Selective recognition of ATP by multivalent nano-assemblies of bisimidazolium amphiphiles through “turn-on” fluorescence response

Article abstract of DOI:10.3762/BJOC.16.223

Bisimidazolium receptors, tagged with chromophoric pyrene at one end and linked to an n-alkyl chain at the other, underwent self-assembly in aqueous media depending on the length of the alkyl segment. The amphiphilic derivatives having n-decyl or longer chains, formed nano-assemblies with cyanic-green emission resulting from the stacked pyrene chromophores in the aggregates. The presence of positive surface charges on the multivalent aggregates led to ATP binding which was accompanied by a significant increase in the excimeric emission intensity. This provided a convenient way of monitoring ATP binding in a “turn-on” mode and an efficient detection of ATP was achieved in aqueous buffer and also in buffer containing 150 mM NaCl at physiological pH value. Furthermore, the multivalent aggregates demonstrated a significant selectivity in ATP detection over ADP, AMP and pyrophosphate.

Full text of DOI:10.3762/BJOC.16.223

Selective recognition of ATP by multivalent nano-assemblies
of bisimidazolium amphiphiles through “turn-on”
fluorescence response
Rakesh Biswas, Surya Ghosh, Shubhra Kanti Bhaumik and Supratim Banerjee*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2020, 16, 2728–2738.
Department of Chemical Sciences, Indian Institute of Science
Education and Research Kolkata, Mohanpur-741246, Nadia, India
https://doi.org/10.3762/bjoc.16.223
Received: 01 August 2020
Accepted: 23 October 2020
Published: 10 November 2020
Email:
Supratim Banerjee* - supratim.banerjee@iiserkol.ac.in
* Corresponding author
This article is part of the thematic issue "Molecular recognition" and is
dedicated to the memory of Carsten Schmuck.
Keywords:
amphiphile; ATP; excimer; multivalency; self-assembly
Guest Editor: J. Niemeyer
© 2020 Biswas et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Bisimidazolium receptors, tagged with chromophoric pyrene at one end and linked to an n-alkyl chain at the other, underwent self-
assembly in aqueous media depending on the length of the alkyl segment. The amphiphilic derivatives having n-decyl or longer
chains, formed nano-assemblies with cyanic–green emission resulting from the stacked pyrene chromophores in the aggregates. The
presence of positive surface charges on the multivalent aggregates led to ATP binding which was accompanied by a significant
increase in the excimeric emission intensity. This provided a convenient way of monitoring ATP binding in a “turn-on” mode and
an efficient detection of ATP was achieved in aqueous buffer and also in buffer containing 150 mM NaCl at physiological pH
value. Furthermore, the multivalent aggregates demonstrated a significant selectivity in ATP detection over ADP, AMP and
pyrophosphate.
Introduction
Supramolecular anion sensing is an active area of research materials. Among the different varieties of anionic species, the
which has developed tremendously in the last few decades selective and sensitive detection of bio-anions is particularly
[1-4]. The importance of anion sensing stems from its impor- important due to their involvement in various biological func-
tance in various practical applications which range from identi- tions. One of the challenges in the bio-anion detection is that the
fying and detecting environmentally toxic anions to medical sensory probes have to be effective under physiological condi-
diagnostics. In recent years, anion responsive materials [5,6] tions. The highly polar nature of the aqueous media consider-
have also emerged as an interesting class of stimuli-responsive ably weakens supramolecular interactions such as H-bonding
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and electrostatic interactions and as a result, it is in general [36-38], conjugated polymers [39-42], quantum dots [43,44]
challenging to design high-affinity synthetic probes for anion and sensors based on organic receptors such as imidazolium,
sensing under such competitive conditions [7]. One of the ammonium, guanidinium [45-52], etc. have also been de-
strategies that has proven to be quite successful in this regard, veloped. Imidazolium based synthetic receptors typically utilize
not only for bio-anions, rather for a variety of several other a combination of electrostatic and (C–H)···O− hydrogen bonds
analytes also, is multivalency [8-11]. It is a principle ubiqui- involving the acidic proton at the 2-position of the imidazolium
tously used in biology to achieve high affinity binding events moiety to bind to ATP and other phosphate analytes [46]. A
with examples ranging from protein–carbohydrate interactions number of cyclophanes and tweezers [53-56] have been re-
to host–pathogen interactions or cell surface adhesion [12-14]. ported possessing the following common structural features:
The high affinity originates from the simultaneous interactions a) the presence of multiple imidazolium groups to overcome the
of multiple sites in the receptor with their complementary high hydration enthalpy of phosphates and b) the imidazolium
binding partners in the target analyte [15]. One of the ways to groups are connected to aromatic moieties such as anthracene,
design multivalent systems is to connect the receptors through pyrene, etc. The variation in the luminescence of the aromatic
covalent linkages. Conjugated polymers and conjugated moieties signals the binding event and furthermore, they
polymer electrolytes are prominent examples of covalently provide additional stacking interactions with the nucleobases
constructed multivalent systems and they have been frequently which contributes to the stability of the complexes. In many
exploited for the highly sensitive detection of a diverse range of cases, these stacking interactions provide binding selectivity
target analytes [16-18]. However, the covalent route often among the nucleotides [53-56].
requires time consuming synthetic steps and hence, in recent
years, self-assembled multivalency has emerged as a suitable al- Most of the aforementioned receptors were predominantly “mo-
ternative [19]. In this methodology, multivalent arrays are built lecular” in nature with the binding of the nucleotides occurring
from comparatively smaller binding sites through self- through multivalent electrostatic interactions in the cavity/cleft
assembly. The smaller molecular units are easier to synthesize created by the spatial orientation of the imidazolium groups. We
and moreover, the morphology and the surface functionalities of herein consider a self-assembly based approach in which
the resultant multivalent structures can be tuned in a modular amphiphilic monomers with bisimidazolium moieties are
fashion [20,21]. A number of elegant examples of self-assem- utilized to create multivalent nano-aggregates for binding to the
bled multivalent systems targeting biological analytes such as nucleotides. For this purpose, four pyrene tagged bisimida-
DNA, heparin, proteins, carbohydrates, etc. have been reported zolium-based receptors PBImN (Figure 1, N = 4, 10, 12 and 14;
in the literature [22-27].
N denotes the number of carbons in the n-alkyl chain) were syn-
thesized. The longer chain derivatives with N ≥ 10 underwent
ATP is an important bio-anion that is the energy currency in self-assembly in aqueous buffer and produced nanoaggregates
cells and is involved in a number of essential biological func- with surface exposed positive charges. Interestingly, the aggre-
tions including active transport and cell division [28,29]. For gates displayed cyanic green luminescence which originated
ATP detection, metal complexes using metal ions such as from the excimeric emission of the stacked pyrenes [57-59]. In
Zn(II), Cu(II) and lanthanide ions have been frequently em- a recent work, we have demonstrated that self-assembled
ployed [30-35]. A variety of other systems such as aptamers nanofibrous aggregates of anthracene tagged imidazolium re-
Figure 1: Chemical structures of (a) PBImN (N = 4, 10, 12 and 14) and (b) ATP, ADP and AMP.
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ceptors responded to ATP through an amplified fluorescence ture procedure [62,63] with slight modifications. Then, BImNs
quenching [60]. Based on these results, we wanted to develop (N = 4, 10, 12, 14) were synthesized from 5 by mono-alkyl-
similar self-assembled systems which would show a “turn-on” ation using the corresponding n-alkyl halides in DMF. Finally,
response in the presence of ATP or other phosphate analytes. PBImNs (N = 4, 10, 12, 14) were obtained through nucleo-
The current design of PBImNs is based on this objective and philic substitution reactions between BImNs (N = 4, 10, 12, 14)
we found that the excimeric pyrene emissions in these aggre- and 3. All synthesized compounds were characterized by NMR
gates indeed exhibited a significant increase in the presence of spectroscopy and high-resolution mass spectrometry.
ATP and thus provided a convenient protocol for its detection
Self-assembly properties of PBImNs in
(Scheme 1). Furthermore, this “turn-on” response was achieved
with an appreciable level of selectivity as the response ob- aqueous buffer
served in the presence of other adenosine phosphates, ADP and The self-assembly properties of the four PBImN derivatives in
AMP and even pyrophosphate (PPi) was comparatively much aqueous buffer (5 mM tris-HCl, 10 mM NaCl, water/DMSO
lower than that observed for ATP in a wide concentration range 99.5:0.5, pH 7.4) were investigated using a combination of
(1–100 µM).
absorption and emission spectral studies, dynamic light scat-
tering (DLS) and field emission scanning electron microscopy
(FESEM). In a “good solvent” such as DMSO, in which all
PBImNs were readily soluble, almost identical absorption
Results and Discussion
Synthesis
The synthesis of PBImNs (N = 4, 10, 12, 14) is depicted in (λmax = 347 nm) and emission (λmax = 395 nm) spectra were
Scheme 2 and the elaborate procedures are given in Supporting observed (Supporting Information File 1, Figure S1). In com-
Information File 1. The preparation of compounds 2 and 3 are parison, in aqueous buffer, certain differences existed between
analogous to the procedures from ref. [60]. The pyrene deriva- the butyl derivative PBIm4 and the three longer chain deriva-
tive 1-(bromomethyl)pyrene (3), which was the common tives PBIm10, PBIm12 and PBIm14, hinting at the possible
starting material for making all the PBImNs, was synthesized self-assembly behavior of the latter three molecules. In order to
from pyrene-1-carbaldehyde (1) following a literature report investigate in a detailed manner, we first carried out absorption
[46]. In brief, pyrene-1-carbaldehyde (1) was first converted to measurements at different concentrations (Figure 2a, Support-
pyren-1-ylmethanol (2) by reduction with sodium borohydride ing Information File 1, Figures S2a and S3a). All derivatives
in ethanol [61] and this was followed by treating of 2 with PBr3 displayed three absorption peaks around 314–316 nm,
in toluene to furnish 3. Di(1H-imidazol-1-yl)methane (5) was 327–329 nm and 343–345 nm. These peaks were assigned to
synthesized from imidazole (4) and CH2Br2 following a litera- 0–0 and its vibronic replicas related to an S0–S2 transition in the
Scheme 1: Schematic representation of ATP sensing by multivalent assemblies of PBImN in aqueous media.
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Scheme 2: Synthetic route for the preparation of PBImNs.
pyrene derivatives [64]. The concentration dependence of the tives exhibited nanometer size aggregates (Table 1) whereas
A0–0/A0–1 ratio (A0–0 and A0–1 are the absorbance of the 0–0 PBIm4 did not exhibit any aggregate formation.
and 0–1 vibronic bands at 343–345 nm and 327–329 nm, re-
spectively) generally indicates a possible mode of organization The emission spectra recorded in aqueous buffer (Figure 2b)
of the pyrene chromophores upon self-assembly [63]. It was also showed a significant difference between PBIm4 and the
found that in the concentration range of 5–50 µM [65], al- three amphiphilic derivatives. For example, the amphiphilic de-
though a decrease in this ratio was observed for PBIm4; much rivatives exhibited structured emission bands around 376 nm,
more pronounced reductions were noticed for the N ≥ 10 deriva- 396 nm and 417 nm as well as a broad structureless band
tives (Figure 2a and Supporting Information File 1, Table S1), centered around 480–490 nm (PBIm10: 481 nm, PBIm12:
with the highest change being exhibited by PBIm14. These ob- 485 nm and PBIm14: 490 nm). These were assigned to the
servations suggested that in going to higher concentrations, characteristic monomeric and excimeric emissions of pyrene,
there was a gradual increase in the aggregation of the longer respectively [58]. The intensity of the excimeric emission was
chain derivatives and in the aggregates, the pyrene chro- found be higher with 365 nm excitation (Supporting Informa-
mophores had ground state interactions and were possibly tion File 1, Figure S5; see Figure S6 for the excitation spectra)
stacked in co-facial manner as found in H-aggregates [63]. The and hence this wavelength was used for all the subsequent
aggregate formation was further supported by FESEM images studies. In comparison, PBIm4 displayed a weaker broad band
(Figure 3a and Supporting Information File 1, Figure S7) which at 475 nm along with comparatively more intense monomeric
revealed the formation of elongated nano-objects for PBIm10, bands. Since no aggregate was observed from the DLS studies
PBIm12 and PBIm14. The existence of the aggregates was also in the studied concentration range, it meant that the excimeric
revealed by the DLS measurements. The three N ≥10 deriva- emission presumably resulted from small clusters of PBIm4.
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Figure 2: (a) Absorption and (b) emission spectra of PBImN (50 µM) derivatives in buffer. (c) Absorption and (d) emission spectra of PBIm12 at differ-
ent concentrations in buffer.
Time-correlated single photon counting (TCSPC) measure- values were significantly reduced and eventually became nega-
ments revealed ≈80 ns (Supporting Information File 1, Table tive (Table 1) indicating a strong interaction of the negatively
S2) lifetimes for the 395 nm monomeric peak in DMSO for all charged ATP with the aggregates. An appreciable increase in
the four derivatives and these values correlated well with the re- the size of the nanoaggregates was also observed upon addition
ported lifetimes for monomeric pyrenes [66,67]. In aqueous of ATP (Table 1) pointing to the formation of larger co-aggre-
buffer, the lifetime of the 395 nm band decreased (≈15–35 ns, gates. Furthermore, a visible change in morphology for the
Supporting Information File 1, Table S3) probably owing to the co-aggregates was observed from FESEM images (Figure 3b).
increment in the solvent polarity [68]. The average lifetime All these results, taken together, indicated that the nanoaggre-
values (τavg) of the excimeric bands of PBIm10, PBIm12 and gates were able to recognize ATP in a multivalent fashion
PBIm14 were found to be 33.97 ns (481 nm), 34.44 ns leading to the formation of larger co-aggregates. On the con-
(485 nm) and 33.27 ns (490 nm), respectively (Supporting trary, PBIm4 did not show the formation of aggregates even
Information File 1, Table S3). The large Stokes shifts and the after the addition of ATP.
absence of vibronic features are typical characteristics of
excimeric emission of pyrene [69,70].
The most interesting feature of the ATP binding was the
remarkable increase in the excimeric emission of the nano-
aggregates (Figure 3c and Supporting Information File 1,
ATP binding studies
The aggregates with N ≥ 10 exhibited positive zeta potential (ζ) Figure S8b and S8c). The sensitivity of the detection was corre-
values under the studied conditions (Table 1) suggesting that lated to the I/I0 ratio (I and I0 are the emission intensity of the
they possessed surface exposed positive charges. Based on this, excimeric bands with and without ATP). PBIm12 (75 µM)
we envisaged that they could be potential ATP binders in displayed the best sensitivity among the three amphiphilic de-
aqueous buffer. Indeed, upon addition of ATP, zeta potential (ζ) rivatives having a significantly enhanced I/I0 value of 10.6 upon
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Figure 3: FESEM images of PBIm12 (a) without and (b) with ATP. (c) Emission spectral changes of PBIm12 (75 µM) in buffer upon addition of ATP.
(d) Comparison plot for I/I0 of PBIm10 (100 µM), PBIm12 (75 µM) and PBIm14 (30 µM) upon addition of different amounts of ATP.
Table 1: DLS data for the average size and zeta potential (ζ) of the self-assembled nanoaggregates of PBIm10, PBIm12 and PBIm14 before and
after ATP addition in aqueous buffer (5 mM tris-HCl, 10 mM NaCl, water/DMSO, 99.5:0.5, pH 7.4).
PBImN derivative
Size (nm) w/o- ATP
Size (nm) w/- ATP
(50 µM)
ζ (mV) w/o- ATP
ζ (mV) w/- ATP
(50 µM)
PBIm10
(150 µM)
619.5
689.2
621.3
1582
1449
816
3.45
2.41
14
–10.1
–8.02
–6.53
PBIm12
(75 µM)
PBIm14
(50 µM)
50 µM ATP addition whereas for PBIm10 (100 µM) and of the lower limit of detection, ATP was detected at 500 nM
PBIm14 (30 µM) the values were 2.3 and 5.8, respectively concentration by the PBIm12 aggregates. A noticeable increase
(Figure 3d). It is noteworthy to mention here that for each of the in the average lifetime (τavg) of the excimeric band was ob-
amphiphiles, the best response was obtained at an optimum con- served after ATP addition (Supporting Information File 1, Table
centration of the amphiphiles. For example, both at a concentra- S4). For example, in case of PBIm12 (75 µM), it increased
tion lower and higher than 75 µM, PBIm12 exhibited lower from 34.44 ns to 41.99 ns upon ATP (50 µM) addition (Sup-
responses (Supporting Information File 1, Figure S9). In terms porting Information File 1, Table S4). ATP also induced sub-
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stantial changes in the absorption spectra for the amphiphilic positive charges either in “molecular” form or as clusters and
derivatives (Supporting Information File 1, Figure S10b and further signified the requirement of a multivalent organization
10c). The absorption maxima red shifted slightly and increased of the charges as provided by the aggregates.
scattering was observed at higher amounts of ATP. The
A0–0/A0–1 values were also found to decrease upon formation of Selectivity in the ATP detection
the co-aggregates with ATP (Supporting Information File 1, One of the key issues of a sensor design is the selectivity for an
Table S5).
analyte in comparison to other interfering analytes. Therefore,
we carried out selectivity studies with the multivalent sensors
The small-chain analogue, PBIm4 (100 µM), on the contrary, for ATP over the other adenosine phosphates ADP and AMP,
showed only nominal changes in its monomeric or excimeric PPi and inorganic phosphate (Pi). We employed PBIm12
emission upon ATP addition (Supporting Information File 1, (75 µM) for this purpose as it exhibited the best sensitivity in
Figure S8a). It has already been pointed out that under these the detection of ATP. A significant selectivity was noticed for
conditions, PBIm4 was mainly present in monomeric form ATP over ADP and AMP, PPi and Pi (Figure 4a and Figure 4b).
along with some proportions of small clusters. Previous litera- For example, the I/I0 value at 485 nm for PBIm12 (75 µM) in-
ture reports have shown that for “molecular” imidazolium- creased to 10.62 upon ATP addition (50 µM), whereas for PPi
based sensors, typically four positive charges [46,54,56] were (50 µM) and ADP (50 µM), these values were 4.8 and 1.87, re-
necessary to accomplish ATP detection in the micromolar con- spectively (Figure 4b). Practically no increments were ob-
centration range. The lack of an ATP detection capability of served for AMP (50 µM) and Pi (50 µM) (Supporting Informa-
PBIm4, therefore, clearly illustrated the point that under these tion File 1, Figure S11a and Figure 4b). As the multivalent
conditions, ATP binding was difficult for a receptor with two binding is predominantly through electrostatic interactions, it
Figure 4: a) Emission changes of PBIm12 (75 µM) upon the addition of ATP, ADP, AMP and PPi in buffer. Bar diagram comparing the I/I0 values
(485 nm) of PBIm12 (75 µM); b) upon addition of ATP, ADP, AMP, PPi and Pi in a buffer (each at 50 µM) and c) with only ATP (50 µM) (A) and a mix-
ture of ATP, ADP, AMP, PPi and Pi (50 µM of each) (B) in buffer. d) Emission spectra of PBIm12 (100 μM) upon the addition of ATP in a buffer con-
taining 150 mM NaCl.
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was not surprising to see a selectivity towards ATP (3 negative and pyrophosphate (PPi). The detection of ATP was also
charges) over ADP and AMP (2 and 1 negative charge(s), re- possible in the presence of 150 mM NaCl. The modular nature
spectively) at physiological pH. However, it was intriguing to of the sensing platform provided by the multivalent nano-
note the selectivity over PPi which had four negative charges. It assemblies of the bisimidazolium amphiphiles can in principle
is likely that in the ATP induced co-assemblies, there is a more be extended for the detection of other biologically important
compact arrangement of the stacked pyrene chromophores and analytes through a structural modulation of the amphiphiles and
this restriction of internal motion might have resulted in a the studies along this direction are currently in progress in our
higher excimer intensity [71]. In order to see whether the pres- group.
ence of other phosphate analytes interfered in ATP detection,
Experimental
(50 µM) and ATP (50 µM) in the presence of a mixture of Materials and methods
fluorescence enhancements of PBIm12 (75 µM), for only ATP
ADP/AMP/PPi/Pi (50 µM of each analyte) were compared. As All starting materials were purchased from commercially avail-
can be seen from Figure 4c, even in the presence of four other able sources and used without further purification. All the
phosphate analytes with a total concentration of 200 µM, the nucleotides, 1-pyrenecarboxaldehyde, sodium borohydride, so-
I/I0 ratio only showed a marginal decrease compared to that ob- dium hydride, phosphorus tribromide and 1-bromobutane,
tained in the presence of only ATP. This experiment thus 1-bromodecane, 1-bromododecane and 1-bromotetradecane
clearly showed that the selectivity towards ATP was retained were purchased from Sigma-Aldrich. Imidazole, dibro-
even in the presence of other phosphates. PBIm12 (75 µM) was momethane and tris(hydroxymethyl)aminomethane were pur-
also able to discriminate ATP from GTP up to 30 µM concen- chased from TCI chemicals. Sodium chloride, 28% ammonia
tration (Supporting Information File 1, Figure S13b). The solution and all the solvents were purchased from Merck. Milli-
response of PBIm12 was also examined in the presence of Q water was used for all the experiments. UV–vis spectroscop-
many other anions such as iodide, bromide, fluoride, acetate, ic measurements were carried out in Agilent Cary 8454 spec-
bicarbonate, sulfate, etc. and all of them exhibited only negli- trophotometer. Emission spectroscopic measurements were
gible changes in the excimeric emission (Supporting Informa- carried out in a Horiba Fluoromax 4 spectrofluorometer. Images
tion File 1, Figure S14).
were taken under a 365 nm UV lamp. DLS and zeta potential
measurements were carried out using a Malvern Zetasizer
NanoZS. A Horiba Jobin Yvon Fluorocube instrument fitted
with a 340 nm diode laser excitation source (with a temporal
ATP detection in the presence of 150 mM
NaCl
In order to demonstrate the practical utility of the sensors, ATP resolution of 70 ps) was used for the time-resolved fluores-
binding studies were investigated in a highly competitive medi- cence experiments applying the time correlated single photon
um, i.e., a buffer containing 150 mM NaCl. In a medium of counting (TCSPC) method. 1H and 13C NMR were performed
such a high ionic strength, the binding affinity typically gets on Jeol 400 MHz and Bruker 500 MHz spectrometers. Mass
diminished by electrostatic screening [72]. Although the spectra were recorded in a Bruker mass spectrometer. SEM
response was comparatively much lower than that observed in images was recorded by using the instrument CARL ZEISS
the plain buffer, ATP detection was possible in the concentra- (model SUPRA 55VP).
tion range of 200 µM to 5 mM using PBIm12 (100 µM,
Figure 4d) and this covered the typical concentration values of Preparation of solutions
ATP in cell (3–5 mM) [73].
A. Preparation of PBImN solutions
Initially, stock solutions of PBImNs (N = 4, 10, 12, 14) were
prepared by dissolving the solid powders in spectroscopic grade
Conclusion
In conclusion, we present here the synthesis, self-assembly and dimethyl sulfoxide (DMSO). These concentrated DMSO solu-
phosphate binding properties of four imidazolium-based recep- tions were then diluted to 5 mM tris-HCl buffer in 10 mM NaCl
tors PBImN (N = 4, 10, 12, 14). The derivatives with N ≥ 10 made in Milli-Q water to get the desired solutions having 1 or
chains were amphiphilic in nature and underwent self-assembly 0.5% DMSO in the final DMSO fraction. Solutions of PBIm4
in aqueous buffer to produce nano-assemblies that displayed in the buffer were equilibrated for 10 min whereas the solutions
excimeric emission from the stacked pyrene chromophores. The of PBIm10, PBIm12 and PBIm14 were equilibrated for
positive surface potential values of the nano-assemblies allowed 15 min.
them to bind to ATP which was accompanied by a noticeable
increase in the intensity of the excimeric emission. Furthermore, B. Preparation of stock solutions of nucleotides
the multivalent assemblies displayed a high-level selectivity Stock solutions of nucleotides (10 mM) were prepared by
towards ATP over other adenosine phosphates (ADP and AMP) dissolving the required amounts of them in Milli-Q water.
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first stabilized for 15 min and then 5 µL of these solutions were
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Funding
IISER Kolkata and INSPIRE are acknowledged for research
fellowships to RB and SKB, respectively. We are thankful to
the Science and Engineering Research Board (SERB), Depart-
ment of Science and Technology (DST), Government of India
(project: EMR/2016/002838) and Indian Institute of Science
Education and Research Kolkata for support of this work.
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