structure–activity relationships for anion-responsive poly(squaramides): support for an...

Structure−Activity Relationships for Anion-ResponsivePoly(squaramides): Support for an Analyte-Induced NoncovalentPolymer Cross-Linking MechanismAli Rostami,† Gerald Guerin, and Mark S. Taylor*

Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada

*S Supporting Information

ABSTRACT: Poly(squaramides) are a novel class of anion-responsive macromolecules that incorporate the diaminocyclobu-tenedione hydrogen bond donor group into the polymerbackbone. Herein, the synthesis and properties of a series offluorene-based poly(squaramides) varying in conformationalrigidity, squaramide content, and propensity for aggregation aredescribed. Structure−activity relationships for the anion sensorybehavior of these polymers (as probed by fluorescence titrations,dynamic light scattering, confocal fluorescence microscopy, andtransmission electron microscopy) indicate that anion-inducedpolymer aggregation leads to a cooperative response with enhanced levels of sensitivity and selectivity. These observations areconsistent with a mechanism involving noncovalent cross-linking of polymer chains through squaramide−anion hydrogen-bonding interactions and point toward new applications of polyamides as stimulus-responsive materials.

■ INTRODUCTIONThe development of synthetic anion-binding agents is aproblem of fundamental interest and one relevant toapplications in biochemistry, medicine, environmental monitor-ing and remediation, food and beverage analysis, and catalysis.1

Anion binding and transport are also essential to biologicalprocesses: proteins capable of high-affinity, selective anionbinding in aqueous solvent have been characterized,2 andstabilization of anionic species or incipient anions is a commonfeature of numerous enzymatic mechanisms.3 Mimicry ofstructural or functional aspects of naturally occurring anionbinders thus provides additional motivation for the design andstudy of such systems. Several classes of interactions (Lewisacid/base, hydrogen bonding, ion pairing, anion-π and halogenbonding) have been shown to be useful as the basis forsynthetic anion hosts, with host preorganization andcomplementarity with guest structure being among the generalprinciples underlying the diverse structures that have beenidentified to date.4

Synthetic macromolecules present unique opportunities inthe area of anion recognition. In addition to practical featuressuch as their suitability for fabrication into thin films ormembranes or for integration into functional devices, polymersexhibit properties that can be exploited for enhanced selectivityand/or selectivity.5 The ability of analytes to influence theelectronic structure, conformation, and/or aggregation behaviorof polymers, and the resulting effects on photophysical orconductivity properties, have been used as signal transductionschemes. Signal amplification resulting from the “molecularwire” effect in conjugated polymers enables detection ofanalytes at low concentrations.6 Multivalency and cooperativity

can also be envisioned, taking advantage of the repeating natureof polymer structures.7 While a range of structurally diverseanion-responsive polymers have been identified in recentyears,8,9 the majority have been developed by appendinganion-binding groups to conjugated (polyphenylene, poly-(phenylenevinylene) or poly(phenylene−ethynylene)) archi-tectures known to give rise to signal amplification throughexciton transport. Structurally novel polymer backbones couldgive rise to unusual anion-induced responses or new trans-duction mechanisms.We recently reported the synthesis of a new class of

polyamides incorporating the 3,4-diaminocyclobutene-1,2-dione (squaramide) functional group.10 A polymer of thistype displayed a turn-on fluorescence response to tetra-n-butylammonium dihydrogen phosphate (Bu4N

+H2PO4−) anion

in a water/N-methylpyrrolidinone (NMP) mixture, and controlexperiments indicated that the macromolecular nature of thepoly(squaramide) had beneficial effects on both the selectivityand the magnitude of its response toward anions. The ability ofthis synthetic polyamide to act as an anion receptor wasnoteworthy,11,12 as was the observation of a “polymer effect” ina system bearing little structural resemblance to the π-conjugated polymers for which such effects are precedentedand well understood. Several types of evidence, including theconcentration dependence of the response to H2PO4

−, dynamiclight scattering experiments, and transmission electronmicroscopy, suggested that an interplay between anion binding

Received: June 18, 2013Revised: July 31, 2013Published: August 15, 2013

Article

pubs.acs.org/Macromolecules

© 2013 American Chemical Society 6439 dx.doi.org/10.1021/ma401263q | Macromolecules 2013, 46, 6439−6450

pubs.acs.org/Macromolecules

and polymer aggregation was responsible for this unusualbehavior. We proposed a model in which noncovalent cross-linking of polymer chains is effected by H2PO4

−−squaramidehydrogen-bonding interactions, giving rise to a cooperativeanion response.To probe structure−activity relationships in these novel

anion-responsive polymers, and to test the model described

above, we have prepared a series of macromolecules varying inconformational rigidity, squaramide content, and propensity foraggregation. Here we discuss details of the synthesis andproperties of these materials as well as their behavior as anionsensors. The latter data (studied by fluorescence titrations,dynamic light scattering (DLS), transmission electron micros-copy (TEM), and confocal fluorescence microscopy) establish a

Scheme 1. Synthesis of Diaminofluorenes with Varied Side Chains and Spacers

Macromolecules Article

dx.doi.org/10.1021/ma401263q | Macromolecules 2013, 46, 6439−64506440

link between the extent to which the poly(squaramides) areable to aggregate and both the degree of cooperativity and theselectivity of their response to anionic analytes. These resultssuggest that the unique properties of polyamides and relatedstructures can be exploited productively in the development ofstimulus-responsive macromolecules.

■ RESULTS AND DISCUSSION

Monomer Synthesis. Diaminofluorenes 4a−c, 8, and 14were targeted as monomers for polycondensation with diethylsquarate, allowing access to polysquaramides varying in thenature of the solubilizing substituents at the fluorenyl C9position, the level of squaramide content, or the rigidity of thepolymer backbone (Scheme 1). Diamines 4a−4c were preparedby palladium-catalyzed couplings of benzophenone imine withthe corresponding dibromofluorenes, followed by iminehydrolysis under acidic conditions.13 Alternative approaches(e.g., direct couplings with ammonia14 or transimination of thebenzophenone imine with hydroxylamine) were complicated bydifficulties in purification of the diaminofluorene products. Thepalladium-catalyzed amination is carried out under mildconditions, results in high yields and compares favorably tothe classical nitration/reduction sequence that is often used toaccess such targets.A 2-fold Suzuki coupling was used to assemble diaminotri-

fluorene monomer 8.15 Unsymmetrical bromofluorene 6 wassynthesized by monoamination of the corresponding dibromo-fluorene with benzophenone imine. It was coupled tofluorenediboronic ester 5 to afford 8 after imine hydrolysis.An asymmetrically substituted bromofluorene was also neededfor the preparation of the tetraethylene glycol-linked diamine14. Friedel−Crafts acylation of monobromofluorene 9 followedby Baeyer−Villiger oxidation and ester hydrolysis furnished 7-bromofluoren-2-ol derivative 11, which was employed in a 2-fold Williamson ether synthesis to yield 14.Polymer Synthesis. Polycondensations of diamines with

diethyl squarate were carried out using a modification of ourrecently developed Lewis acid-catalyzed protocol.16 Metaltriflates capable of two-point binding to dicarbonyl substrateswere found to increase the rates of condensation betweenanilines and squarate esters and to suppress formation of the1,3-squaraine regioisomers, with Zn(OTf)2 providing optimalresults. In adapting this method to the copolymerization of 4aand diethyl squarate, we found that catalytic Sc(OTf)3 in atoluene/N,N-dimethylformamide (DMF) solvent mixtureprovided superior results. As shown in Table 1, the number-average molecular weight Mn and polydispersity Mw/Mn, asjudged by gel permeation chromatography (GPC) in 0.2 wt %LiCl/N-methylpyrrolidinone (NMP) against poly(methyl

methacrylate) standards, depended on the composition of themixed solvent system; using a solvent ratio of 2:1toluene:DMF, poly1a was obtained with Mn = 1.8 × 104 g/mol and Mw/Mn = 1.7. In the absence of the deaggregant LiCl,GPC analysis of poly1a revealed a broad chromatogram with anapparent Mn of 2.0 × 106 g mol−1, indicating that thispoly(squaramide) is prone to aggregation in NMP. Thisbehavior is consistent with the properties of oligo- andpoly(aramides), which are known to aggregate throughintermolecular hydrogen-bonding and arene−arene interac-tions.17,18

To control the degree of polymerization and to use end-group analysis for independent determinations of Mn,polycondensations were carried out in the presence of 4-tert-butylaniline (Scheme 2). Values of Mn for poly1b and poly1c

determined by 1H NMR spectroscopy (1.3 × 104 and 5.0 × 103

g/mol, respectively) and GPC (1.5 × 104 and 7.0 × 103 g/mol,respectively) were in good agreement.Polycondensations of 4b,c, 8, and 14 with diethyl squarate

proceeded smoothly under the conditions optimized for poly1,yielding poly2−poly5 (Figure 1 and Table 2). Each of theobtained polymers was soluble in amide solvents such as DMFand NMP. The proposed structures of these macromoleculeswere supported by 1H NMR, 13C NMR, and FTIRspectroscopic analysis. The latter revealed N−H stretchingfrequencies near 3000 cm−1, along with medium intensity

Table 1. Effect of Solvent Composition on the Polycondensation of Diethyl Squarate and 4aa

solvent ratio (toluene:DMF) Mn (× 104 g mol−1) Mw/Mn

1:1 1.2 1.52:1 1.8 1.719:1 2.5 3.9

a0.2 wt % LiCl/NMP was used as the eluent. Calibration was carried out using poly(methyl methacrylate) standards.

Scheme 2. Preparation of Polysquaramides with DefinedEnd Groups



carbonyl bands at roughly 1790 cm−1. These features, and theabsence of signals near 1600 cm−1, were consistent with 1,2-(squaramide) rather 1,3-(squaraine-type) linkages.19 Both thesolubility of these materials and their regioregular nature arenoteworthy in light of previous attempts to prepare poly-(squaramides).19 The polymers show good thermal stabilities,as assessed by thermogravimetric analysis (TGA), with onsettemperatures for decomposition ranging from 377 °C for poly3to 450 °C for poly5 (see the Supporting Information). Theseare higher than those reported for structurally relatedpoly(amide ureas)20 but lower than those generally observedfor polyaramides.21

Computational Modeling of Oligo(fluorenesquar-amides). The vinylogous amide moieties present in squar-amides give rise to syn/anti conformational isomerism similar tothat of substituted ureas. Conformational preferences ofsquaramides derived from both primary and secondary22

amines have been studied:23 for the former class, the anti/anti conformation predominates in solid-state cocrystals withhydrogen bond acceptors, while NMR studies indicate thatboth syn/anti and anti/anti conformations are accessible insolution.24 To obtain a preliminary picture of the conforma-

tions available to fluorene-based poly(squaramides), we carriedout computational modeling (Gaussian 09,25 B3LYP/6-31G(d), gas phase) of aminofluorene-based mono- and bis-squaramides 16 and 17 (Figure 2).

The syn/anti (sp,ap) conformation of bis(fluorenyl)-squaramide 16 was calculated to be lower in energy than theanti/anti (ap,ap) conformation (ΔE = 1.4 kcal/mol). Thismodest difference in energy was consistent with previouscomputational and experimental studies. As expected, the syn/syn conformer was significantly higher in energy than the syn/anti (ΔE = 3.3 kcal/mol). Likewise, the squaramide groupswere predicted to adopt syn/anti arrangements in the two

Figure 1. Poly(squaramides) poly1−poly5 and model compound 15prepared by Lewis acid-catalyzed condensation.

Table 2. Molecular Weight and Polydispersity Data forpoly1−poly5a

polymer Mn (× 104 g/mol) Mw/Mn

poly1a 1.8 1.7poly2 1.6 1.6poly3 1.4 1.8poly4 1.5 1.6poly5 3.3 2.3

a0.2 wt % LiCl/NMP was used as the eluent. Calibration was carriedout using poly(methyl methacrylate) standards.

Figure 2. Calculated structures and relative energies (B3LYP/6-31G(d)) of conformers of squaramide 16 and bis-squaramide 17.



conformers of bis(squaramide) 17 calculated to be lowest inenergy ((sp,ap,sp,ap-17 and sp,ap,ap,sp-17)). Here thepreference over the “extended” (ap,ap,ap,ap) conformationwas significant (ΔE = 5.6 kcal/mol). These calculations suggestthat poly(squaramide) chains of this type are likely to adoptfolded conformations in isolation.To investigate the possibility of delocalization through the

squaramide functional group, the energies of the highest-energyoccupied molecular orbitals (HOMO) and lowest-energyunoccupied molecular orbitals (LUMO) were calculated for16, 17, and the corresponding tris-squaramide 18. TheHOMO−LUMO gaps (B3LYP/6-31G(d)) were calculated tobe 3.6, 3.3, and 3.2 eV for 16, 17, and 18, respectively. Theslight decrease in HOMO−LUMO gap predicted across thisseries of oligomers hints at a degree of short-rangedelocalization in poly(squaramides). Kilbinger and co-workershave observed that the optical absorption spectra of oligo-(aramides) are consistent with delocalization of this type acrossthe amide functional group.26

Optical Properties. Normalized optical absorption (UV−vis) spectra of poly1−poly5 and model compound 15 in 9:1NMP/water, each at a concentration of 8 × 10−6 M based onthe squaramide repeat unit, are collected in Figure 3a. The

fluorene-based polysquaramides and bis(fluorene)-squaramide15 absorb strongly between 374 and 422 nm with vibronicallyunstructured bands. Figure 3b compares the UV−vis spectrumof compound 15 with those of polymers poly1a−poly1c,revealing a molecular-weight-dependent bathochromic shift:absorption maxima (λmax) of 2.93, 3.04, and 3.17 eV wereobserved for 15, poly1c (Mn = 5.0 × 103 g/mol), and poly1a

(Mn = 1.8 × 104 g/mol), respectively. The observation of ahigher λmax for poly1c relative to model compound 15 appearsto be consistent with the DFT calculations of frontier orbitalenergies discussed above. On the other hand, the difference inλmax between poly1a and poly1c most probably reflects anaggregation-induced red-shift (see below), since the effectiveconjugation length is likely to be significantly less than thedegree of polymerization of these macromolecules.The results of GPC analysis described above indicated that

poly1a undergoes aggregation in NMP at room temperature.This behavior was further probed by variable-temperature UV−vis spectroscopy in DMF (Figure 4). The reversible blue-shift

of λmax upon heating is consistent with polymer deaggregationat higher temperatures. Poly4 is presumably less prone toaggregation, due to the presence of flexible tetraethylene glycollinkers between squaramide groups. Consistent with this idea,λmax of poly4 is blue-shifted relative to those of poly1−poly3,appearing close to that of bis(fluorenesquaramide) 15. Poly5,in which the squaramide groups are linked by a trifluorenemoiety, also displayed a blue-shift in λmax in comparison topoly1−poly3, despite the presence of a more extensivelyconjugated fragment. This property is solvent dependent:addition of water to a solution of poly5 in NMP resulted inblue-shift (Δλmax = 18 nm), a property observed to a muchlesser extent for poly1−poly3. These observations appear tosupport the formation of H-aggregates of poly5, consistent withreports of oligoaramides displaying spectral properties charac-teristic of aggregates of this type.21

Chiral, enantioenriched polysquaramide poly2 was furtherstudied by circular dichroism (CD) spectroscopy. The absenceof pronounced Cotton effects in the CD spectrum, and thesimilarity between the CD spectrum of poly2 and that of anonpolymeric chiral squaramide bearing the same tetrahy-drogeranyl substituent (see the Supporting Information),

Figure 3. (a) Normalized absorption spectra of model compound 15and polymers poly1a−poly1c in NMP/H2O (9:1) at a repeat unitconcentration of 8 × 10−6 M. (b) Effect of molecular weight on theabsorption spectra of squaramide derivatives.

Figure 4. Variable temperature UV/vis spectra of poly1a in DMF: (a)heating from 25 to 85 °C; (b) cooling from 85 to 25 °C.



suggest that this macromolecule does not display long-rangehelical order or form helical aggregates in NMP.Anion Recognition by Polysquaramides. As described

in our preliminary communication, poly1a displayed anincrease in fluorescence quantum yield upon addition oftetrabutylammonium dihydrogen phosphate (Bu4N

+H2PO4−)

in a 9:1 NMP/H2O solvent mixture (Figure 5a). Changes inthe UV−vis spectrum of poly1a were also observed in thepresence of Bu4N

+H2PO4−: as depicted in Figure 5b, a new

band appeared at 437 nm, with an isosbestic point at 430 nm.Addition of trifluoroacetic acid (TFA) resulted in a decrease ofemission intensity to its initial level, reflecting the reversiblenature of the polymer−dihydrogen phosphate interaction, andsequential additions of Bu4N

+H2PO4− and TFA enabled

repeated cycling between “on” and “off” states (Figure 5d).1H NMR spectroscopy in d7-DMF solvent revealed thebroadening and eventual disappearance of the signalscorresponding to the squaramide N−H groups, along withthe appearance of new signals corresponding to aromatic C−Hresonances, upon addition of Bu4N

+H2PO4−. Again, the spectral

changes could be reversed by addition of TFA. Theseobservations may reflect deprotonation of the squaramideN−H groups, a behavior consistent with earlier studies ofmonomeric squaramides by our group16 and by that ofFabbrizzi.27 NMR experiments in d9-NMP/D2O or d7-DMF/D2O mixtures were not possible due to solubility limitations,and so it is unclear whether there is a connection between thedeprotonation behavior in pure DMF and the absorbance/emission spectral changes observed in mixed organic/aqueoussolvent.The polymeric nature of poly1a influences its response to

H2PO4−. The magnitude of the emission response of poly1a

was higher than those of nonpolymeric 15 and lower-molecular-weight poly1b and poly1c (Figure 5c). Althoughquantum yields of poly1a−1c and 15 in the presence andabsence of H2PO4

− were not determined, it appears that thiseffect is a result of the poorly emissive nature of poly1a relativeto 15 in NMP/H2O in the absence of anion. A roughly 4-foldincrease in the sensitivity of poly1a relative to 15 was evidentfrom the titration curves. Furthermore, a sigmoidal plot ofemission intensity versus Bu4N

+H2PO4− concentration was

observed for poly1a. This behavior was less pronounced forpoly1b−1c and absent for 15. The latter displayed a hyperbolicresponse that could be fit to a 1:1 binding isotherm withassociation constant Ka = 2.8 × 103 M−1. The sigmoidaltitration curves for the polysquaramides were interpreted asarising from a cooperative anion binding process.28,29

Accordingly, differences between poly1a−1c were quantifiedusing the Hill coefficient (nH, the slope at half-saturation of alog/log plot of binding-site occupancy versus guest concen-tration): values of nH were 2.5, 2.0, and 1.9 for poly1a, 1b, and1c, respectively. In a still more competitive medium (8:2 NMP/H2O), poly1a displayed a similar H2PO4

−-induced enhance-ment in emission and a slight increase in the degree ofcooperativity (nH = 2.8). While the proposed binding model,which involves a given anionic guest interacting with more thanone squaramide moiety (see below), is not compatible with astraightforward application of the Hill analysis,30 the use of nHprovides at least a semiquantitative measure by which tocompare the extent of cooperativity.The anion selectivity of poly1a was also influenced by its

macromolecular nature. Whereas model compound 15 showedthe highest emission response to fluoride, poly1a was selectivetoward dihydrogen phosphate (Figure 6).31,32 Previous work

Figure 5. Changes in emission (a, λex = 415 nm) and absorption (b) spectra of poly1a upon addition of Bu4N+H2PO4

− in 9:1 NMP/H2O. (c)Concentration dependence of the fluorescence response to Bu4N

+H2PO4− (λem = 453 nm) for poly1a−1c and model compound 15. (d) Reversal of

the Bu4N+H2PO4

−-induced emission response of poly1a by addition of trifluoroacetic acid (TFA).



has highlighted the challenges in achieving selectivity forH2PO4

− over F− due to the high basicity of the latter.33

Selectivity over other monovalent anions (HSO4−, AcO−) was

also improved for poly1a in comparison to 15. Thepoly(squaramide) was able to discriminate dihydrogenphosphate from other structurally related analytes, includingpyrophosphate (P2O7

4−), adenosine mono-, di-, and triphos-phate (AMP, ADP, and ATP), and sulfate (SO4

2−).Structure−Activity Relationships on the Polymer

Response to H2PO4−. The effects on sensitivity and selectivity

upon incorporating the squaramide group into a polymer wereunusual given the lack of long-range delocalization in thissystem and unprecedented for a polyamide-type material. Thesigmoidal shape of the titration curve for poly1a, along withDLS and microscopy data to be discussed in the sections thatfollow, appear to be consistent with synergistic anion bindingand polymer aggregation, giving rise to cooperative behaviorand enhanced analyte selectivity. Structure−activity relation-ships for the anion-responsive behavior of poly2−5 wereinvestigated to further test this hypothesis, since differences in

Figure 6. (a) Normalized fluorescence response (I/I0) of poly1a (black bars) and model compound 15 (gray bars) to Bu4N+X− in 9:1 NMP/H2O.

Anion concentration was 140 μM for poly1a and 1200 μM for 15. (b) Normalized fluorescence response (I/I0) of poly1a to divalent and multivalentanions Xn− (140 μM in 9:1 NMP/H2O).

Figure 7. Concentration dependence of the emission response of polysquaramides to Bu4N+H2PO4

− in 9:1 NMP/H2O. Increase in emissionintensity (a) and normalized increase in emission intensity (b) are plotted as a function of binding site occupancy. (c) Selectivity of the anion-induced fluorescence response of polysquaramides poly1a−poly4 (140 μM Bu4N

+X− in 9:1 NMP/H2O). (d) Emission spectra of poly1a−poly4and model compound 15 in the presence of Bu4N

+H2PO4− in 9:1 NMP/H2O.



polymer backbone or side-chain structure were expected tomodulate the extent of polymer aggregation.The emission responses of poly2−5 toward H2PO4

− werestudied in 9:1 NMP/H2O. Tris(fluorenylene)-based poly5 waspoorly emissive both in the presence and in the absence ofH2PO4

−, suggesting that aggregation of this rigid andhydrophobic macromolecule was too favorable to permitH2PO4

− binding. This hypothesis is consistent with the UV−vis spectroscopic studies of poly5 discussed above. In contrast,polysquaramides poly2−4 displayed increases in emissionintensity upon addition of anion analogous to those observedfor poly1a. However, the four polymers differed in both themagnitude of the anion-induced fluorescence changes and theshape of the titration curve (sigmoidal versus hyperbolic). Tofacilitate comparisons between poly1−4, the relative emissionintensity is plotted versus binding site occupancy34 for eachpolymer in Figure 7a (poly1b is used in this graph so thatpolymers with similar degrees of polymerization are compared).In Figure 7b, the emission intensity changes are normalized(that is, Imax/I0 is set to 1 for each polymer) to further facilitatevisual comparisons of the graphs. The two sets of graphsindicate that the dioctylfluorene-derived polymer displayed thehighest relative increase in emission intensity of the polymersstudied, and its titration curve (in particular, that of the highermolecular weight sample poly1a) showed the most pronouncedsigmoidal shape. The lower molecular weight congener poly1bdisplayed a less obviously sigmoidal curve, and the same wastrue of poly2 and poly3, which bear β-branched andoxygenated substituents on the fluorenyl moieties, respectively.Each of these modificationsdecreased molecular weight andthe incorporation of branched or ethylene glycol side chainswould be expected to decrease the tendency of these

macromolecules to aggregate relative to poly1a.35 The extremeof this behavior was exhibited by poly4, which showed noevidence of a cooperative response to H2PO4

−, but rather atitration curve that fit well to a standard 1:1 binding isothermlike that of nonpolymeric 15. Thus, in the case where a flexiblelinker was employed between the squaramide groups,incorporation into a macromolecule did not significantlyperturb the behavior of the anion-responsive functionalgroup. The series of polysquaramides also differed in theirselectivity toward anions. The selectivity toward H2PO4

−

decreased in the order poly1a > poly2 > poly3 > poly4,mirroring the trend in apparent cooperativity discussed above.The observation that the degree of cooperativity and the levelof selectivity for H2PO4

− decrease for polymers that are lessprone to aggregation is consistent with the hypothesis thatfavorable interpolymer interactions accompany anion binding.

Studies of Anion-Induced Polymer Aggregation byDynamic Light Scattering and Microscopy. In addition tothe indirect evidence provided by the fluorescence titrationdata, further support for the hypothesis of anion-inducedpolymer aggregation was obtained by DLS, TEM, and confocalfluorescence microscopy experiments. Upon addition ofBu4N

+H2PO4− to a DMF solution of poly1a, a significant

increase in light scattering intensity was observed (Figure 8a).An increase in the size (from a mean hydrodynamic radius of50 nm to one of 120 nm) and an accompanying decrease in thepolydispersity (from a PDI of 0.3 to 0.1) of aggregates ofpoly1a resulted from addition of H2PO4

−. A study of the timedependence of the anion-induced change in light scatteringrevealed that this process was largely complete within 2 min,although some evolution in aggregate size was evident after themixture had been allowed to stand for 3 h (Figure 8c). Addition

Figure 8. (a) Autocorrelation functions from DLS measurements at 90° of poly1a (2.4 × 10−5 M): in the absence (red circles) and the presence(blue circles) of Bu4N

+H2PO4− (2.4 × 10−3 M) in DMF. (b) Normalized CONTIN plots for poly1a−poly4 in the presence of Bu4N

+H2PO4− in

DMF. (c) Time course of the progression of autocorrelation functions for poly1a after addition of Bu4N+H2PO4

− in DMF. (d) Autocorrelationfunctions for poly1a (2.4 × 10−5 M) in the presence of Bu4N

+F− (red circles) and Bu4N+HSO4

− (blue triangles) in DMF (2.4 × 10−3 M Bu4N+X−).



of the Bu4N+ salts of HSO4

− and F−, anions that did not inducechanges in the emission of poly1a in NMP/H2O (see above),resulted in no significant changes in scattering intensity. SimilarDLS experiments on poly2−poly4 in the presence of H2PO4

−

revealed aggregates having lower mean hydrodynamic radiithan those obtained from poly1a (Figure 8b). As noted above,these polymers displayed lower degrees of cooperativity in theirfluorescence response to H2PO4

− and lower levels ofdiscrimination toward H2PO4

− over other anions.Upon drop-casting a DMF solution of poly1a (2.4 × 10−5 M

per repeat unit) and Bu4N+H2PO4

− (2.4 × 10−3 M), sphericalaggregates approximately 500 nm in diameter could bevisualized by TEM (Figure 9b). Structures of this type derivedfrom poly1a were not evident in the absence of H2PO4

−

(Figure 9a). Further evidence for the formation of supra-molecular polysquaramide/dihydrogen phosphate-based assem-blies was gained by confocal fluorescence microscopy imagingin DMF solution (Figure 9c). While an exact correspondencebetween particle sizes as judged by DLS and microscopy wouldnot be expected, the data from the two techniques are roughlyconsistent. Similar aggregates were generated from poly2−4and Bu4N

+H2PO4−, as judged by TEM (Figures 9d−f).

Particles generated from the polymers bearing oligoethyleneglycol moieties (poly3 and poly4) appear to be less dense thanthose from poly1a and poly2.Binding Model: Anion-Mediated Noncovalent Poly-

mer Cross-Linking. We proposed that dihydrogen phosphate-induced noncovalent cross-linking of polymer chains could be

responsible for the enhancements in sensitivity and selectivityobtained for poly1a relative to nonpolymeric squaramide 15.Analyte-induced aggregation has been described for otherclasses of stimulus-reponsive macromolecules, especiallyconjugated polymers and polyelectrolytes,36 including examplesin anion detection.7d,37 A model of this type is consistent withthe sigmoidal dependence of emission on anion concentration,as it provides a mechanism by which interactions withphosphate can trigger the assembly of additional anion-bindingsites. Furthermore, direct evidence for the formation ofpolymer-based aggregates in the presence of H2PO4

− hasbeen obtained by DLS and microscopy. As described above, thetrends in anion binding data, DLS, and microscopy results forpoly1a−poly4 lend additional support to this hypothesis, asthey establish semiquantitative relationships between thedegree of cooperativity, anion selectivity, and propensity foraggregation of this set of structurally similar polymers.Possible modes of interpolymer interaction between

squaramide groups, and of 2:1 squaramide:H2PO4− complex-

ation, were assessed by computation (B3LYP/6-31G(d), Figure10) using the bis(dimethylfluorenyl)squaramide 16 (Figure 2)as a simplified model compound. The predicted geometry for2:1 anion complexation involves both squaramide groups intheir anti/anti conformation, as expected based on previousstudies of squaramides as “double” hydrogen bond donors23 aswell as reported examples of 2:1 urea:H2PO4

− complexes.38 Onthe other hand, favorable squaramide dimerization waspredicted to arise from hydrogen-bonding interactions between

Figure 9. TEM images of poly1a in the absence (a) and presence (b) of Bu4N+H2PO4

−. (c) Laser confocal microscopy image of a DMF solution ofpoly1a (2.4 × 10−5 M per repeat unit) and Bu4N

+H2PO4− (2.4 × 10−3 M). TEM images of poly2 (d), poly3 (e), and poly4 (f) in the presence of

Bu4N+H2PO4

−. Samples for TEM were prepared by drop-casting DMF solutions of the polymers (2.4 × 10−5 M per repeat unit) and Bu4N+H2PO4

−

(2.4 × 10−3 M). The lacy pattern is the copper framework of the carbon-coated copper TEM grid.



individual molecules in their syn/anti conformation. Whereassolid-state structures of squaramides derived from aliphaticamines usually display DD/AA-type hydrogen bondingbetween molecules having the anti/anti conformation, thispacking motif appears to be less prevalent for aniline-derivedsquaramides: rather, the latter have been found to crystallize assolvates, incorporating a hydrogen bond acceptor (e.g., DMSO)that interacts with the squaramide NH groups.16 We also noteCosta and co-workers’ work demonstrating that squaramidemonoesters undergo favorable dimerization in CDCl3 throughhydrogen bonding between syn conformers.24a

While the calculations discussed above suggest that H2PO4−

is capable of noncovalent cross-linking of polymer chains, andthat interchain hydrogen-bonding interactions between poly-squaramide chains are feasible, a rationale for the relatively highselectivity of the polymer toward H2PO4

− is not readilyapparent. For example, 2:1 squaramide:SO4

2− complexation hasbeen employed by Costa and co-workers in their design of abis(squaramide) host that undergoes sulfate-induced dimeriza-tion, reinforced by intermolecular hydrogen-bonding inter-actions between squaramide groups.39 Computational modelingat the level of theory described in the preceding paragraphrevealed analogous 2:1 16:F− and 16:HSO4

− complexeswherein the anion accepts four hydrogen bonds (Figure 11),reinforcing the idea that this mode of interaction is feasible inthe gas phase for anions other than H2PO4

−. In the case of F−,

the two squaramide groups adopt a bisected conformation, witha dihedral angle of 82° between squaramide planes, aconformation that may not be optimal for polymer cross-linking. Presumably, the coplanar 2:1 complex is destabilized bysteric strain between fluorenyl groups due to the small size ofthe anion and the short H---F− hydrogen bonds (averagedistance 1.64 Å). The calculated structures of the HSO4

− andH2PO4

− 2:1 complexes are roughly similar, and in neither caseis an interaction between the OH group of the oxoanion and ahydrogen bond acceptor site on the fluorene−squaramideapparent. Contacts of this type have been proposed to play arole in the selective complexation of HPO4

2− over SO42− by

phosphate-binding protein.2a Shorter hydrogen bond distanceswere calculated for the more basic H2PO4

− anion (1.82 versus1.90 Å, respectively). Presumably, desolvation also contributesin an important way to the selectivity observed for the polymersquaramides.

■ CONCLUSIONSIncorporation of the squaramide group into a rigid, polymericarchitecture thus has significant effects on the nature and

Figure 10. Geometries of (a) hydrogen-bonded bis(fluorenyl)-squaramide dimer [16]2 and (b) 2:1 16:H2PO4

− complex, as modeledby computation in the gas phase (B3LYP/6-31G(d)).

Figure 11. Geometries of (a) the 2:1 16:F− complex and (b) the 2:116:HSO4

− complex, as modeled by computation in the gas phase(B3LYP/6-31G(d)).



selectivity of its response to anionic hydrogen bond acceptors.The structure−activity relationships explored in this studyindicate that the magnitude of these effects can be tunedthrough modifications that alter the propensity of thepoly(squaramides) toward aggregation. The results suggestthat binding of H2PO4

− to these macromolecules incompetitive solvent is assisted by thermodynamically favorableinterpolymer interactions. Contributions of intrareceptorinteractions to the thermodynamics of host−guest complex-ation have been identified as an important feature of bindingevents involving biomolecules, but mimicry of this phenomen-on in synthetic anion-binding systems has not beenstraightforward. A compelling example is found in the workof Kubik, Otto, and co-workers, who provided evidence thatintrareceptor interactions, driven by the hydrophobic effect,enhance the anion affinity of cyclopeptide-based hosts thatfunction in aqueous solvent.40,41 Macromolecular systemsappear to offer particular advantages in this regard, as drivingforces for polymer collapse or aggregation can be significant,and changes in polymer aggregation state or conformationoften give rise to measurable changes in optical properties orconductivity. Exploring such effects in the context of diversetypes of polymeric architectures, and elucidating the detailedthermodynamic properties of these complex binding events,may help to fuel further advances in this direction.

■ ASSOCIATED CONTENT*S Supporting InformationExperimental details and characterization data; computationaldetails and full citation data for ref 25. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected] (M.S.T.).

Present Address†A.R.: Department of Chemistry, Shahid Beheshti University,Evin, Tehran 1983963113, Iran.

Author ContributionsG.G. carried out and interpreted data from the dynamic lightscattering experiments.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSFinancial support from NSERC (Discovery Grants Program)and the Canada Foundation for Innovation is gratefullyacknowledged. M.S.T. is a Fellow of the A.P. Sloan Foundation.The authors thank Prof. Mitch Winnik (University of Toronto)for access to instrumentation for GPC and DLS experimentsand for valuable discussions. Mike Chudzinski (University ofToronto) is gratefully acknowledged for assistance with thecomputational modeling.

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structure–activity relationships for anion-responsive poly(squaramides): support for an...

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