Novel pyrene-calix[4]arene derivatives as highly sensitive sensors for nucleotides, DNA and RNA

Covalent functionalization of a calix[4]arene with one or two pyrene arms at one rim and two imidazoles at the opposite rim of the macrocyclic basket, yields fluorescent conjugates characterized by intramolecular pyrene-calixarene exciplex emission of a mono-pyrene conjugate, whereas the bis-pyrene derivative exhibits pyrene excimer fluorescence. The pyrene emission in these novel compounds is shown to be sensitive to non-covalent interactions with both mono- and polynucleotides. Pyrene-calixarene conjugates, acting as host molecules, strongly interact with nucleotides, as monitored by moderate emission quenching, reaching 0.1 μM affinities, comparable to some of the most effective supramolecular sensors for nucleotides. These compounds are efficiently inserted into ds-DNA/RNA grooves, with a high, 0.1–1 μM affinity, not influencing significantly any of the ds-polynucleotide native properties, whereby complete emission quenching allows the detection of DNA at nM concentration.


Introduction
6][7][8] Some of these systems were recently studied by us as possible candidates for DNA/RNA binding in a frame of a wider project oriented towards the research of DNA/RNA recognition by specially designed calixarene derivatives. 9,10][13] The covalently linked pyrene functional group, belonging to a group of polycyclic aromatic hydrocarbons (PAH), is especially interesting to us due to its unique properties like intense blue emission, high uorescence quantum yield, long-lived singlet excited state, long emission lifetime (>100 ns), as well as its pronounced hydrophobicity.5][16][17][18] Pyrene can form variety of non-covalent interactions with DNA/RNA, like aromatic stacking intercalation into DNA/RNA, binding into the DNA minor groove via a combination of hydrophobic and edge-to-face aromatic interactions, or by forming pyrene excimer within the DNA minor or RNA major groove.Pyrene is also prone to form exciplex in combination with other chromophores. 19,202][23][24][25][26][27] In a design of uorescent sensors, an interesting approach is to attach two aromatic uorophores close enough so that the electronic excitation of one ring can cause an enhanced interaction of its neighbour, 28 leading to an excited-state dimer or excimer. 29In particular, calix [4]arene derivatives bearing two highly p-delocalized planar systems such as pyrenes, display an efficient excimer signal due to the intra-or intermolecular p-p interactions between the two pyrenes, and this excimer emission can be perturbed in presence of guest ions/molecules. 30For these reasons, pyrene-armed calix [4]arenes are widely used as uorescent chemical sensors capable of selectively recognizing toxic cations, 31,32 uoride ions, 33 and biologically and environmentally relevant anions such as cyanide, lactate, nitrite, nitrate and borate, and show potential analytical applications in environmental and biological areas. 34n order to enhance the capability of our calixarene based systems to bind to DNA or RNA, and to ensure efficient tracking of this binding by means of uorometric methods, we have designed and synthesized a new series of calixarene derivatives, by graing different uorophores to the calixarene basket.We have previously demonstrated that particular cationic calixarene dimers bind into ds-DNA major groove, while monomeric cationic calixarenes revealed different DNA-binding proles, likely to be positioned within the DNA minor groove. 10However, at that point, being focused on dimeric calixarenes, we did not study the interactions between the monomeric calixarenes and DNA/RNA systematically.Only recently we partially addressed that issue by studying interactions of neutral and cationic calixarenes with nucleotides and DNA, revealing the importance of positive charge for efficient binding to DNA. 9 In this paper, we report on the synthesis, photochemical and photophysical characterization of pyrene-calixarene conjugates 2 and 3 (Scheme 1), including quantum yield and uorescence lifetime measurements, as well as the binding affinity of these compounds towards mononucleotides and DNA/RNA chains (ctDNA, p(dAdT) 2 , pApU, p(dGdC) 2 ).The stability constants of the formed complexes were determined from UV-Vis and/or uorescence titration data.The classical molecular dynamics simulations were carried out to gain a detailed insight into the structure of calixarene-pyrene conjugates.Circular dichroism (CD) spectropolarimetry was used to study conformational changes in the secondary structure of polynucleotides upon the addition of calixarenes, while the effect on the thermal stability of polynucleotides was investigated by calculating the melting temperature from thermal denaturation curves.To the best of our knowledge, this is the rst study on the recognition of mono-and polynucleotides by calixarene-pyrene conjugates.

Synthesis
Novel compounds were synthesized as described in Scheme 2, by a one-step coupling of pyrene-butyric acid activated by HOBT/ HBTU with calix(4)arene diamine 1, 35

Spectrophotometric characterisation of 2 and 3
Compounds 2 and 3 are poorly soluble in water, hence for the purpose of a biorelevant study, we have prepared their stock solutions in DMSO (5 mM), which were stable at 4 °C.For all further experiments, aliquots of stock solutions were diluted in buffer (sodium cacodylate buffer, I = 50 mM, pH 7.0) prior to measurement.It should be noted that at pH 7, two imidazole units are partially protonated (pK a = 7), thus compounds have a net +1 positive charge.The absorbances of aqueous solutions of 2 and 3 were proportional to their concentrations up to c = 2 × 10 −5 M (ESI, Fig. S7 †).The absorption maxima and their corresponding molar extinction coefficients are given in Table 1.
Comparison of the UV/Vis spectra (Fig. 1a) reveals a strong bathochromic and hypochromic effect of conjugates 2 and 3 in respect to the referent chromophore (1-pyrenebutyric acid, A).As A is free of any hydrophobic or aromatic interaction, obtained spectra strongly suggest intramolecular aromatic stacking interactions in 2 and 3.The analogous UV/Vis spectra collected in DMSO (Fig. 1b) reveal less pronounced but still present bathochromic and hypochromic effects in 2 and 3, thus demonstrating that DMSO did not completely cancel intra-or inter-molecular interactions of pyrene in 2 and 3.
Both studied derivatives show uorescence emission in aqueous and DMSO solutions (Fig. 2), with emission intensities proportional to their concentration up to c = 2 × 10 −6 M (ESI, Fig. S8 †), thus excluding intermolecular interactions.Intriguingly, 2 and 3 show the strongest bathochromic shi of emission maximum (+70 nm) with respect to the referent 1-pyrenebutyric acid (A), and similar to the previously noted effect of referent peptide B (Fig. 2, top), suggesting the formation of either pyrene excimer or exciplex between pyrene and calixarene aromatic moieties. 36The temperature variation induced pronounced changes in the uorescence spectra of the conjugates 2 and 3 (ESI, Fig. S9 and S10 †), conrming intramolecular interactions of the chromophores.However, mono-pyrene analogue 2 showed minor peaks in the 370-430 nm range (in a good agreement with spectrum of A), suggesting that part of the pyrene in 2 is freely exposed to water, thus not in the excimer/exciplex form.Adversely to 2, bis-Scheme 1 Molecular structures of novel calix [4]arene mono-(2) and bis-pyrene (3) derivatives studied in this work, and of referent compounds: previously studied analogue with two cationic triazole pendant arms (4), 35 1-pyrenebutyric acid (A) and pyrene-tryptophane peptide (B). 36heme 2 Synthesis of calix [4]arene-pyrene conjugates 2 and 3 from 1.
pyrene analogue 3 reveals a spectrum in water with only a single maximum at 470 nm, whereas its uorescence spectrum in DMSO coincides to a great extent with the spectrum of reference A, with only a minor emission at 480 nm, pointing out that even in DMSO not all pyrene chromophores in 3 are void of excimer/exciplex interactions.
At this point, question arose whether the observed strongly shied emissions of 2 and 3 are due to the pyrene excimer formed between two pyrene units or due to the pyrenecalixarene exciplex formation.For mono-pyrene derivative 2, linear dependence of emission intensity on c(2) excluded intermolecular stacking of two molecules of 2 for pyrene excimer, thus observed effect is likely the same as noted for referent peptide B: 36 formation of intramolecular pyrene exciplex with another aromatic unit of the same molecule.
If pyrene forms intramolecular aromatic stacking interactions with imidazole, the stability and emission response of such exciplex should be strongly affected by protonation/ deprotonation of imidazole (pK a 7).Emission spectra of 2 and 3 collected at pH = 6-8.5 (ESI, Fig. S11 †) reveal only a minor difference in intensity but the shape of the emission spectrum remains unchanged, suggesting that imidazole is not in interaction with pyrene(s).
In order to obtain a deeper insight into the photophysical processes in solutions of 2 and 3, we have collected timeresolved uorescence decay data (s in Table 1, ESI, Fig. S12 and S13 †) and compared them with two referent compounds, A (free, non-stacked pyrene) and B (pyrene exciplex with another aromatic unit). 36The data were collected at two emission wavelengths: 475 nm (characteristic for excimer/exciplex) and 400 nm (characteristic for non-stacked pyrene), and in two solvents, DMSO and water (only the latter stimulates aromatic stacking and hydrophobic interactions).
Analysis of time-resolved uorescence decay data (s in Table 1) shows (at 400 nm) the presence of long-lived emitting species in all cases, attributed to the free pyrene uorophore (in comparison to reference A), thus not in the interaction with another aromatic moiety (e.g.calixarene or another pyrene).Data collected at 475 nm reveal the presence of several shorterliving emissive species, which are by comparison to reference B and literature data 37 attributed to the pyrene excimer or exciplex.In an aqueous medium, analogues 2 and 3 show a similar distribution of three species, whereby two values of similar  magnitude dominate (12.66 and 47.23 ns for 2; 26.69 and 64.65 ns for 3).In DMSO, at 475 nm both 2 and 3 show very similar dominant values, 47.60 or 49.57ns, respectively, pointing out that upon exclusion of water, one dominant type of pyrene stacked with another aromatic moiety remains.For monopyrene analogue 2, this can only be a pyrene stacked with a calixarene aromatic system (exciplex similar to reference B), and an almost identical s value observed for bis-pyrene 3 suggests similar intramolecular interaction.However, in water, bis-pyrene 3 might form, besides an exciplex, an intramolecular pyrene excimer with calixarene aromatics.Thus in both, 2 and 3, the pyrene uorophore(s) is/are in contact with the calix [4]  arene core and, consequently, the uorescence of 2 and 3 should be very sensitive to any binding event with DNA/RNA targets.

Molecular modelling
In order to clarify whether the dominant interaction is formation of excimer (stacking between two pyrene units) or exciplex (pyrene-benzene p-p interaction), we have performed molecular dynamics simulations of 2 and 3. Attaching one or two relatively large pyrene groups to calixarene scaffold can induce conformational changes in solution due to a free rotation of these "legs" around the linkers (in 2 and 3 these linkers contain three methylene groups).Some previous studies have revealed the possibility of an inclusion of single pyrene unit into the calixarene 25,28 or cyclodextrine 34 macrocyclic cavity which signicantly inuences the uorescence sensing.
The initial structures of calixarenes 2 and 3 for molecular dynamics simulations were prepared in cone conformation with pyrene rings stretched below the calixarene lower rim.Aer 10 ns MD simulation (Fig. 3a) in water, in conjugate 2 pyrene forms aromatic stacking interactions with the benzene ring from the calixarene basket, which supports the hypothesis of exciplex formation (vide supra).The structure of its bis-pyrene analogue 3 aer 10 ns MD simulation (Fig. 3b) was characterized by two mutually parallel pyrene arms with average distances between corresponding carbon atoms in the range of 3-4 Å, supporting pyrene excimer formation.Hence, although both conjugates have the emission maxima at 475 nm, the origin of this emission can be attributed to non-covalent interactions between different moieties.
The encapsulation of pyrene uorophore(s) between pendant "legs" or into the cavity was not observed because of   steric hindrances.Peptide bond is planar without possibility of rotation limiting the possibility of bending a calix-pyrene linker.Furthermore, the energy penalty for reorientation of functional groups and extending the calix [4]arene cavity for inclusion of pyrene would be too large.Hence, the formation of exciplex for 2 is achieved on the outside of the cavity meaning that pyrene is partially exposed to water molecules which correlates with the experimental minor uorescence peaks at 370-430 nm for free pyrene.
Both 2 and 3 are versatile macrocycles with plethora of possibilities to accommodate guests like ions, small molecules, solvents, etc. Conformations of the ligands are expected to be altered upon the complexation since the upper and lower rim groups orientate inwards to encapsulate the guest in the binding site, while in the host-guest complex, due to variety of non-covalent interactions induced by inclusion, the structure of calixarene becomes rigidied upon inclusion.In other words, aromatic stacking interactions as well as hydrogen bonds of electron-donating atoms (e.g.oxygen and nitrogen) with guest molecules may determine the structure and size of the binding site.
Due to the steric hindrance of methylimidazoles on the upper and pyrene and tert-butyl groups on the lower rim, the binding sites at the two rims are asymmetric and the relative orientation of the functional subunits is different in conjugates 2 and 3. 2 forms stacking interactions with benzene ring causing signicantly larger distance between methylimidazoles as compared to 3. For instance, the distances between oxygen -OCH 2 Im atoms for calixarene 2 are 5.0 Å and 3.5 Å for 3. On the other hand, the methoxy oxygen atoms are signicantly closer in 2 as compared to 3: d(2) = 3.6 Å, d(3) = 5.7 Å.The distances between free N-atoms of methylimidazole are approximately the same for 2 and 3, around 5.2 Å.On the lower rim, in the binding site of 2 the N-N amino groups are separated for approximately 11.5 Å, while for 3 peptide bond oxygen atoms are oriented towards each other due to p-p interaction between pyrenes: d(O-O) = 3 Å.Tert-butyl groups are practically parallel in monopyrene derivative while for bis-pyrene derivative they are oriented outside of the calix-rim to reduce steric hindrance.Considering all these structural effects, the upper rim of calixarene 2 is narrow and the lower rim is extended, while for 3 stacking of pyrenes reduces the size of binding site and induces the rotation of benzene and imidazole moieties on upper rim giving a wider binding site.
Calculations were also performed for cationic +1 forms of 2 and 3 since at pH = 7 two imidazole moieties are partially protonated forming an imidazolium ion.The MD simulations started from the same initial structures as for the neutral conjugates, but with a proton added to one of the free N-atoms of imidazoles.The results revealed that protonation of imidazoles does not induce signicant conformational changes when considering the orientation of pyrene subunit relative to calixarene scaffold.This is especially true for bis-pyrene derivative where the protonation of imidazole cannot disrupt very strong hydrophobic interactions between pyrenes on the opposite rim.However, the positively charged imidazolium arm of monopyrene tends to move towards p-electron rich pyrene subunit during the simulation but the relative orientation of rings is not so favourable for aromatic stacking interactions, as for pyrenebenzene ring.This supports the emission spectra of 2 and 3 which change only slightly when changing pH from 6 to 8.5 (ESI, Fig. S11 †) suggesting that imidazolium is not in direct interaction with pyrene.
Non-covalent interactions of 2 and 3 with various mononucleotides (AMP, CMP, GMP, and UMP) and ds-DNA/ ds-RNA Spectrophotometric titrations.As many pyrene derivatives exhibit captivating biorelevant interactions, and, as strong chromophores and uorophores, act as probes for various DNA/ RNA sequences, we performed preliminary studies on interactions of derivatives 2 and 3 with several mononucleotides and with the most commonly used ds-DNA representativesnaturally isolated calf thymus (ct)-DNA characterized by typical Bhelix and approximately equimolar number of G-C and A-T base pairs, synthetic polynucleotides poly(dAdT) 2 , and poly(dGdC) 2 also having B-helical structure, as well as with the analogous synthetic ds-RNA representative: poly A-poly U, characterized by A-helical structure.
In general, the addition of any mononucleotide or ds-DNA/ ds-RNA to 2 or 3 resulted only in moderate hypochromic effect in 2 or 3 UV/Vis spectrum (ESI, Fig. S15-S22 and S26-S33 †), typical for the engagement of the pyrene chromophore in aromatic stacking interactions. 37UV/Vis spectra changes of both studied conjugates show no selectivity towards any of the mono-/polynucleotides employed (ESI, Fig. S15-S22 and S26-S33 †).
The titrations result with dominant changes of 2 and 3 UV/ Vis spectra at excess of each studied dye to mono-/ polynucleotide, which is unfavourable for the determination of single molecule interaction with DNA/RNA.Namely, at such conditions, dyes tend to aggregate along the polynucleotide, 42 which can result in the apparent increase of binding affinity caused by monitoring the spectroscopic changes of two events (de-aggregation and nucleotide binding) simultaneously.
Fortunately, strong uorescence of 2 and 3 allowed us to perform uorometric titrations at much lower molar ratios r = [dye]/[polynucleotide] (r # 1), and at a concentration of dye at which no intermolecular aggregation is present (5.0 × 10 −7 M).The incubation aer every addition was two minutes.It was determined experimentally that aer one minute upon each aliquot addition no further changes in spectrum occurred.Thus, double time (two minutes) was considered adequate for the incubation to ensure thermodynamic equilibrium.These conditions allowed accurate processing of titration data by non-linear tting procedure to Scatchard equation, [43][44][45] or for the mononucleotides the best t was obtained for 1 : 1 stoichiometry of dye/ NMP complex formed (Table 2).The addition of any mononucleotide or ds-DNA/ds-RNA to 2 and 3 resulted in quenching their emission (Fig. 4 and 5; ESI; Fig. S23-S25 and S34-S36 †).
Upon addition of nucleotide or DNA/RNA, emission of mono-pyrene 2 was strongly quenched only at exciplex maximum at 475 nm, while emission at 390 nm (attributed to the free pyrene) only negligibly changed.Such changes would imply that a complexation event causes major disruption of pyrene-calixarene exciplex.The pyrene-excimer emission of bispyrene analogue 3 was partially quenched by nucleotides (Fig. 4) and completely abolished by ds-DNA/RNA (Fig. 5).
Detailed analysis of titration experiments reveal that the binding constants obtained by UV/Vis titrations (ESI; Table S2, † log K) differ somewhat in comparison to those resulting from uorometric titrations (Table 2), likely due to the unfavourably high concentrations of 2 or 3, causing partial aggregation of dye at high rations r[dye]/[nucleotide or DNA].Thus, the log K values obtained by uorometric titrations are more reliable and could be compared with data previously obtained for 1 and 4.
The comparison of obtained data for 2 or 3 with those previously published 9 for referent calixarene 1 and its permanently 2+ charged analogue 4, reveals several intriguing points.The pyrene-containing calixarenes 2 and 3 bind nucleotides with similar affinity (Table 2), but at least an order of magnitude stronger in comparison to referent 1 or permanently 2+ charged analogue 4. Particularly signicant differences are observed for pyrimidine nucleotides (UMP, CMP), which 2 and 3 bind two or even three orders of magnitude stronger than 1 or 4. Taking into account that only 2 and 3 contain pyrene moieties, such differences strongly support aromatic stacking interactions between pyrene and nucleobase as the main reason for increased affinity as compared to 1 or 4; agreeing well with   previous binding studies of pyrene-containing derivatives with nucleotides under comparable conditions, giving values of log K ∼ 2. 46 Summarised results are implying a general rule for hereby studied series of calixarenes: graing the pyrene subunits to calixarene analogue 1 increases the stability constant of the calixarenemononucleotide non-covalent complex by a factor of 10-100.The interactions of studied calixarenes with ds-DNA or ds-RNA are signicantly different in comparison to those with nucleotides, by means that nucleotides "insert" into the structure of calixarenes, whereas calixarenes "insert" into grooves of polynucleotides. 10We showed previously 9 that referent calixarene 1 does not interact with DNA/RNA unless a positive charge is introduced into the structure, as is the case in the analogue 4.However, hereby obtained results (Table 2) reveal that pyrenecontaining conjugates 2 and 3 strongly interact with DNA/RNA even if they have no permanent positive charge whatsoever.
The question remains which type of interaction with DNA/ RNA pyrenes attached to 2 and 3 provide: (i) intercalation between DNA-base pairs; or (ii) hydrophobic-driven insertion into DNA/RNA grooves with possible edge-to-face aromatic interactions between pyrene and nucleobases.Pyrene UV/vis and uorescence spectra cannot give accurate cues on this aspect, thus more structurally informative methods are needed.Insufficient solubility of 2 and 3 in water, however, hampered NMR experiments with oligomeric DNA.

Circular dichroism (CD) experiments
To gain an additional insight into the structural mode of binding of our conjugates 2 and 3 to ds-DNA and ds-RNA we have employed circular dichroism (CD) spectroscopy.CD spectroscopy is a useful analytical tool to study the interactions of small molecules with chiral macromolecules such as DNA, 47 since it can provide information on the mode of binding to a polynucleotide, with distinctive spectral differences for intercalators and groove-binding derivatives. 48,49oth 2 and 3 are achiral with no measurable CD bands within the 240-400 nm range (ESI, Fig. S37 †), thus not interfering with the CD bands of DNA/RNA in the 240-290 nm range, nor with the eventually induced CD bands of the pyrenes, which could appear at >300 nm upon binding to ds-DNA/RNA.
The results of the titration data (ESI, Fig. S38-S43, † the molar ratios r(dye/DNA) = 0.1, 0.3 and 0.5) reveal the negligible inuence of the studied calixarene derivatives 2 and 3 on the helicity of studied polynucleotides, thus excluding classical intercalation of pyrene between base pairs. 48Moreover, in the range >300 nm no induced CD bands were observed, suggesting that upon binding to DNA/RNA, pyrene moieties of 2 and 3 are not uniformly oriented in respect to the polynucleotide chiral axis.Thus, obtained results support the non-specic binding of 2 and 3 within DNA/RNA grooves.

Thermal denaturation of ds-DNA/ds-RNA
Thermally-induced dissociation of the ds-polynucleotides into two single-stranded polynucleotides occurs at a well-dened temperature (T m value), thus being used for the characterization of various ds-DNA or ds-RNA-related processes.Non-covalent binding of small molecules to ds-polynucleotides usually increases the thermal stability of the ds-helices, thus resulting in an increased T m value, and this increase (DT m ) can (in corroboration with other methods) be related to the various binding modes. 50For example, most pyrene analogues by intercalating into ds-DNA/ds-RNA, cause stabilization for DT m > +5 °C due to aromatic stacking interactions with polynucleotide base pairs, whereas the binding of pyrenes within the polynucleotide groove, usually driven mostly by a hydrophobic effect accompanied by weak H-bonding, should have a negligible stabilizing outcome (DT m < 2). 51In our previous study of referent calixarenes 1 and 4, 9 we demonstrated that electrostatic interactions are controlling the thermal stabilization effect, neutral calixarene 1 having no impact on ds-DNA/RNA thermal denaturation, unless it is in cationic form (achieved by adding positively charged substituentsanalogue 4).
The thermal denaturation results revealed that adding 2 or 3 did not stabilize ct-DNA or ds-RNA against thermal denaturation (ESI; Table S3, Fig. S44 and S45, † the molar ratios, r(dye/ DNA) = 0.2 and 0.3) which supports the hypothesis that these conjugates bind to the grooves, rather than by intercalation.17a,50

Conclusions
Newly designed and prepared pyrene-containing calix [4]arene conjugates 2 and 3 are characterized by studying their distinct intramolecular aromatic stacking interactions in an aqueous medium.Strong bathochromic shi of mono-pyrene derivative 2 uorescence emission relative to the free pyrene maximum is attributed to the formation of pyrene-calixarene exciplex, whereas a similar effect observed for bis-pyrene derivative 3 is a consequence of the intramolecular pyrene-excimer formation.
Since the emissions of 2 and 3 are controlled by intramolecular aromatic stacking interactions, it is necessarily exceedingly sensitive to any kind of interaction with other molecules, which would cause a conformational change of calixarene and/or modify the microenvironment around pyrene uorophores.
Pyrene-calix [4]arene conjugates 2 and 3 strongly interact with mononucleotides, combining hydrophobic interactions of parent calix [4]arene 1 (ref.8][39] Although no selectivity between recognition of various nucleotides by 2 and 3 was observed, these systems, with appropriate additional structural modications to enable selective recognition of different mononucleotides, thanks to their extraordinary efficient nucleotide binding capacity, can easily evolve to new lead compounds. Novel pyrene-calix [4]arene conjugates 2 and 3 bind into ds-DNA or ds-RNA grooves non-specically, with high (0.1-1 mM) affinity, not inuencing signicantly any of the dspolynucleotide native properties (secondary structure and thermal denaturation point).The intrinsic emission of 2 and 3 is sturdily quenched upon binding, thus allowing the detection of interaction with DNA at as low as nM concentration.These ndings invite for further biological studies, focusing on the intrinsic cytotoxicity of 2 and 3, as well as the photo-induced cytotoxicity of pyrene tether, well-known for ability of singlet oxygen sensitization. 37
Nucleotides (AMP, GMP, UMP and CMP) were purchased from Sigma, dissolved in stock solutions of c = 0.01 M and used.Polynucleotides were purchased as noted: poly dAdT-poly dAdT, poly A-poly U, poly dGdC-poly dGdC, (Sigma), calf thymus (ct)-DNA (Aldrich) and dissolved in sodium cacodylate buffer (I = 0.05 M, pH = 7.0), as described by producer.The presence of double stranded helix was conrmed by single, well-dened transition in the thermal denaturation experiment, giving T m value agreeing well with the literature; as well as by collecting CD spectra of free ds-DNA or ds-RNA, which also agreed well with the literature. 48,49The ct-DNA was additionally sonicated and ltered through a 0.45 mm lter to obtain mostly short (ca. 100 base pairs) rod-like B-helical DNA fragments.Polynucleotide concentration was determined spectroscopically as the concentration of phosphates (corresponds to c(nucleobase)). 52neral synthetic procedure for coupling reactions The suspension of a 4-(1-pyrenyl)butyric acid (0.1 mmol) in dry CH 3 CN (2 mL) was activated by means of HOBt (0.1 mmol) and HBTU (0.1 mmol), in the presence of bis-amino calix(4)arene (0.1 mmol) and Et 3 N (0.2 mmol) in an argon atmosphere.The reaction mixture was stirred at room temperature overnight.The solvent was removed under reduced pressure and the crude material was puried by preparative chromatography (CH 2 Cl 2 / MeOH 9 : 1) to obtain the desired product (Scheme 2).All characterization data of derivatives 2 and 3 ( 1 H and 13 C NMR spectra, HRMS spectra) are given in ESI (Fig. S1-S6 †).

Photophysical properties
TC-SPC measurements were performed on an Edinburgh FS5 spectrometer equipped with a pulsed LED at 340 nm.Fluorescence signals at 400 and 475 nm were monitored over 1023 channels with a time increment of 488 ps per channel.The decays were collected until they reached counts of range 3000 in the peak channel.A suspension of silica gel in H 2 O was used as a scattering solution to obtain instrument response function (IRF).Absorbances at 340 nm were 0.07-0.09.Prior to the measurements, the solutions were purged with a stream of argon for 20 min.The measurement was performed at rt (25 °C).Decays of uorescence were t to a sum of exponentials according to eqn (1): R(t) = A + B 1 e −t/s1 + B 2 e −t/s2 + B 3 e −t/s2 (1) Using soware implemented with the instrument; absolute quantum yields were determined by the Integrating sphere SC-30 of the Edinburgh FS5 spectrometer in the quartz cuvette of 10 mm path length, to avoid the scattering of incident light at the liquid-air interface, testing solutions with a 2 mL volume were used.

b
Samples were excited by pulsing diode at 340 nm.The measurements were performed three times and the average values are reported.The associated errors correspond to the maximum absolute deviation.c Solutions were purged by argon.d Done in water.e Done in DMSO.f Negative pre-exponential factor, without relative representation characteristic for excimer formation.g Absolute uorescence quantum yield was determined by integrating sphere SC-30, Edinburgh Inst., for argon purged solutions, by l exc = 353 nm.

Fig. 1
Fig. 1 Comparison of UV/Vis spectra: A (1-pyrenebutyric acid), 36 2 and 3 at concentration c = 1 × 10 −5 M in: (a) water, (b) DMSO.The Dl values are shown only for 342 nm maximum of A but bathochromic shift is present for complete spectra of 2 or 3.