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. Author manuscript; available in PMC: 2014 May 20.
Published in final edited form as: Inorg Chem. 2013 Apr 26;52(10):5838–5850. doi: 10.1021/ic302798u

Integrated and Passive 1,2,3-Triazolyl Groups in Fluorescent Indicators for Zinc(II) Ions – Thermodynamic and Kinetic Evaluations

J Tyler Simmons †, John R Allen ‡, Deborah R Morris §, Ronald J Clark †, Cathy W Levenson §,*, Michael W Davidson ‡,*, Lei Zhu †,*
PMCID: PMC3687791  NIHMSID: NIHMS473752  PMID: 23621758

Abstract

In addition to being a covalent linker in molecular conjugation chemistry, the function of a 1,2,3-triazolyl moiety resulting from the copper(I)-catalyzed azide-alkyne cycloaddition reaction as a ligand for metal ions is receiving considerable attention. In this work, we characterize the thermodynamic and kinetic effects of incorporating a 1,2,3-triazolyl group in a multidentate ligand scaffold on metal coordination in the context of fluorescent zinc(II) indicator development. Ligands L14, BrL14, and FL14 (1,4-isomers) contain the 1,4- disubstituted-1,2,3-triazolyl group that is capable of binding with zinc(II) in conjunction with a di(2-picolylamino) (DPA) moiety within a multidentate ligand scaffold. The 1,2,3-triazolyl in the 1,4-isomers is therefore “integrated” in chelation. The 1,5-isomers L15, BrL15, and FL15 contain 1,2,3-triazolyls that are excluded from participating in zinc(II) coordination. These 1,2,3- triazolyls are “passive linkers”. Zinc(II) complexes of 2:1 (ligand/metal) stoichiometry are identified in solution using 1H NMR spectroscopy and isothermal titration calorimetry (ITC), and in one case, characterized in the solid state. The 1:1 ligand/zinc(II) affinity ratio of L14 over L15, which is attributed to the affinity enhancement of a 1,2,3-triazolyl group to zinc(II) over that of the solvent acetonitrile, is quantified at 18 (−1.7 kcal/mol at 298 K) using an ITC experiment. Fluorescent ligands FL14 and FL15 are evaluated for their potential in zinc(II) sensing applications under pH neutral aqueous conditions. The 1,4-isomer FL14 binds zinc(II) both stronger and faster than the 1,5-isomer FL15. Visualization of free zinc(II) ion distribution in live HeLa cells is achieved using both FL14 and FL15. The superiority of FL14 in staining endogenous zinc(II) ions in live rat hippocampal slices is evident. In summation, this work is a fundamental study of 1,2,3-triazole coordination chemistry, with a demonstration of its utility in developing fluorescent indicators.

Keywords: 1,2,3-triazole; zinc sensor; fluorescence; isothermal titration calorimetry; stoppedflow; hippocampus; photoinduced electron transfer

Introduction

The [3+2] cycloaddition of azide and alkyne affords 1,2,3-triazoles,1 which have recently been utilized in a wide variety of research fields including catalysis,2–7 sensing,8,9 bioconjugation,10 and magnetic materials development.11,12 The renaissance of 1,2,3-triazole chemistry is primarily due to the discovery by Fokin, Sharpless,13 and Meldal14 of regiospecific synthesis of 1,4- disubstituted-1,2,3-triazoles via the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). It was later shown that 1,5-disubstituted-1,2,3-triazoles can be prepared also regioselectively using Ru- or base-catalyzed approaches (Scheme 1).15–18

Scheme 1.

Scheme 1

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Three approaches to the syntheses of 1,2,3-triazoles via azide-alkyne cycloaddition.

The 1,2,3-triazolyl moiety resulting from the CuAAC reaction has been used primarily as a passive covalent linker in bridging together two molecular components. More recently, the functionality of 1,2,3-triazole itself has been appreciated, in particular as a metal coordination ligand.19–21 To this end, the ability of a 1,2,3-triazole to act as integrated components in multidentate ligand scaffolds (e.g., see Figure 1, left22) has begun to receive considerable attention. With the increasing role of 1,2,3-triazolyl moiety in coordination chemistry, a quantitative analysis of the thermodynamic and kinetic benefits of incorporating a 1,2,3-triazolyl group in a multidentate ligand would be valuable to the future designs of 1,2,3-triazolylcontaining ligands. Herein, we report such a study that was undertaken in the context of developing fluorescent indicators for zinc(II) ions.

Figure 1.

Figure 1

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1,2,3-Triazolyl group assists in metal (M) coordination (left), or acts only as a covalent linkage (right) between the aryl (Ar) group and the metal coordination site in 1,4- and 1,5- isomeric ligands, respectively. L: a monodentate ligand or solvent.

Zinc(II) ion is known to be a key regulator of a number of important biological processes.23–25 Due to the lack of spectroscopic signature of zinc(II) ions, the use of a zinc(II)-reporting fluorescent indicator under physiological conditions has become the method of choice for detecting and tracking zinc(II) ions in cell- and neurobiological experiments.26 Ideally, zinc(II) indicators undergo a significant change in either fluorescence intensity and/or frequency upon zinc(II) binding. Other desirable properties of a zinc(II) indicator include facile synthesis, visible light excitation, high water solubility, tunable affinity for zinc(II) via convenient ligand modification, fast but reversible binding kinetics, photostability, and high brightness.26,27 Reflecting the extensive activities in the pursuit of suitable zinc(II) indicators in various applications, numerous reviews have been published on this topic over the past decade.26,28–37

1,2,3-Triazolyl-containing compounds 1–3 have been developed as fluorescent indicators for zinc(II) ions. In these compounds, the 1,2,3-triazolyl group acts as both a ligand for helping chelate zinc(II) ion and a linker to join a fluorophore and the coordination site, which constitutes an integrated design.19 Our group reported compound 1, which includes an anthryl fluorophore and a 1,2,3-triazolyl-containing acyclic tetradentate ligand.22a, 38 Compound 1 shows potential in imaging zinc(II) distribution in live mammalian cells.38 However, the moderate brightness of the zinc(II) complex (ε × φF = 711 at the excitation wavelength of 300 nm) and high energy excitation of the anthryl fluorophore limit the utility of this compound in bioimaging experiments. Watkinson and Todd showed that a 1,2,3-triazolyl group acts as an integrated binder for zinc(II) ions in their cyclam-based sensors (e.g. compound 2).39,40 The visualization of zinc(II) in zebrafish embryos was accomplished in their work. In another notable study, Belfield and coworkers developed compound 3 as a two-photon excited probe for zinc(II).41a Finally, a zinc(II) sensor that applied only 1,2,3-triazolyl groups for coordination was reported recently.41b

In the current study, the properties that we seek to improve upon the previous contributions are the inherent brightness (εZnL × φZnL) and the fluorescence contrast between free- and zinc(II)- bound forms (φZnL/φL) of 1,2,3-triazolyl-containing indicators under pH neutral aqueous conditions. Furthermore, by comparing isomeric 1,2,3-triazolyl-containing ligands (Figure 1), in which the 1,2,3-triazolyl group is either an “integrated binder” (1,4-isomer on the left) or a “passive linker” (1,5-isomer on the right), the thermodynamic and kinetic contributions of the 1,2,3-triazolyl group to zinc(II) coordination are deduced.

Results and Discussion

Experimental Design

The zinc(II) coordination chemistry of both 1,4- (L14 or BrL14) and 1,5-isomers (L15 or BrL15) were studied in CH3CN using isothermal titration calorimetry (ITC) and 1H NMR spectroscopy. These ligands are easier to prepare in large quantities than the fluorescent analogs FL14 and FL15. Therefore, they were used to complete the concentrationdemanding ITC and 1H NMR experiments. The brominated ligands BrL14 and BrL15 carry the benefit of enhanced propensity to crystalize, which aids the acquisition of single crystal structures of the zinc(II) complexes. From ITC, all thermodynamic parameters of a ligand/receptor equilibrium (ΔG°, ΔH°, and ΔS°) can be obtained,42 and the difference in binding affinities associated with 1,2,3-triazolyl binding can be quantified. Fluorescent ligands FL14 and FL15 were investigated as potential fluorescent indicators for zinc(II) ions. Zinc(II)-dependent fluorescence of ligands FL14 and FL15 was studied under pH neutral aqueous conditions. Competitive binding experiments were used to compare their affinities for zinc(II). Stopped-flow kinetic measurements were conducted to determine the second order rate constants kon of both isomers. The viability of FL14 and FL15 as fluorescent zinc(II) indicators in imaging zinc(II) ions in both mammalian cells and rat hippocampal slices was demonstrated.

Synthesis

L14 was synthesized from azidobenzene and N,N-di(2-picolyl)propargylamine via a CuAAC reaction in 60% yield (Scheme 2) as reported earlier.22a L15 was prepared in three steps starting with the thermal 1,3-dipolar cycloaddition of azidobenzene and propargyl alcohol to give a mixture of the 1,4- and 1,5-triazole isomers, 4a and 4b, with 89% total conversion from azidobenzene. The ruthenium(II)-catalyzed procedure to give 1,5-disubstituted-1,2,3- triazoles17,43 could be used as well. However, aromatic azides have low reactivities under these conditions.17 Due to the low cost of the starting materials, high product conversion, and ease of separation of the regioisomers in a later step, the thermal cycloaddition method was used in the current work. Bromination of the mixture of 4a and 4b with PBr3, followed by chromatographic separation gave the 1,5-isomer 5 in 49% yield (from the mixture of 4a and 4b). Finally, SN2 substitution with di(2-picolyl)amine yielded L15 in 95%. Other ligands were synthesized in a similar fashion starting from 1-azido-4-bromobenzene (BrL14 and BrL15, Scheme S6) or 3- azido-7-methoxycoumarin (FL14 and FL15, Schemes S7 and S8).

Scheme 2.

Scheme 2

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Reagents and conditions: (a) Cu(OAc)2·H2O (5 mol %), sodium ascorbate (25 mol %), CH3OH, rt, 20 h, 60%; (b) toluene, 70 °C, 12 h, 89%; (c) PBr3, CH2Cl2, rt, 6 h, 49%; (d) DIPEA, CHCl3, rt, 16 h, 95%.

Solid state structures

Single crystals of formula [Zn(BrL14)(CH3CN)](ClO4)2 and [Zn(L15)(H2O)]n(ClO4)2n (Figure 2) were obtained from vapor diffusion of diethyl ether into CH3CN solutions of the complexes. The distances of coordinative bonds are shown in the caption of Figure 2. Similar to our previously reported 1,4-isomer/zinc(II) complexes,22,38 the structure of [Zn(BrL14)(CH3CN)](ClO4)2 contains a distorted trigonal bipyramidal zinc(II) center that is bound with the N3 position of the 1,2,3-triazolyl group (Figure 2B). The trigonal bipyramidal core of [Zn(L15)(H2O)]n(ClO4)2n also consists of two pyridyl groups and a 1,2,3- triazolyl group (from a different molecule) on the trigonal plane, while the axial positions are occupied by the tertiary amino group and a H2O molecule (Figure 2D). The incorporation of a 1,2,3-triazolyl group from a nearby ligand allows for the formation of a polymeric chain (Figure 2E).

Figure 2.

Figure 2

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(A) ChemDraw44 structure and (B) ORTEP45 diagram of [Zn(BrL14)(CH3CN)](ClO4)2 (30% ellipsoids). Selected distances: N1-Zn = 2.035(5) Å; N2-Zn = 2.038(5) Å; N3-Zn = 2.025(5) Å; N4-Zn = 2.233(5) Å; N5-Zn = 2.056(6) Å. (C) ChemDraw44 structure and (D) ORTEP45 diagram of [Zn(L15)(H2O)]n(ClO4)2n (30% ellipsoids). The intermolecular attachments to N and Zn are highlighted in blue in (C). Selected distances: N1-Zn = 2.035(1) Å; N2-Zn = 2.044(1) Å; N3-Zn = 2.023(2) Å; N4-Zn = 2.228(1) Å; O-Zn = 2.086(2) Å. (E) Extended view of [Zn(L15)(H2O)]n(ClO4)2n showing the coordination polymer chain. Non-coordinating solvent molecules and counter ions are omitted for clarity. Hydrogen and carbon: black; nitrogen: blue; oxygen: red; and zinc: brown.

The single crystals of the 2:1 (ligand/metal) complex between BrL15 and zinc(II) were afforded using the same vapor diffusion approach. The bond distances in the zinc(II) coordination sphere are shown in the caption of Figure 3. A fac isomer of C2 symmetry was observed (Figure 3), in which the two 2-pyridylmethyl groups in the same molecule are no longer equivalent. This feature is also seen in the 1H NMR spectrum of the 2:1 complex in solution (vide infra). The 2:1 (ligand/zinc(II)) octahedral complexes involving two N,N-di(2- picolyl)amino (DPA) tridentate ligands are relatively rare.46–49 Despite the difference in the Nsubstituent of the DPA ligands, all but one46 reported cases share the same fac/C2-symmetric stereoisomer, suggesting a thermodynamic preference for its formation.

Figure 3.

Figure 3

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The cationic part of the (A) ChemDraw44 structure and (B) ORTEP45 diagram of [Zn(BrL15)2](ClO4)2 (30% ellipsoids). Selected distances: N1-Zn = 2.171(2) Å; N2-Zn = 2.105(2) Å; N3-Zn = 2.171(2) Å; N4-Zn = 2.247(2) Å; N5-Zn = 2.105(2) Å; N6-Zn = 2.247(2) Å. Hydrogen and carbon: black; nitrogen: blue; and zinc: brown.

Characterization of zinc(II) coordination in CH3CN

Isothermal titration calorimetry (ITC) is a valuable tool for investigating the thermodynamics of small molecule/metal coordination systems.12,38,50–55 We developed an experimental procedure based on Scheme 3 that allows for determining the affinity of N3 of 1,2,3-triazolyl group (see numbering in Scheme 3A) to zinc(II), which accounts for the difference in zinc(II) affinities of L14 and L15.

Scheme 3.

Scheme 3

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Binding equilibria envisioned for 1,4-isomer L14 (red LT in A) and 1,5-isomer L15 (blue LT in B) upon titrating them into a solution of Zn(ClO4)2 at 298 K. L represents the tridentate N,N-di(2-picolyl)amino component, T represents the 1,2,3-triazolyl group, S is the coordinating solvent, in this case CH3CN.

With L14, we envision a sequence of binding events where a 1:1 complex would form first as L14 is titrated into a solution of Zn(ClO4)2. The 1:1 complex is shown as [Zn(L)(T)(S)] in Scheme 3A, in which L, T, and S represent N,N-di(2-picolyl)amino, 1,2,3-triazolyl, and CH3CN, respectively. The 1,4-isomer L14 would allow for incorporation of the 1,2,3-triazolyl group (T) into the coordination sphere of the 1:1 complex (Scheme 3A). Therefore, L, T, and S are all bound with zinc(II). The ITC data in Figure 4A confirms the initial formation of a 1:1 complex. The binding affinity of this complex (K1) is too strong to be accurately determined (Scheme 4A).56 Upon further addition of L14 in the system, the 2:1 ligand/metal complex ([Zn(L)2](T)2 in Scheme 3A) forms with a K2 value of (4.0 ± 0.48) ± 104 M−1 (Scheme 4B). The two tridentate N,N-di(2-picolyl)amino groups likely saturate the octahedral coordination sphere of zinc(II), leaving the 1,2,3-triazolyl unbound. This scenario, as depicted in Scheme 3A, is consistent with the 1H NMR titration data (vide infra).

Figure 4.

Figure 4

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(A) A solution of L14 (2.0 mM) was titrated into Zn(ClO4)2 (0.090 mM) in CH3CN at 298 K. The one-site model was used to fit the data of the second transition. n (molar ratio) = 1.7 ±0.07, K = (4.0 ± 0.48) ×104 M−1, ΔH = −(12 ± 0.83) kcal/mol, and TΔS° = −6.2 kcal/mol. (B) A solution of L15 (2.0 mM) was titrated into a solution of Zn(ClO4)2 (0.090 mM) in CH3CN at 298 K. The one-site model was used to fit the data of the second transition. n (molar ratio) = 1.9 ± 0.01, K = (7.0 ± 0.7) × 105 M−1, ΔH = −(11 ± 0.02) kcal/mol, and TΔS° = −3.0 kcal/mol.

Scheme 4.

Scheme 4

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Equilibria involving L14 (red) and L15 (blue), and the calculation of the affinity between the N3 of a 1,2,3-triazolyl group with zinc(II) ion (black). N.D.: not determined, because the affinity is higher than the upper limit of the range of determinable values.

The ITC trace of the titration of L15 into Zn(ClO4)2 (Figure 4B) at the initial stage is similar to that of L14, even though the 1,2,3-triazolyl is not coordinating in the 1:1 complex ([Zn(L)(S)2](T) in Scheme 3B). The 1:1 binding constant K3 is also too large to accurately measure (Scheme 4C). The equilibrium constant K4 (Scheme 4D) of the reaction from the 1:1 to the 2:1 complex of L15 is (7.0 ± 0.7) ×105 M−1, over an order of magnitude larger than that of L14 (K2 in Scheme 4B). This difference is explained by the affinity lost from the 1,2,3-triazolyl (T) coordination in the 1:1 complex with L14 ([Zn(L)(T)(S)]), which is not present in the 1:1 complex of L15 ([Zn(L)(S)2](T)). Using Hess’s Law, the equilibrium constant of the coordinative exchange between the N3 of the 1,2,3-triazolyl and the solvent molecule CH3CN to zinc(II) was derived to be 18 (Scheme 4E). This difference appears to largely arise from an entropic effect (see ΔH° and TΔS° values in the caption of Figure 4), which suggests that the entropic gain of releasing coordinated solvent molecules accounts for a significant portion of the chelation effect. The coordinative exchange value of 18 (−1.7 kcal/mol at 298 K) can be interpreted as the additional binding affinity of the 1:1 zinc(II) complex of 1,4-isomer L14, gained from 1,2,3-triazolyl binding, over that of 1,5-isomer L15. Comparing to the zinc(II) affinity gain of three orders of magnitude via adding a pyridyl ligand to N,N-di(2-picolyl)amine to afford N,N,N-tris(2-picolyl)amine,57 the effect of a 1,2,3-triazolyl group is rather moderate.

The solution binding properties of BrL14 and BrL15 were also studied using 1H NMR spectroscopy (data of L14 and L15 are available in Figures S1 and S2). Upon addition of Zn(ClO4)2 to a CD3CN solution of BrL14,58 peaks associated with the pyridyl protons (a–d in Figure 5, aliphatic methylene protons (e and f), and 1,2,3-triazolyl proton (T) are broadened. The two methylene Hf protons shift upfield up to the 2:1 ligand/zinc(II) ratio, similar to the observations made with compound 1.38 At 2:1 ligand/zinc(II) ratio, the pyridyl and aliphatic protons, comparing to 4-phenyl-1,2,3-triazolyl protons, are disproportionally broadened, which is an indication that the N,N-di(2-picolyl)amino group preferentially binds zinc(II) at the early stage of the titration to form a 2:1 ligand/zinc(II) complex (see structures on the right margin of Figure 5). As the concentration of zinc(II) increases, the peaks resharpen and are downfield shifted. The four methylene protons He (Figure 5) are now split into two doublets under the constraints of a five-membered chelating ring with increased kinetic stability, while Hf protons of the 1,2,3-triazolyl group remain a singlet. This observation indicates that the dissociation of pyridyl/zinc(II) coordination is slower than that of 1,2,3-triazolyl N3 and zinc(II) ion.59

Figure 5.

Figure 5

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1H NMR spectra (500 MHz, CH3CN) of BrL14 in the presence of increasing concentrations of Zn(ClO4)2. The assignments are based on 2D-COSY spectra (Figures S3, S4). S: solvent CD3CN. Selective ratios of ligand/metal are shown in the parentheses.

The 1H NMR spectra of BrL15 collected during the titration experiment (Figure 6) with Zn(ClO4)2 are strikingly different from that of BrL14. Throughout the titration, all peaks remain sharp, and separate sets of peaks can be assigned to species of different coordinated states (i.e., free ligand, 2:1 and 1:1 complexes). This is an indication that the coordination kinetics of the 1,5-isomer, the tridentate ligand BrL15, is slower than that of 1,4-isomer tetradendate BrL14. It appears that the 2:1 complex of BrL14 constitutes a shallower minimum on the potential energy surface than that of BrL15. The chelatable 1,2,3-triazolyl group in BrL14 may aid the ligand exchange, i.e. accelerate the equilibrium between the 2:1 and the 1:1 complexes.

Figure 6.

Figure 6

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Partial 1H NMR spectra (500 MHz, CD3CN) of BrL15 in the presence of increasing concentrations of Zn(ClO4)2. The assignments are based on 2D-COSY spectra (Figures S5–S7). For the assignment of the 2:1 ligand/zinc(II) complex, see a detailed analysis in the Supporting Information. The ChemDraw structures are based on the single crystal structures of similar 2:1 (Figure 3) and 1:1 complexes (Figure 2). Selective ratios of ligand/metal are shown in the parentheses.

Early in the titration, two emerging AB systems at 3.4 and 4.3 ppm of two 2-pyridylmethyl groups in BrL15, and an apparent singlet (actually a very tight AB system of two diastereotopic 5-(1,2,3-triazolyl)methyl protons, see similar examples in Figures S2 and S8) at 3.2 ppm of equal integrated intensity are assigned to the 2:1 ligand/zinc(II) complex (Figure 6, see Supporting Information for the rationale of peak assignments). Clearly, the two pyridyl rings in BrL15 are no longer chemically equivalent in the 2:1 complex. The differentiation of the two pyridyl groups can be accounted for in the 2:1 complex structure depicted in Figure 3, which is a fac stereoisomer of C2 symmetry. The two pyridyl groups of the same ligand are placed in different chemical environments, which are coded red (axial) and blue (equatorial) in the 1H NMR spectrum of the 2:1 complex in Figure 6. Other stereoisomers of the 2:1 complex are possible,46 but the thermodynamic dominance of the fac/C2 isomer is apparent in the 1H NMR titration experiment. Despite the prevalent use of N,N-di(2-picolyl)amino group in zinc(II) indicator development and other metal coordination constructs,60,61 to the best of our knowledge the structural differentiation of the two 2-picolyl groups has not been reported. The 1:1 BrL15/zinc(II) complex formed at the end of the titration experiment only shows one set of pyridyl signals, suggesting the return to equivalency of the two pyridyl groups in the 1:1 complex. A singlet and two doublets of an AB system in the aliphatic region are assigned to 5- (1,2,3-triazolyl)methyl Hf and 2-pyridylmethyl He, respectively (Figure 6), which is similar to the assignments applied to the 1,4-isomer BrL14 (Figure 5).

The viability of FL14 and FL15 as fluorescent indicators for zinc(II) ions

Fluorescent indicators for zinc(II) ions have received considerable attention as a viable platform for tracking biological zinc(II).26,29,31,35,62,63 Our group22a,38 and others39–41,64 are interested in incorporating the 1,2,3-triazolyl group into the recognition component of the indicator, because in addition to being a metal binding site, the 1,2,3-triazolyl group allows for facile CuAAC-based synthesis, hence easy modification of the fluorophore/ionophore pair. Extending our study on multidentate 1,2,3-triazolyl-containing ligands for zinc(II),22a,38,55 fluorescent ligands FL14 and FL15 were synthesized containing 7-methoxycoumarin as the fluorophore. Coumarin derivatives have been widely used in the sensing community, including sensors for zinc(II),65–68 because of their photostability, high fluorescence quantum yields, and water solubility,69 all of which are superior to the anthryl fluorophore in compound 1. The N,N-di(2-picolyl)amino ionophore is known to bind strongly with zinc(II),57 and has been consistently utilized by the zinc(II) sensing community. The zinc(II)-dependent fluorescence of FL14 in CH3CN is shown in Figure S11. A 28-fold fluorescence intensity enhancement from the free ligand (off) state to the zinc(II)-bound (on) state was observed, accompanying a minimal change in its absorption spectrum (Figure S12). These observations are expected for indicators operating via a metal binding affected photoinduced electron transfer (PET) process.70

The interpretation that zinc(II)-coordination to FL14 or FL15 slows down or eliminates the PET nonradiative decay process is supported by the fluorescence lifetime measurements. Due to the relevance in bioimaging, data collected from samples under pH neutral aqueous conditions are shown in Figure 7. Similar conclusions are drawn from experiments in CH3CN. Both FL14 and FL15 show biexponential decays in the free ligand forms (red circles in Figures 7A and 7B). The short (τ1 = 0.27 ns for FL14 and 0.13 ns for FL15) and long (τ2 = 2.4 ns for FL14 and 2.7 ns for FL15) components are attributed to the excited state population (e.g. conformer) that is prone to intramolecular PET and the population that is not, respectively.71,72 Upon formation of the zinc(II) complex, the fast components of both ligands disappear as the electron transfer process is no longer thermodynamically favorable, and only the single exponential decays of 2.7 ns for FL14-Zn2+ and 2.4 ns for FL15-Zn2+ are observed (Figures 7A and 7B, blue triangles).

Figure 7.

Figure 7

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Fluorescence decay profiles monitored at 420 nm by exciting samples using a 370-nm nanoLED excitation source. (A) Fluorescence decay of instrument response function (IRF, ■), FL14 (●), and the zinc(II) complex of FL14 (▲). (B) Fluorescence decay of IRF (■), FL15 (●), and the zinc(II) complex of FL15 (▲). The concentration of the indicator was 12 μM. All measurements were obtained in an aqueous NTA-HEPES buffer at pH 7.3. See details in the Supporting Information.

Zinc(II)-dependent fluorescence enhancement of FL14 is observed also under physiological conditions buffered by the metal chelator nitrilotriacetic acid (NTA, Figure 8A).73 FL14 undergoes a 16-fold enhancement in fluorescence quantum yield upon zinc(II) binding, and competes well with the NTA buffer (NTA Kd(Zn) = 14 nM). The Kd of the FL14/zinc(II) complex (assuming a 1:1 stoichiometry) was determined to be 5.5 nM by plotting the fluorescence enhancement vs the “free” zinc(II) concentration.74 An indicator with a low nanomolar Kd is appropriate for zinc(II) visualization in most biologically relevant settings, where the free zinc(II) concentration is low. The fluorescence quantum yield of FL14 under the simulated physiological conditions (as described in the caption of Figure 8A) is 0.04, which increases to 0.64 upon binding with zinc(II). The 16-fold enhancement and the high fluorescent quantum yield of the zinc(II) complex of FL14 are significant improvements over the previously reported compound 1 based on anthryl fluorescence (8-fold enhancement, φZnL = 0.14).22a,38

Figure 8.

Figure 8

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(A) Fluorescence spectra of FL14 (6.6 μM, λex = 350 nm) in the presence of Zn(ClO4)2 (0–7.2 mM) in an aqueous solution ([HEPES] = 100 mM, [NTA] = 4 mM, [KNO3] = 100 mM, pH = 7.4). (B) Fluorescence spectra of FL15 (5.7 μM) in the presence of Zn(ClO4)2 (0–5 mM) in an aqueous solution ([HEPES] = 100 mM, [NTA] = 3 mM, [KNO3] = 100 mM, pH = 7.4). (C) Fluorescence spectra of FL15 (5.7 μM) in the presence of Zn(ClO4)2 (0–14 mM) in an aqueous solution ([HEPES] = 100 mM, [ADA] = 8 mM, [KNO3] = 100 mM, pH = 7.4). NTA = nitrilotriacetic acid; ADA = N-(2-acetamido)iminodiacetic acid. The initial and final spectra of each titration experiment are coded green and red, respectively.

The fluorescence of FL15 undergoes a 36-fold fluorescence intensity enhancement upon addition of increasing amounts of Zn(ClO4)2 in CH3CN (Figure S11). It retains a 19-fold enhancement in fluorescence quantum yield under aqueous conditions. FL15 has lower fluorescence quantum yields of 0.01 and 0.19 for the free ligand and zinc(II) complex, respectively, than those of FL14. Competition titrations of FL15 with NTA (Kd(Zn) = 14 nM) show that the ligand is unable to compete for zinc(II) with the metal chelator (Figure 8B). FL15 competes for binding to zinc(II) with the weaker metal chelator, N-(2-acetamido)iminodiacetic acid (ADA, Kd(Zn) = 83 nM)75 effectively (Figure 8C), thus allowing for the determination of the zinc(II) affinity of FL15 at 90 nM (the dissociation constant of the apparent 1:1 ligand/metal complex), which is on par with the reported zinc(II) affinity of the N,N-di(2-picolyl)amino group.57 The affinity difference between FL14 and FL15 in water in conjunction with the ITC results in CH3CN shows that the 1,2,3-triazolyl group strengthens the binding affinity of the ligand for zinc(II) in both organic and aqueous solvents. The metal ion coordination preferences of both FL14 and FL15 generally follow the Irving-Williams series, similar to those of reported polyaza fluorescent ligands.22a,76 The fluorescence-based selectivity data is shown in Figure S14. As a side note and evidence of FL14 to form complexes with other metal ions, the structures and utilities of copper(I/II) complexes of similar 1,4-isomer-containing tetradentate ligands have been reported.5, 22b–c

The kinetics of ligand/zinc(II) binding in a pH neutral aqueous solution was investigated at 9.7 °C employing the stopped-flow method similar to those described by Lippard and coworkers 77 and Nagano and coworkers.78 The zinc(II) concentration was kept at least 10 times higher than the ligand (FL14 and FL15) concentration to assure pseudo-first order kinetics. Under these conditions, the 1:1 ligand/metal complex presumably forms directly without the intermediacy of a 2:1 complex that was observed in the 1H NMR titration experiments. At least five different zinc(II) concentrations for each ligand were used, and multiple experiments were conducted on different days to ensure reproducibility. The experimentally determined kobs (rate = kobs[L]) values were plotted vs zinc(II) concentration, the slope of which yields the second order rate constant kon (kobs = kon[zinc(II)], see Figure S15). The stronger binder FL14 also binds with zinc(II) 17 times faster than the weaker binder FL15 (see Table 1). Figure 9A shows how the time course of the coordination reaction of FL15 changes with respect to zinc(II) concentration. Figure 9B shows the difference in the rates of zinc(II) binding between FL14 and FL15 at a zinc(II) concentration of 18 μM. The kobs value for FL14 is 81 s−1 while FL15 has a lower kobs of 4.0 s−1, a 20-fold difference. The properties of FL14 and FL15 with respect to zinc(II) binding under aqueous conditions are summarized in Table 1.

Table 1.

Summary of the photophysical and zinc(II)-coordination properties of FL14 and FL15 under pH neutral aqueous conditions, and their pKa values.

λabs/nma λfl/nmb φLc φZnLd kon/M−1s−1e Kd/nMf pKa τL/nsg τZnL/nsh
FL14 345 421 0.04 0.64 3.4 × 106 5.5 5.5 0.27 (82%)
2.4 (18%)		 2.7
FL15 343 410 0.01 0.19 2.0 × 105 90 ~3.5 0.13 (75%)
2.7 (25%)		 2.4
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a

Wavelength of maximal absorption;

b

wavelength of maximal emission intensity;

c

fluorescence quantum yield of free ligand;

d

fluorescence quantum yield of 1:1 ligand/zinc(II) complex;

e

second order rate constant of 1:1 ligand/zinc(II) complex formation;

f

dissociation constant of 1:1 ligand/zinc(II) complex;

g

fluorescence lifetime of the free ligand, amplitude of each component is in the parentheses;

h

fluorescence lifetime of the 1:1 ligand/zinc(II) complex.

Figure 9.

Figure 9

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(A) Stopped-flow traces of FL15 (1.0 μM) with Zn(ClO4)2 concentrations of 18 μM, 38 μM, 76.5 μM, 114.5 μM, and 153 μM at 9.7 °C in an aqueous buffer. [HEPES] = 50 mM; [KNO3] = 100 mM; pH = 7.4. The kobs values are 4.0 s−1, 8.0 s−1, 15 s−1, 22 s−1, and 30 s−1 obtained from single exponential fits. (B) Overlaid stopped-flow traces of FL14 and FL15 with [Zn] = 18 μM in HEPES buffer. The kobs for FL14 is 81 s−1 and kobs = 4.0 s−1 for FL15.

Visualizing zinc(II) ions in live HeLa cells

Ligands FL14 and FL15 were evaluated for potential in visualizing zinc(II) ions in live HeLa cells. All cells were incubated in a medium containing 2 μM of indicator for 30 min. The indicator-containing medium was then replaced with fresh medium that contained either basal or enriched levels of zinc(II) ions (ZnCl2 was used in the experiments). The confocal microscopic images (λex = 405 nm, Figure 10) were acquired after a further 10-min incubation. The differential interference contrast (DIC) and fluorescence images are labeled A–C and D–F, respectively. Cells incubated with media devoid of supplemental zinc(II) afforded images A and D. The fluorescence intensity of image D, presumably from the emission of the free ligand and/or complex formed with pockets of endogenous zinc(II), was minimal. After incubating the cells in the presence of 50 μM ZnCl2, a large increase in fluorescence was observed in image E relative to that of image D. Finally, image F shows the reversibility of FL14 as a zinc(II) binder under physiological conditions by adding the high affinity zinc(II) ligand, tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, pKd(Zn) = 15.6).79 Zinc(II) ions were sequestered by TPEN from the fluorescent zinc(II)/FL14 complex. Consequently, the emission intensity decreased to almost the level shown in Figure 10D. Without organelle-directing functional groups, the subcellular localization of FL14 is undefined, and follows a similar pattern to that of other previously reported neutral polyazabased indicators.74

Figure 10.

Figure 10

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Images of HeLa cells incubated with FL14 (2 μM) for 30 min. A–C: DIC images. (A) [Zn] = 0 μM. (B) [Zn] = 50 μM. (C) [Zn] = 50 μM; [TPEN] = 50 ZM. D–F: corresponding fluorescence images (λex = 405 nm, emission window 420–520 nm). Scale bar (left in D–F) = 10 μm; intensity bar (right in D–F) = 0–255.

Similar results were obtained with compound FL15 (Figure 11). But FL15 shows overall dimmer fluorescence than that of FL14 using the identical set of imaging parameters, consistent with the characterization that fluorescence quantum yield of FL15/zinc(II) complex is lower than that of FL14. Apart from that, both indicators show potential to report intracellular [zinc(II)] changes fluorometrically under live-cell imaging conditions. There was no change of cell morphology (e.g. balling up) over the course of the microscopic experiment, indicating a minimal level of toxicity of FL14 or FL15 at the prescribed dosage. The fluorescence enhancement was reversed upon addition of TPEN, corroborating that the enhanced fluorescence under zinc(II)-enriched conditions originates solely from the zinc(II)/indicator complex, not any other biochemical transformations that might have given a fluorescent readout.

Figure 11.

Figure 11

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Images of HeLa cells incubated with FL15 (2 μM) for 30 min. A–C: DIC images. (A) [Zn] = 0 μM. (B) [Zn] = 50 μM. (C) [Zn] = 50 μM; [TPEN] = 50 μM. D–F: corresponding fluorescence images (λex = 405 nm, emission window 420–520 nm). Scale bar (left in D–F) = 10 μm; intensity bar (right in D–F): 0–255.

Visualizing endogenous zinc(II) in hippocampal slices

The effectiveness of FL14 and FL15 to stain zinc(II)-rich areas of live rat hippocampal slices was investigated. A commercially available zinc(II) indicator, ZP1, developed by Lippard and coworkers,80,81 was also used in comparison under the same experimental conditions. The preparation of the slices and relevant experimental protocols are included in the Supporting Information. Briefly, the slices were stained via incubation in oxygenated artificial cerebrospinal fluid (aCSF) containing 10 μM of indicator for 30 min. Slices were imaged using a confocal microscope equipped with a 405-nm diode laser line for exciting FL14 and FL15. ZP1 requires a 488-nm excitation, which is suitable for the fluorescein fluorophore of ZP1. FL14 resulted in clear and reproducible staining of the zinc(II)-rich dentate gyrus and CA3 regions of the hippocampus (see Figure S17 for an anatomical map) that was comparable to that of ZP1 (Figure 12). Based on physiological experiments, there is a consensus that free zinc(II) ions are concentrated in the dentate gyrus and CA3 areas of the hippocampus.82 The zinc(II) staining experiments using fluorescent indicators have thus far corroborated this conclusion.78,83–89 FL15, the zinc(II) complex which has a lower fluorescence quantum yield and a higher dissociation constant than FL14 (Table 1), was not effective in staining zinc(II) in the hippocampal slices (data not shown). The specificity of FL14 for zinc(II) was confirmed by use of the zinc(II) chelator TPEN. Treatment of hippocampal slices with 100 μM TPEN prior to staining eliminated FL14 fluorescence (Figure S18).

Figure 12.

Figure 12

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The indicator FL14 stains zinc(II) in the dentate gyrus (DG) and CA3 regions of the hippocampus. Confocal microscopy images are shown comparing live rat hippocampal slices (250 μm thick) stained with 10 μM indicators FL14 and ZP1. Intensity bar in the lower right frame: 0–255.

In our hands, ZP1 stains both the DG and CA3 regions, whereas FL14 also stains both of those regions but with less fluorescence intensity. FL15 does not appreciably stain either the DG or CA. The difference in zinc(II) staining between FL14 and ZP1 may be attributed to the lower excitation energy applied in the ZP1 case (488-nm laser), which not only suppresses autofluorescence but is not attenuated as much by the optics setup of the microscope as a higher energy 405-nm laser excitation source. Therefore, creating 1,2,3-triazolyl-integrated indicators that are excitable by lower energy lasers is our next objective.

Conclusion

We have compared the coordination properties of isomeric 1,2,3-triazolyl-containing ligands (e.g. L14 and L15) in both solid state and solution. In the solid state, L15 is a tridentate ligand for zinc(II) in the 1:1 ligand/metal complex, while an intermolecular 1,2,3-triazolyl coordination is maintained in the single crystals to result in a coordination polymer. The 2:1 BrL15/zinc(II) complex crystallizes into a fac stereoisomer with C2 symmetry, which is shown to be the thermodynamically stable form in solution. The equilibrium constant of N3 1,2,3- triazolyl/CH3CN exchange for zinc(II) binding is 18 (−1.7 kcal/mol at 298 K), as determined in an ITC experiment. This value represents the benefit of enlisting 1,2,3-triazolyl in cooperative binding to the overall zinc(II) affinity in CH3CN. 1H NMR titrations of 1,4- and 1,5-isomers with Zn(ClO4)2 reveal the structural details of the 2:1 and 1:1 ligand/zinc(II) complexes, which are corroborated by the conclusion from the single crystal X-ray data. Competition binding experiments and stopped-flow data show that the 1,4-isomeric ligand (FL14) with an integrated 1,2,3-triazolyl binds stronger and faster with zinc(II) ions than the 1,5-isomer FL15. FL14 and FL15 are viable fluorescent indicators for zinc(II) ions in live-cell imaging experiments. Both indicators report a large intracellular fluorescence enhancement when zinc(II) in the growth medium is enriched, while no toxic effect was observed over the course of the staining/imaging experiments. FL14 is capable of staining endogenous zinc(II) in the dentate gyrus and CA3 regions of rat hippocampus, which is known to accumulate free zinc(II) ions. FL15 is ineffective due to a low affinity to zinc(II) and overall low achievable fluorescence quantum yield. Both the brightness at the excitation wavelength (ε × φF = 4672 at 350 nm) and fluorescence quantum yield of the zinc(II) complex of FL14 (φF = 0.64) are significant improvements over those previously reported for compound 1 (ε × φF = 711 at 300 nm, φF = 0.14), thus achieving the objective stated in the Introduction section. The conclusion from this study shall aid in the designs of future 1,2,3-triazolyl-containing multidentate ligands.

Supplementary Material

1_si_002
2_si_001

Chart 1.

Chart 1

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Fluorescent zinc(II) indicators that integrate 1,2,3-triazolyl groups.

Chart 2.

Chart 2

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Structures of 1,4- and 1,5-isomeric ligands.

Acknowledgments

Funding Sources

R01GM081382 (NIGMS).

This work was supported by the National Institute of General Medical Sciences (R01GM081382). The authors thank Dr. Kesavapillai Sreenath for helpful discussions regarding the interpretation of the lifetime data, and the Institute of Molecular Biophysics (IMB) at FSU for providing access to a VP-ITC microcalorimeter (MicroCal) and an Applied Photophysics SX20 double-mixing stopped-flow apparatus. Dr. Claudius Mundoma at IMB is acknowledged for technical assistance.

Footnotes

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Supporting Information.

Procedures for syntheses, characterizations of new compounds, detailed 1H NMR assignment of the 2:1 BrL15/zinc(II) complex, and additional spectra and images. This material is available free of charge via the Internet at http://pubs.acs.org.

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Supplementary Materials

1_si_002
NIHMS473752-supplement-1_si_002.cif (96.1KB, cif)
2_si_001
NIHMS473752-supplement-2_si_001.pdf (1.4MB, pdf)

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