the dynamic assembly process by spectroscopic and
microscopic methods. Moreover, the selective binding of the ligand to
the wheat germ agglutinin (WGA) could be assessed by a turbidity
assay.
2. Results and discussion
2.2. Spectroscopic and microscopic studies
2.1. Syntesis of the thioGlcNAc-pyrene conjugate 7
The absorption spectra recorded for compounds 6 and 7, showed the
characteristic pattern of the pyrene systems (Figs. S1 and S2).
Whereas the deacetylated probe 7 was fully soluble in water
(maximum concentration of 1 mM, obtained by dilution of a 25 mM
methanolic stock solution), its precursor 6 was soluble in chloroform
and other organic solvents, as expected. Monitoring the variations of
excimer emission at 480 nm in chloroform solution (for 6) or water (for
7), spanning a range of concentrations between 0.25 and 10.0 Mm or
5 × 10−3 – 1.0 mM, respectively, accounted for the self-assembly of
both, the acetylated precursor 6, and the free derivative 7 in solution
(Figs. S3 and S4).
For the construction of the GlcNAc pyrene conjugate, the azide-
pyrene functionalized precursor 3 was needed. The synthesis started
with a reductive amination of pyrene carbaldehyde and 5-azido-1-
pentilamine (Scheme 1) [27]. The latter was synthesized by standard
methods from commercial 5-aminopentanol. In this way, compound 3
was obtained in 70% yield. An alternative route comprising the re-
ductive amination of 1 with 5-aminopentanol to obtain 4, followed by
transformation of the OH group into the azide, produced 3 in 23% yield
(see Supporting information file).
On the other hand, the alkynyl β-thioglucosamine precursor 5 was
prepared according to reported procedures developed by our group
[28]. Briefly, compound 5 was synthesized by a one-pot methodology
from 1,3,4,6-tetra-O-acetyl N-acetylglucosamine by treatment succes-
sively with a) BF3. Et2O/thiourea and b) TEA/propargylbromide in
CH3CN. The β-configuration of the major product 5 was confirmed by
1H-NMR spectroscopy, as the H-1 appeared at 4.80 ppm, as a doublet
A first inspection of the self-assembly behavior of 7 due to ground
state aggregation can be derived from the decrease of the ratio of the
absorption peak (341 nm) and valley (331 nm) value (Apeak/Avalley) in
water (2.23) compared to the value in methanol (2.75) or acetonitrile
(2.80) (Fig. S5). Concentration-dependent fluorescence spectra of
compound 7 in the range 5 × 10−3 – 1.0 mM showed a decrease of the
Scheme 1. Synthesis of the azide-pyrene precursor 3.
7

H.O. Montenegro, et al.
CarbohydrateResearch479(2019)6–12
Scheme 2. Synthesis of the thioGlcNAc-pyrene con-
jugate 7.
Fig. 1. Concentration-dependent emission of the monomer (red circles) and the
aggregates (black circles) of probe 7 in aqueous medium (4% MeOH). (For
interpretation of the references to colour in this figure legend, the reader is
referred to the Web version of this article.)
Fig. 3. CD spectra of compound 7 at 5 × 10−5 M (black line), 1 × 10−4 M (red
line) and 5 × 10−4 M (blue line) in aqueous medium (maximum 0.4% me-
thanol). (For interpretation of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)
emission at 375 nm corresponding to the monomer with a concomitant
enhancement of intensity at 490 nm, reflecting an emissive associated
state (Fig. 1).
electronic energy transitions, evidencing more than one emissive ag-
gregated state of 7. This spectroscopic behavior is in agreement with
observations made in the emission profile of similar pyrene-sugar
structures aggregated in water solution [cf. 6]: the longer the excitation
wavelength, a blue-shift in the emission maximum of the aggregate and
in the ratio of the intensities monomer/aggregate were detected.
The self-assembly behavior of 7 in water was also characterized by
circular dichroism (CD) spectroscopy and the concentration-dependent
spectra are shown in Fig. 3. At low concentrations (5 × 10−5 and
1 × 10−4 M), no evidence of a CD signal was observed. Nevertheless, a
5 × 10−4 M solution of 7 in water showed a moderate but evident,
positive Cotton effect with maximum positive and minimum negative
CD signals at 241 nm and ca. 229 nm respectively. This CD signal ac-
counts for the formation of a chiral assembly of supramolecular nature
in a concentration dependent way.
Changing the excitation wavelength affects not only the fluores-
cence intensity but also shifts the emission maximum of the aggregates
and introduces variation of the ratio monomer/aggregate (375 nm/
490 nm) (Fig. 2). Ruling out the purity of the probe, this deviation from
the Kasha's rule introducing different emission profiles along with ex-
citation wavelength, corresponds to the presence of different emissive
species according to the aggregation equilibrium with distinct
When the fluorescence intensity of the probe 7 was scanned in the
pH range 7.7–11.0, a decrease of the emission at 375 nm was observed
along with an increase at 490 nm, this indicates that the self-assembly
process is also pH dependent (Fig. 4). From the plot of I375 nm vs pH,
fitted with the Henderson-Hasselbalch equation, the pKa of the probe
was computed to a value of 8.8 (Fig. S6). Therefore, the changes ob-
served correlates with the protonation-deprotonation of the amine
present in the probe, inferring that the protonated species reduces the
efficiency of the assembly due to charge repulsion.
We performed a DLS (Dynamic Light Scattering) experiment to de-
termine the hydrodynamic diameter of the aggregates formed at pH 7
and 10. At pH 7 a diameter of 0.4 μm was determined for the smaller
aggregates and a relatively constant population of bigger clusters is
present with diameters over 2 μm. The size of the aggregates of 7 in-
creased at pH 10 and two clear populations of particles are present. The
smaller one with a very narrow size dispersion has a mean diameter of
1.3 μm (Fig. S7), and a second population with higher size dispersion, in
the range of 6 y 12 μm. This result is in accordance with those obtained
Fig. 2. Fluorescence spectra of 7 (1.0 × 10−3 M) in aqueous medium (4%
methanol) with the variation of excitation wavelength.
8

H.O. Montenegro, et al.
CarbohydrateResearch479(2019)6–12
Fig. 4. Variation of the emission intensity with pH. of the free (375 nm) and
assembled (490 nm) probe 7 at 0.5 mM in aqueous medium (2% methanol).
from fluorography experiments, confirming that the aggregates in-
creased their size when the pH increased.
To get an insight into the morphology of the self-assembled system
we also performed SEM microscopy to the solids obtained after lyo-
philization of aqueous solutions of 7 at pH 7 and 10 (Fig. 5). From the
images it is evident that at pH 10 a self-assembled fibrillar system is
present. This cross linked fibrillar system may result from the self-as-
sembly of 7 in water into tubular micelles where the sugar heads are
pointing out to the exterior and the non-polar pyrene inside the as-
sembly protected from water. The thinner tubes have a diameter of 70 Å
and a length of hundreds of microns (Fig. 5a). At pH 7 the solid is
amorphous, so in this case, the assembly is poorer than at pH 10, due to
the protonation of the probe, and is not observed after lyophilization.
These results are in agreement with the pH dependent fluorescence
experiments where the emission at 490 nm increases with the pH ac-
counting for a favored self-assembly of the non-protonated probe. In
order to have an insight into the dimensions of the molecular ag-
gregates, computational simulation of an isolated and completely
elongated molecule of 7 was performed on Hyperchem (semiempirical
AM1 in vacuum). It gave a molecular length value of 31.8 Å (Fig. 6).
Even though in the self-assembled aggregates the elongated con-
formation may not be the most stable one, this length is the maximum
value possible. Taking into account that the pyrene moiety involved in
the π-π stacking measures approximately 6.3 Å length, the value of 70 Å
observed in the SEM photographs (measured with the ImageJ) is in
accordance with the calculated maximum diameter of a fiber con-
sidering the aromatic rings superimposed through π-π stacking inter-
actions (Figs. S9 and S10).
2.3. Protein binding assay
Fig. 5. SEM images of a sample of 7 obtained from lyophilization of aqueous
solutions at pH 7 (panel a) and pH 10 (panels b and c).
In order to investigate the performance of probe 7 to study the specific
lectin-carbohydrate interaction in real time, turbidity assays were carried
out using wheat germ agglutinin (WGA). The WGA is a well-known plant
lectin which recognizes α- and β-GlcNAc terminal residues. The WGA
structure consists in a 36 kDa heterodimer with a total of eight hevein
domains, the 43-aminoacid sequence responsible for the GlcNAc recogni-
tion [29,30]. The simultaneous occupancy of its eight sites was verified by
R-X diffraction of crystals obtained by co-crystallization of WGA with a
divalent α-GlcNAc derivative [31]. Thus, WGA provides an excellent tool to
verify if 7 could act as a supramolecular multivalent ligand of the lectin.
Thus, a solution of WGA (20 μM) in PBS buffer (10 mM, pH 7.4) was added
to a solution of 7 (0.1 mM in PBS) and the concomitant increase in turbidity
was registered by monitoring the absorbance at 677 nm [26]. By dimin-
ishing the concentration of WGA at 10 μM, the signal also decreased,
showing that this effect was concentration dependent. The reversibility of
these interactions was proved by displacing the assembled glycocluster
from the binding sites of the lectin by adding an excess of free GlcNAc
(Fig. 7). A control experiment carried out with bovine serum albumin
(BSA) showed no change, indicating absence of binding. This result ac-
counts for a carbohydrate-protein interaction between 7 and WGA, which
does not exist in the case of 7 and BSA.
9

H.O. Montenegro, et al.
CarbohydrateResearch479(2019)6–12
on silica gel 60 F254 plates (Merck). The spots were detected with 5%
(v/v) sulfuric acid in EtOH, containing 0.5% p-anisaldehyde. Column
chromatography was performed on silica gel 60 (230–400 mesh,
®
Merck) or on reverse-phase silica gel LiChroprep RP-18 (40–63 μm)
MERCK, by elution with the solvents indicated in each case. Pyrene was
purchased from Sigma-Aldrich. Strata C18-E cartridges were purchased
from Phenomenex. A CEM Discover MW instrument with a System
Internal IR probe type, at 70 °C (power max 300 W), was used for the
click reaction. 1H and 13C Nuclear Magnetic Resonance (NMR) spectra
were recorded at 25 °C at 500 and 125.7 MHz, respectively, in a Bruker
Avance II 500 spectrometer. For 1H, 13C chemical shifts are reported in
parts per million relative to tetramethylsilane or a residual solvent peak
(CHCl3: 1H: δ 7.26 ppm, 13C: δ 77.2 ppm). Assignments of 1H and 13C
were assisted by 2D 1H COSY and 2D 1H-13C HSQC experiments. High
resolution mass spectra (HRMS) were obtained by Electrospray
Ionization (ESI) and Q-TOF in a Bruker micrOTOF-Q II spectrometer.
Optical rotations were determined in a PerkinElmer 343 polarimeter, at
20 °C in a 1 dm cell. Melting points were measured in a Fisher-Johns
apparatus.
Fig. 6. Proposed self-assembly mode of two molecules of 7 and molecular di-
mensions.
Absorption spectra were measured on a Cary UV–Vis 50 spectro-
photometer equipped with a full spectrum Xe pulse lamp. Corrected
fluorescence emission spectra were obtained on a Cary Eclipse spec-
trophotometer equipped with two Czerny-Turner monochromators and
a 15 W Xenon pulse lamp (pulse width: 2–3 us, power: 60–75 kW).
Circular dichroism experiments were performed on a Jasco J-815 ORDE
402/15 spectrometer with a Jasco PFD-425S/15 temperature con-
troller. DLS experiments were performed on an Anton-Parr Litesizer™
500 apparatus at 25 °C. SEM pictures were taken on a Carl Zeiss NTS
SUPRA 40FEG scanning electron microscope. A small portion of the
solid sample was attached to the holder by using a conductive adhesive
carbon tape. Prior to examination they were coated with a thin layer of
platinum.
Fig. 7. Turbidity assay of 7 (at a final concentration = 73 μM) at 677 nm with
WGA and BSA proteins. At t = 0.8 min, WGA (at a final concentration = 20 μM
in PBS pH = 7.4, black line), or WGA (at a final concentration = 10 μM in PBS
pH = 7.4, red line), or BSA (at a final concentration = 16 μM in PBS pH = 7.4,
grey line), were added. At t = 10 min or t = 7.8 min a solution of GlcNAc (final
concentration = 35 mM) was added. (For interpretation of the references to
colour in this figure legend, the reader is referred to the Web version of this
article.)
4.2. Synthesis
4.2.1. Synthesis of 3
5-Azido-1-pentanamine hydrochloride (497 mg, 3.02 mmol) was
dissolved in absolute EtOH (40 mL) and TEA (262 μL) was added. After
addition of pyrene carboxaldehyde 1 (695 mg, 3.02 mmol), the mixture
was refluxed for 18 h. An extra addition of TEA (200 μL, 1.44 mmol)
and reflux for 2 additional h, was required for the complete consump-
tion of the starting materials, as detected by TLC analysis
(hexane:EtOAc 1:1). The solution reached room temperature and then,
sodium borohydride (123 mg, 3.25 mmol) was added at 0 °C. The re-
action proceeded for 18 h, and 1 M HCl solution was added at 0 °C until
pH = 5. The solution was concentrated and the product was purified by
silica gel column chromatography, by elution with hexane:EtOAc
(3:7)→ EtOAc → EtOAc:MeOH (8:2), containing 1% TEA. Compound 3
was eluted with EtOAc:MeOH (8:2). Yield: 724 mg, 70%. Rf = 0.5
(EtOAc:MeOH:TEA 8:2:0.01); λmax (nm), 375, 395, 417; 1H NMR
(500 MHz, CDCl3): δ 8.30–7.97 (m, 9H, H-aromatic), 4.48 (s, 2H,
CH2Ar), 3.90 (brs, 1H, NH), 3.19 (t, 2H, J = 6.9 Hz, CH2N3), 2.78 (t,
3. Conclusions
Reductive amination in combination with click chemistry resulted a
convenient methodology for the synthesis of a pyrene-thioGlcNAc de-
rivative. This probe was characterized spectroscopically by UV–Vis,
fluorescence and CD. It showed to be an excellent fluorescent building
block for the formation of a multivalent supramolecular ligand. SEM
microscopy revealed a fibrillar system with 70 Å width fibers, in
agreement with the presence of tubular micelles where the pyrene ring
interact through π−π stacking. This assembly is favored at pH 10, due
to deprotonation of the amine group present in the probe. At lower pH
the self-assembly resulted less effective due to the protonation – de-
protonation equilibrium and consequent repulsion among positive
charges. Still, the aggregation seems sensitive enough to effectively
form crosslinked networks with the WGA at pH 7.4.
a
c
2H, J = 7.4 Hz, CH2NH), 1.65 (m, 2H, CH2), 1.53 (m, 2H, CH2), 1.35
(m, 2H, bCH2); 13C NMR (125.7 MHz, CDCl3): δ 131.3, 131.2, 130.8,
129.4, 128.2, 127.7, 127.6, 127.4, 126.1, 125.5, 125.4, 125.0, 124.9,
124.8, 122.8 (C-aromatic), 51.3 (CN3), 50.4 (CH2Ar), 48.7 (CH2NH),
28.7 (bCH2), 28.4 (aCH2), 24.4 (bCH2). HR-MS(ESI) m/z [M − N3]+
calcd for C22H22N 300.1747, found 300.1743.
The use of the multivalent ensemble formed by the pyrene-
thioGlcNAc amphiphile could be useful for the analysis and detection of
lectins with high selectivity and sensibility. Improvement in the design
on the chemical structure to enhance the self-assembly process in
neutral medium is currently under investigation.
4.2.2. Synthesis of 6. Click reaction
4. Experimental
Compound 3 (350 mg, 1.022 mmol), 2-propynyl 2-acetamido-3,4,6-
tri-O-acetyl-2-deoxy-1-thio-β-D-glucopyranoside
5
[28] (410 mg,
4.1. General procedures and apparatus
1.022 mmol), CuI (97 mg, 0.5 mmol) and DIPEA (890 μL, 5.11 mmol)
were dissolved in DMF (10 mL) and the mixture was stirred at 70 °C
under microwave irradiation during 50 min. The mixture was evapo-
rated, resuspended in CH2Cl2 (150 mL) and extracted with water
Solvents used in the column chromatographic separations were
distilled before use. Thin layer chromatography (TLC) was performed
10

H.O. Montenegro, et al.
CarbohydrateResearch479(2019)6–12
(3 × 50 mL). The organic phase was dried (anh. MgSO4), filtered and
evaporated. Compound 6 was purified by column chromatography
eluting with chloroform → chloroform:MeOH (93:7). Yield: 550 mg,
72%. [α]D20 − 33.5 (c = 1.0, CHCl3); Rf = 0.22 (EtOAc:MeOH:TEA
8:2:0.01); λmax (nm), 375, 395, 417; 1H NMR (500 MHz, CDCl3): δ 8.33
(d, 1H, J = 9.2 Hz), 8.17 (t, 2H, J = 7.5 Hz), 8.12 (d, 2H, J = 8.5 Hz),
8.02 (brs, 2H), 7.99 (m, 2H) (H-aromatic), 7.37 (s, 1H, H-triazole), 6.08
(d, 1H, J2,NH = 9.2 Hz, NHAc), 5.14 (dd, 1H, J2,3 = 9.9, J3,4 = 9.5 Hz,
H-3), 5.08 (dd, 1H, J3,4 = 9.5, J4,5 = 9.8 Hz, H-4), 4.73 (d, 1H,
Acknowledgments
Support for this work from the Universidad de Buenos Aires, the
Agencia Nacional de Promoción Científica y Tecnológica, ANPCyT and
the Consejo Nacional de Investigaciones Científicas
y Técnicas,
CONICET is gratefully acknowledged. Hugo Orlando Montenegro is a
PhD fellow from CONICET. Pablo H. Di Chenna, Carla S. Spagnuolo and
María Laura Uhrig are research members of CONICET.
J
1,2 = 10.4 Hz, H-1), 4.49 (brs, 2H, CH2Ar), 4.25 (t, 2H, J = 7.1 Hz,
Appendix A. Supplementary data
CH2-triazole), 4.20 (dd, 1H, J5,6 = 4.6, J6,6’ = 12.4 Hz, H-6), 4.12 (dd,
1H, J1,2 = 10.4, J2,3 = 9.9, J2,NH = 9.1 Hz, H-2), 4.09 (dd, 1H,
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.carres.2019.04.010.
J
J
J
5,6’ = 2.3, J6,6’ = 12.4 Hz, H-6′), 4.00, 3.79 (2 d, 1H each,
gem = 14.7 Hz, CH2S), 3.64 (dd, 1H, J5,6’ = 2.3, J5,6 = 4.6,
4,5 = 9.8 Hz, H-5), 2.77 (t, 2H, J = 7.1 Hz, CH2N), 2.50 (bs, 1H, NH),
References
2.06, 2.00, 1.99, 1.86 (4s, 3H each, 4 CH3CO), 1.84 (m, 2H, J = 7.4 Hz,
cCH2), 1.60 (m, 2H, J = 7.2 Hz, CH2), 1.36 (m, 2H, J = 7.8 Hz, bCH2);
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a
13C NMR (125.7 MHz, CDCl3): δ 171.1, 170.8, 170.4, 169.5 (CO), 145.2
(C-triazole), 131.4, 131.0, 130.9, 129.3, 127.9, 127.5, 127.4, 126.1,
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12