Article pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Steric Effects on the Binding of Phosphate and Polyphosphate Anions by Zinc(II) and Copper(II) Dinuclear Complexes of m‑Xylyl-biscyclen Catarina V. Esteves,† David Esteban-Gómez,§ Carlos Platas-Iglesias,§ Raphael̈ Tripier,‡ and Rita Delgado*,† †
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780−157 Oeiras, Portugal § Departamento de Química, Facultade de Ciencias & Centro de Investigaciones Científicas Avanzadas, Universidade da Coruña, 15071 A Coruña, Spain ‡ UFR des Sciences et Techniques, Université de Bretagne Occidentale, UMR-CNRS 6521, IBSAM, 6 avenue Victor le Gorgeu, C.S. 93837 29238 Brest Cedex 3, France S Supporting Information *
ABSTRACT: The triethylbenzene-bis-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) compound (tbmce) was designed with an imposed structural rigidity at the m-xylyl spacer to be compared to a less restrained and known parent compound (bmce). The framework of both compounds differs only in the substituents of the m-xylyl spacer. The study was centered in the differences observed in the acid−base reactions of both compounds, their copper(II) and zinc(II) complexation behaviors, as well as in the uptake of phosphate and polyphosphate anions (HPPi3−, ATP4−, ADP3−, AMP2−, PhPO42−, and HPO42−). On the one hand, the acid−base reactions showed lower values for the third and fourth protonation constants of tbmce than for bmce, suggesting that the ethyl groups of the spacer in tbmce force the two cyclen units to more conformational restricted positions. On the other hand, the stability constant values for copper(II) and zinc(II) complexes revealed that bmce is a better chelator than tbmce pointing out to additional conformational restraints imposed by the triethylbenzene spacer. The binding studies of phosphates by the dinuclear copper(II) and zinc(II) complexes showed much smaller effective association constants for the dicopper complexes. Single-crystal X-ray and computational (density functional theory) studies suggest that anion binding promotes the formation of tetranuclear entities in which anions are bridging the metal centers. Our studies also revealed the dinuclear zinc(II) complex of bmce as a promising receptor for phosphate anions, with the largest effective association constant of 5.94 log units being observed for the formation of [Zn2bmce(HPPi)]+. Accordingly, a colorimetric study via an indicator displacement assay to detect phosphates in aqueous solution found that the [Zn2bmce]4+ complex acts as the best receptor for pyrophosphate displaying a detection limit of 2.5 nM by changes visible to naked eye.
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(bmce; Chart 1).14 The protonated forms of this compound did not reveal especially good for the uptake of phosphate and polyphosphate anions, namely, for orthophosphate (HPO42−), pyrophosphate (HP 2 O 7 3− or HPPi 3 − ), triphosphate (HP3O104−),15 adenosine monophosphate (AMP2−), adenosine diphosphate (ADP3−), and adenosine triphosphate (ATP4−).16 However, when used in the form of dinuclear zinc(II) complexes the association with nitrophenylphosphate14 and phenylphosphate (PhPO42−)17 appeared more promising. The complexation behavior of the p-xylyl-bis-cyclen derivative (bpce) with zinc(II)18 and copper(II)19,20 was also undertaken, but studies of association with phosphate anions were not reported.
INTRODUCTION Dinuclear complexes can act as receptors to bind anionic substrates through formation of cascade species.1,2 The search for new receptors that selectively bind anions in water is a challenging and important task with applications in biological systems, medical diagnostics, and environmental monitoring.3−8 Achieving this goal for phosphorylated anions, which are crucial anions in energy storage and transcriptional activity in biological systems and that play important roles on the environment, has an enormous scientific relevance.9−12 Bis-macrocyclic compounds with a variety of spacers13 are interesting receptors, both when protonated or forming dinuclear complexes, to establish stable supramolecular associations with anions, especially m-xylyl-bis-cyclen (cyclen = 1,4,7,10-tetraazacyclododecane) derivatives such as 1,1′-[1,3phenylenebis(methylene)]bis(1,4,7,10-tetraazacyclododecane) © XXXX American Chemical Society
Received: March 1, 2018
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DOI: 10.1021/acs.inorgchem.8b00539 Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry
Article
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Chart 1. Compounds Prepared in This Worka and the Studied Phosphates
RESULTS AND DISCUSSION Synthesis. Compound tbmce was obtained quantitatively by addition of 2,4-bis(bromomethyl)-1,3,5-triethylbenzene24,37 to an excess of cyclen (see Scheme 1). The excess of cyclen aims to Scheme 1. Synthetic Procedure to Obtain tbmcea
a (i) (CH2O)n, AcOH, HBr/AcOH 33%, 80 °C, 8 h; (ii) cyclen, Et3N, CHCl3, rt, 5 d.
a
avoid the formation of higher alkylated products, and the unreacted amount was recovered by extraction. The bmce compound was obtained through the same synthetic procedure with an improved yield (68%) in comparison to the described one (35% only).14 Acid−Base Behavior of tbmce and bmce. The protonation constants of compounds tbmce and bmce were determined by potentiometry in aqueous solution at 298.2 K and ionic strength 0.10 M in KNO3. The potentiometric titration curves of tbmce and bmce are plotted in Figure S1, their refinement is presented in Figures S2 and S3, and the results from their refinement are collected in Table 1 together with the
Namely, tbmce and bmce.
Dinuclear complexes can be also applied as receptors for substrates of interest through a competitive assay in the presence of an indicator, leading to chemosensing systems. The receptor is in this case able to bind both the indicator and the substrate with different affinities, which leads to diverse responses.21 The chemosensing ensemble strategy was used to develop indicator displacement assays (IDA) for the detection of different anions of interest in aqueous media.22−25 The sensing of HPPi3− is of great interest given the relevance of this anion in several biological processes and diseases.9 Furthermore, while a large number of studies reporting HPPi3− sensing was published, only a few of them were actually performed using physiological conditions.26−34 Moreover, the use of dinuclear complexes derived from nonrigid bis-macrocyclic structures as receptors for HPPi3− has very few precedents in the literature and was performed in mixed solvents media.35,36 Given the promising behavior of dinuclear metal complexes in the recognition of phosphates, we sought to study the impact that small backbone modifications on the spacer of bis-cyclen compounds has on the complexation properties and in the uptake of anions. With this in mind, a new member of the mxylyl-bis-cyclen family with a 1,3,5-triethylbenzene as spacer (tbmce, Chart 1) was synthesized and compared with the already known receptor bmce. The dinuclear copper(II) and zinc(II) complexes of both ligands were studied in parallel, and their association with phosphate anions (Chart 1) was evaluated. However, the copper(II) complexes of bmce and tbmce present very similar association constants with phosphate anions, while in the case of zinc(II) the two receptors provide a markedly different behavior. The reasons behind this surprising behavior are thoroughly analyzed using X-ray diffraction studies and density functional theory (DFT) calculations. Finally, the high affinity of the zinc(II) complex of bmce toward HPPi3− allowed us to develop an IDA assay for this anion.
Table 1. Overall (βiH) and Stepwise (KiH) Protonation Constants of tbmce and bmce in Aqueous Solution at 298.2 ± 0.1 K in 0.10 ± 0.01 M KNO3 and of Cyclen for Comparison equilibrium reactiona
tbmce
L + H+ ⇄ HL+ L + 2 H+ ⇄ H2L2+ L + 3 H+ ⇄ H3L3+ L + 4 H+ ⇄ H4L4+
10.81(1) 20.63(1) 28.86(1) 35.96(1)
L + H+ ⇄ HL+ HL+ + H+ ⇄ H2L2+ H2L2+ + H+ ⇄ H3L3+ H3L3+ + H+ ⇄ H4L4+
10.81(1) 9.82(1) 8.23(1) 7.10(1)
bmce log βiH 10.66(2) 20.58(1) 29.40(2) 37.52(1) log KiH 10.66(2) 9.92(1) 8.82(1) 8.13(1)
cyclenb 11.27 21.23 23.41 25.15 11.27 9.96 2.18 1.74
a
This work; L denotes the ligand in general. bT = 298.2 K, I = 0.5 M in KNO3.38
literature values for cyclen38 for comparison reasons. Four protonation constants were determined corresponding to the protonation of two secondary amines in opposite positions of each cyclen subunit to minimize the electrostatic repulsions in the small macrocycle. The remaining protonation constants were too low and cannot be accurately determined by potentiometry, as already happens in cyclen alone. When these constants were compared with those of cyclen, it is possible to assume that the B
DOI: 10.1021/acs.inorgchem.8b00539 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
Table 2. Overall (βMmHhLl) and Stepwise (KMmHhLl) Stability Constants of the Complexes of tbmce and bmce with Cu2+ and Zn2+ Cations at 298.2 ± 0.1 K and I = 0.10 ± 0.01 M KNO3 in Aqueous Solution tbmce equilibrium reaction
a
Cu
2+
bmce Zn
2+
Cu
2+
Zn2+
log βMmHhLl M2+ + 2 H+ + L ⇄ [MH2L]4+ M2+ + H+ + L ⇄ [MHL]3+ M2+ + L ⇄ [ML]2+ M2+ + L ⇄ [ML(OH)]+ + H+ M2+ + L ⇄ [ML(OH)2] + 2 H+ 2 M2+ + H+ + L ⇄ [M2HL]5+ 2 M2+ + L ⇄ [M2L]4+ 2 M2+ + L ⇄ [M2L(OH)]3+ + H+ 2 M2+ + L ⇄ [M2L(OH)2]2+ + 2 H+ 2 M2+ + L ⇄ [M2L(OH)3]+ + 3 H+
35.6(1) 28.1(1) 18.0(1) 6.8(1)
28.35(9) 23.17(5) 13.97(7) 2.82(8)
37.0(1) 29.2(1) 19.5(1) 9.1(1)
33.7(1) 23.2(1)
21.74(4) 13.85(7) 2.90(9)
36.4(1) 25.7(1)
31.75(5) 24.49(9) 15.84(9) 5.66(9) −5.22(6) 30.19(9) 25.74(3) 18.90(5) 10.48(5) −0.86(7)
log KMmHhLl [MHL] + H ⇄ [MH2L] [ML]2+ + H+ ⇄ [MHL]3+ M2+ + L ⇄ [ML]2+ [ML(OH)]+ + H+ ⇄ [ML]2+ [ML(OH)2] + H+ ⇄ [ML(OH)]+ [M2L]4+ + H+ ⇄ [M2HL]5+ [ML]2+ + M2+ ⇄ [M2L]4+ [M2L(OH)]3+ + H+ ⇄ [M2L]4+ [M2L(OH)2]2+ + H+ ⇄ [M2L(OH)]3+ [M2L(OH)3]+ + H+ ⇄ [M2L(OH)2]2+ [M2L]4+ + OH− ⇄ [M2L(OH)]3+ 3+
a
+
4+
7.5(1) 10.1(1) 18.0(1) 11.2(1)
5.18(9) 9.20(4) 13.97(7) 11.15(4)
7.8(1) 9.7(1) 19.5(1) 10.4(1)
15.7(1) 10.5(1)
7.77(3) 7.89(5) 10.95(6)
16.9(1) 10.7(1)
3.28
5.89
3.08
7.26(8) 8.65(7) 15.84(9) 10.18(5) 10.88(6) 4.45(9) 9.90(9) 6.84(4) 8.42(4) 11.34(4) 6.94
This work; L denotes the ligand in general.
Figure 1. Species distribution diagrams calculated for the copper(II) complexes of tbmce (left) and bmce (right) at 2:1 Cu2+/L stoichiometry. CCu = 2 × CL = 2.0 × 10−3 M. L denotes the ligands.
tbmce appearing at lower pH, and the entire diagram displaced to lower pH compared to that of bmce. Copper(II) and Zinc(II) Complexation Behavior of tbmce and bmce. The ability of tbmce and bmce to form complexes with Cu2+ and Zn2+ cations in aqueous solution was evaluated, and their thermodynamic stability constants were determined in the conditions already used for the protonation constants. The potentiometric titration curves of the copper(II) and zinc(II) complexation studies are plotted in Figure S1, their refinement is presented in Figures S2 and S3, and the values found are collected in Table 2. The corresponding species distribution diagrams for the complexes of both ligands are shown for the 2:1 M/L stoichiometry in Figures 1 and 2 and for the 1:1 in Figures S5 and S6.
protons were added alternatively in equivalent positions in each cyclen subunit. Additionally, the stepwise constants decrease with the increasing protonation state of the bis-macrocycles due to both higher electrostatic repulsion between positive charges and statistical factors. However, in tbmce the third and fourth stepwise constants are 0.59 and 1.03 log units lower than the corresponding ones of bmce, pointing to an increase of electrostatic repulsions between both macrocyclic moieties. This indicates that the ethyl groups of the spacer in tbmce introduce some conformational strain around the cyclen units, so they behave in a more dependent way. The speciation diagrams for the protonation of both compounds (see Figure S4) illustrate well these features showing the starting formation and the corresponding maximum percentage of the most acidic species of C
DOI: 10.1021/acs.inorgchem.8b00539 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
Figure 2. Species distribution diagrams calculated for the zinc(II) complexes of tbmce (left) and bmce (right) at 2:1 Zn2+/L stoichiometry. CZn = 2 × CL = 2.0 × 10−3 M. L denotes the ligands.
phosphates were determined (see below). The copper(II) complexes were also studied for comparison. Binding Constants of the Dinuclear Copper(II) and Zinc(II) Complexes of tbmce and bmce with Phosphate and Polyphosphate Anions. The dinuclear complexes of copper(II) were evaluated as receptors of different phosphate anions. Potentiometric titrations were impossible to undertake due to the slowness of reactions. Therefore, absorption spectroscopy was used instead, following the effect of addition of the substrates to the receptors on the visible region of the spectrum at 298.2 K and pH 7.4 in aqueous solution buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) (Figures S7−S9). The effective association constants (Keff) were obtained using nonlinear least-squares fittings of the titration data with the HYPSPEC software.43 The results are collected in Table 4.
The values obtained for the formation of ML complexes (M = Cu2+, Zn2+ and L = tbmce, bmce) are lower than the corresponding values for cyclen taken from the literature (log β = 23.2939 or 23.440 at 0.5 or 0.1 M in NaNO3, respectively, for [Cu(cyclen)]2+, and log β = 16.240 or 15.7441 at 0.1 M in NaNO3 or 0.1 M in NaCl, respectively, for [Zn(cyclen)]2+, all values at T = 298.2 K). The decreased stability of the complexes of tbmce and bmce may be attributed to steric effects. The comparison of stability constants of metal complexes of ligands with different basicities might be misleading, and therefore the pM = −log[M+] values42 were also determined at physiological pH on the basis of all the constants given in Tables 1 and 2, and they are presented in Table 3. These values highlight Table 3. pMa Values Calculated for Metal Complexes of the Discussed Ligands at pH 7.4 pMa 2+
pCu pZn2+
tbmceb
bmceb
cyclenc
14.20 8.90
14.61 10.17
16.97 9.77
Table 4. Effective Association Constant (Keff) for the Indicated Equilibriuma
a
4+
Values calculated for 100% molar excess of ligand over metal ion with CM = 1.00 × 10−5 M, based on the protonation and stability constants of Tables 1 and 2 or in literature ones. bThis work. cData from refs 39−41.
[Cu2L] + [Cu2L]4+ + [Cu2L]4+ + [Zn2L]4+ + [Zn2L]4+ + [Zn2L]4+ + [Zn2L]4+ + [Zn2L]4+ + [Zn2L]4+ +
the relative strength of the complexes with both ligands. The observed stability constants for the complexes of Zn2+ are smaller than those of Cu2+, as would be expected. The pM values for copper(II) and for zinc(II) complexes reveal that bmce is a better chelator than tbmce, pointing to additional conformational restraints imposed by the triethylbenzene spacer in tbmce. It is noteworthy to observe that the monohydroxo complex [Cu2L(OH)]3+ only starts to form above pH 8 for both ligands (Figure 1), and the values for the binding constant of OH− to [Cu2tbmce(OH)]3+ and [Cu2bmce(OH)]3+ complexes is 3.28 and 3.08 log units, respectively (see Table 2), indicating a weak and similar interaction for both receptors. However, the [Zn2tbmce(OH)]3+ and [Zn2bmce(OH)]3+ complexes start to form at much lower pH values of 5.5 and 5.0 (Figure 2), with calculated association constants of the OH− anion of 5.89 and 6.94 log units, respectively (see Table 2), values much higher than the corresponding ones for the copper(II) monohydroxo complexes. A value of 5.50 log units was found for [Zn(cyclen)(OH)]+,41 which is not far from the value reported here for [Zn2bmce(OH)]3+. These features suggest that the dinuclear zinc(II) complexes of these ligands may be good receptors for anions, and therefore the binding constants of them with
equilibrium reaction
tbmceb
bmceb
HPO42− ⇌ [Cu2L(HPO4)]2+ PhPO42− ⇌ [Cu2L(PhPO4)]2+ HPPi3− ⇌ [Cu2L(HPPi)]+ H2Ind2− ⇌ [Zn2L(Ind)] PhPO42− ⇌ [Zn2L(PhPO4)]2+ AMP2− ⇌ [Zn2L(AMP)]2+ ADP3− ⇌ [Zn2L(ADP)]+ ATP4− ⇌ [Zn2L(ATP)] HPPi3− ⇌ [Zn2L(HPPi)]+
2.69(1) 3.03(1) 4.24(1) 5.05(1) 1.87(1) 1.98(2) 3.05(1) 4.00(1) 4.01(1)
2.67(1) 2.96(8) 3.69(2) 5.75(1) 3.03(1) 4.29(1) 5.18(1) 5.32(3) 5.94(1)
a
At pH = 7.4 buffered with 20 mM MOPS and at 298.2 K. bValues in parentheses are standard deviations in the last significant figures.
Direct determination of the association constants of the HPPi3− anion with both dinuclear copper(II) receptors was not possible; consequently, competition spectrophotometric titrations with HPO42− were performed. A value of 4.24 log units was found for the association constant of HPPi3− with the dinuclear copper(II) receptor of tbmce, and a lower value of 3.69 log units was found for the bmce receptor. This result represents further evidence that the strain imposed by the ethyl groups of the spacer in tbmce is forcing the two cyclen units to adopt restricted conformational positions that in this case led to a more favorable accommodation of HPPi3− and consequently to stronger binding when compared to the receptor of bmce. The binding constants for the other phosphate anions are in the 2.67−3.03 log units range (see Table 4). The monophosphates, such as HPO42− and D
DOI: 10.1021/acs.inorgchem.8b00539 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry PhPO42−, have very similar effective constant values, pointing to similar binding modes. The dinuclear zinc(II) complex of bmce is expected to be a good receptor for anions due to the high association constant calculated for OH− (log K = 6.94, Table 2). Thus, the dinuclear zinc(II) complexes with both tbmce and bmce were also assessed as receptors for phosphates. Batch solutions were prepared for absorption spectroscopy in visible region using an IDA in aqueous solution at pH = 7.4 buffered with MOPS and at 298.2 K. In this case the [Zn2tbmce(Ind)] and [Zn2bmce(Ind)] ensembles were studied in the presence of HPPi3−, ATP4−, ADP3−, AMP2−, and PhPO42−; Ind = bromopyrogallol red (see below). The effective association constants (Keff) were obtained once again by fitting of the titration batch data with the HYPSPEC software.43 The results are collected in Table 4. In agreement with the binding constants obtained for the uptake of OH− anion by the [Zn2L]4+ complexes as receptors, the association constants of the studied phosphates are significantly higher when [Zn2bmce]4+ is the receptor compared with [Zn2tbmce]4+. Additionally, the values increase with the charge of the anion indicating the weight of the electrostatic interactions in the overall binding process in such a way that the uptake of HPPi3− by [Zn2bmce]4+ is very strong. The binding constants reported for the [Zn2bmce]4+ receptor with the PhPO42− anion of 4.6 log units,17 (and of 4.0 log units for nitrophenylphosphate14) are ∼1.57 log units higher than the value obtained by us using this receptor. We do not have an explanation for this discrepancy, which is so high that it cannot be attributed to the different methods used in the determination. To the best of our knowledge, dinuclear bis-cyclen complexes at physiological conditions were not used before as receptors for HPPi3−.9−12,44 Moreover, our results quantitatively demonstrate that subtle modifications of the receptor can lead to significant differences on the binding of the anionic substrate. Single-Crystal X-ray Structure of [(Cu2tbmce)2(μNO3)2]6+ and of [(Cu2bmce)2(μ-PhPO4)2]4+ Complex Cations. A view of the crystal structure of [(Cu2tbmce)2(μ-NO3)2]6+ is presented in Figure 3, while bond distances of the metal coordination environments are collected in Table 5. The structure revealed the presence of the [(Cu2tbmce)2(μNO3)2]6+ dimeric complex in the solid state, instead of the
Table 5. Bond Distances (Å) of the Metal Coordination Environments Observed for [(Cu2tbmce)2(μ-NO3)2]6+ and [(Cu2bmce)2(μ-PhPO4)2]4+ in the Solid State [(Cu2tbmce)2(μ-NO3)2]6+ Cu1−N11A Cu1−N12A Cu1−N13A Cu1−N14A Cu1−O1A Cu2−N21 Cu2−N22 Cu2−N23 Cu2−N24 Cu2−O2A
2.050(7) 2.012(7) 2.014(6) 2.034(7) 2.117(5) 2.057(6) 2.021(7) 2.002(6) 2.020(6) 2.077(10)
[(Cu2bmce)2(μ-PhPO4)2]4+ Cu1−N1 Cu1−N2 Cu1−N3 Cu1−N4 Cu1−O1 Cu2−N5A Cu2−N6A Cu2−N7A Cu2−N8A Cu2−O2A
2.042(7) 2.027(7) 2.024(7) 2.026(7) 2.077(6) 2.023(12) 2.054(14) 1.994(16) 1.92(2) 2.099(6)
expected monomer. Two nitrate anions are bridging two bismacrocyclic copper(II) complexes by interaction with two copper centers of each ditopic backbone through a μ2-η1:η1 coordination. This binding mode of nitrate is rather common in complexes of copper.45 The asymmetric unit is composed by a half [(Cu2tbmce)2(μ-NO3)2]6+ entity, three perchlorate anions, and water molecules. The four Cu centers define a parallelogram with sides of 5.42 and 8.43 Å, which correspond to the distances between metal centers bridged by nitrate anions and that involve two metal ions of a Cu2tbmce unit. The sides of the parallelogram define angles of 76.1 and 104.9°. The crystal structure of [(Cu2bmce)2(μ-PhPO4)2]4+ is shown in Figure 4, and the corresponding bond distances of the
Figure 4. View of the crystal structure of [(Cu2bmce)2(μ-PhPO4)2]4+ complex cation determined from single-crystal X-ray diffraction data. Hydrogen atoms bound to carbon atoms are omitted for clarity. The ORTEP plots are at the 30% probability level.
copper(II) coordination sphere are listed in Table 5. The structure also revealed the presence of a dimeric entity in solid state due to the presence of bridging trans μ2-η1:η1 PhPO42− anions joining together two dimetalated bis-cyclen units with a face-to-face orientation.46 The four copper(II) centers define again a parallelogram of nearly rectangular shape, with sides of 9.58 and 6.44 Å and angles of 85.4 and 94.6°. X-ray crystal structures of xylyl-bis-cyclen dinuclear copper(II) complexes are very scarce. The examples reported in the literature contain either discrete dinuclear entities20,47 or a coordination polymer formed due to the presence of bridging thiocyanate anions.19 The formation of tetranuclear entities supported by bridging anions is to the best of our knowledge unprecedented. The coordination polyhedra around the Cu2+ ions in the two structures can be described as square pyramidal, with the donor atoms of the cyclen fragment defining the basal planes (mean deviations from planarity
