J Solution ChemDOI 10.1007/s10953-009-9456-6

Complexation Studies of Pyridyl Sulfonamide Ligandsfor Sensing Zinc and Copper Ions

Ritu Kataky · Mark A. Knell

Received: 12 November 2008 / Accepted: 15 August 2009© Springer Science+Business Media, LLC 2009

Abstract Ligand protonation and stepwise dissociation constants, formation constants andspeciation of four pyridyl sulfonamide ligands (Congreeve et al., New J. Chem. 27:98–106,2003) were assessed, using potentiometric and UV/Visible spectrophotometric pH titrations(in 80% MeOH − 20% H2O). The suitability of these ligands as Cu(II) and Zn(II) sensors forphysiological applications was assessed. Two ligands L1 and L4 were p-toluenesulfonamidederivatives while L2 and L3 were triflurosulfonamide derivatives. Additionally L3 and L4

were appended with α-methyl groups. The most stable complex was formed by L1 withCu(II) owing to the fact that this complex was square planar (log10 K1 = 12.15 ± 0.004 andlog10 β2 = 15.42±0.006). The rest of the complexes invariably formed distorted tetrahedrongeometry and complexation was weaker. Speciation diagrams show the effect of ligand tometal concentration, revealing that the L2 and L3 ligands are the most suitable for formingML2 complexes at physiological pH.

Keywords pH · Potentiometric · Spectrophotometric · Titrations · Speciation ·Pyridylsulfonamide · Copper · Zinc · Biological

1 Introduction

The binding and speciation in systems containing Cu(II) and Zn(II) ions and suitable lig-ands is of interest in diverse fields including: medical diagnostics, toxicological studies andenvironmental pollution. Copper complexes are being investigated as reagents for the cleav-age of nucleic acids and their biological activity for inducing apoptotic processes [2, 3].The biological role of Zn in synaptic neurotransmission [4] has been widely reported. Zncomplexes are of interest as potential Zn metalloenzyme mimics [5]. There are several publi-cation where 8-aminoquinoline derivatives have been used for making sensors for Zn(II) andCu(II) [6]. Quinoline based compounds appended with sulfonamide ligands have been re-ported as possible ligands for Zn(II) sensors [7, 8]. Several fluorescent probes for Zn(II) have

R. Kataky (�) · M.A. KnellDepartment of Chemistry, University of Durham, Durham, DH1 3LE, UKe-mail: ritu.kataky@durham.ac.uk

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Fig. 1 Chemical structures of the ligands used in this work: 2-(p-toluene sulfonylaminomethyl)pyridine (L1), 2(trifluoromethylsulfonylaminomethyl) pyridine, (L2), 2-(trifluoromethyl sulfony-laminomethyl)-6-methyl pyridine (L3), 2-(p-toluene sulfonylaminomethyl)-6-methyl pyridine (L4)

been reported based on 8-p-toluenesulfonamidoquinoline systems (TSQ) [9]. O’Halloranand co-workers [9] reported a series of TSQ analogues, including 2-Me-TSQ, and demon-strated that 2-Me-TSQ formed a neutral bidentate distorted [Zn(2-Me-TSQ)] tetrahedralcomplex, the methyl group on the quinoline inhibited square planar and octahedral com-plex formation, thereby creating a ‘steric selectivity’ leading to reduced copper and nickelcomplex stability. Nakamura and co-workers [10] studied protonation and binding equilib-ria of TSQ and trifluoromethylsulfonamidoquinoline analogues with cobalt(II), nickel(II),copper(II) and zinc(II). The protonation constant of the sulfonamide, log10 K1, was foundto decrease from 11.9 to 7.6 with the CF3 ligand, and binding stability for all metal ions alsodecreased. The methyl appendage decreased the stability of Ni(II) from log10 βML2 = 16.0to 11.8, while zinc(II) stability is enhanced with 2-Me-CF3SQ from 14.0 to 15.7.

These ligands are however prone to form mixed complexes [11]. This work was under-taken to evaluate the binding and speciation of these metal ions to four pyridyl sulfonamideligands (Fig. 1) using potentiometric and spectrophotometric titrations for preliminary in-vestigations of the potential use of these ligands for biological sensing. Consequently, theaim was to design and evaluate the performance of ligands at physiological pH. Fundamen-tal studies, such as the one reported here, should precede the consideration of ligands foruse in optical and electrochemical sensors.

2 Experimental

The syntheses and crystal structures of the pyridyl sulfonamide ligands have beendescribed previously [1]. The ligands used were: 2-(p-toluene sulfonylaminomethyl)pyridine (L1), 2(trifluoromethylsulfonylaminomethyl) pyridine (L2), 2-(trifluoromethylsulfonylaminomethyl)-6-methyl pyridine (L3), and 2-(p-toluene sulfonylaminomethyl)-6-methyl pyridine (L4) (see Fig. 1).

Acid dissociation constants, pKa stepwise binding constant and speciation studies wereperformed in 80% methanol–water mixtures (MeOH + H2O) using potentiometric titrations.Stock solutions of 0.05 mol·dm−3 metal ions were prepared with reagent grade copper (II)and zinc(II) nitrate salts. Tetramethylammonium nitrate, (NMe4NO3), and potassium ni-trate (KNO3), 0.1 mol·dm−3 solutions were used as background electrolyte. All solutionswere prepared with deionized water (Purite STILL plus system). The titrant was degassed0.1 mol·dm−3 tetramethylammonium hydroxide (NMe4OH) in 80% MeOH + 20% watersolutions. The solutions were made up from 10%, 1.0 mol·dm−3 stock solutions (Sigma–Aldrich, UK) and the exact molarity of the solution determined by titration against ‘Convol’0.01 mol·dm−3 HNO3.

The titration apparatus was comprised of a glass pH semi-micro combination electrode(BDH 309-1025-02, VWR International Ltd, Lutterworth, UK). The titrator was a custombuilt Molspin titrator (Molspin Ltd, Newcastle-upon Tyne, UK) with the capability of small

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volume titrations. The dispenser (burette) was a 1 mL gas-tight Hamilton syringe. All titra-tions were performed in a themostated cell at 25 °C under nitrogen. A 3 mL glass bulbpipette was used to transfer solutions to the titration vessel, the pipette was calibrated usingthe density of the 80% MeOH + 20% H2O mixture; the volume used in subsequent massbalance equations was 3.0947 mL.

Stock phosphate buffer (pH = 7.0) consisted of 30.5 mL 0.2 mmol·dm−3 disodium hy-drogen phosphate Na2HPO4 and 19.5 mL 0.2 mmol·dm−3 monosodium dihydrogen phos-phate, NaH2PO4 per 100 mL. Fresh stock solutions of sodium and disodium phosphateswere made from the dried salts. Potassium hydrogen phthalate buffer (pH = 4.008) wasmade from high purity salt (99.9%, Sigma Aldrich).

The ionic strength of the solutions was calculated using the Davies equation [12]:

logγ(±) = −0.51z2I 1/2

1 + 3.28aI 1/2(1)

where γ(±) is the mean activity coefficient, I the ionic strength, z the ionic charge and α theion size parameter.

Stepwise acid dissociation constants, Ka , and binding constants, β , were evaluated usingHyperquad (Version 2003) [12]. The stepwise formation constant is defined as:

βMLH = [MmLlHh][M]m[L]lah

H

(2)

where βMLH is the stepwise formation constant, m and l are positive integers or zero and h

is positive for protonated species, negative for hydroxo-complexes, or zero. [MmLlHh] is theconcentration of the complex species. [M] and [L] are concentrations of the uncomplexedreactant species and aH is the measured proton activity.

Formation constants are calculated by fitting potentiometric data according to relevantmass and charge balance equations using the Hyperquad program [13]. In this program theconstants are varied in order to minimize the difference between calculated and observedvalues of − log10[H+].

For each experimental titration point, the following mass balance equations are valid:

TM = [M] +j∑

m=1

mβMLH [M]m[L]l[H]h, (3)

TL = [L] +j∑

m=1

lβMLH [M]m[L]l[H]h, (4)

TH = [H] +j∑

m=1

hβMLH [M]m[L]l[H]h + aH

γ − Kw/aH, (5)

where TM , TL, and TH are the analytical concentrations of metal, ligand and proton respec-tively, and are known from the quantities used to make up the solutions. γ is the activitycoefficient for H+, Kw is the dissociation constant of water, j represents the number oftitration points and aH is obtained from potentiometric titration. The unknown parametersare [M] and [L] for each titration point and the formation constants, βMLH . The [M] and[L] variables are calculated and βMLH values are estimated. Hyperquad calculates the best

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fit obtained by minimizing a weighted sum of squared residuals. Each residual is the differ-ence between the calculated e.m.f (electromotive force), μ, and the observed e.m.f. EX . Thecalculated e.m.f. is given by Eq. 6 and the residuals by Eq. 7:

μ = EQ − EQ

n1[L]

(j∑

h=1

βMLH [[M]m][L]l[H]h + aH

γ − Kw/aH

)+ Ex (6)

σ =∑

(X − μ)2

N(7)

where EQ is the equivalence point, n1, the number of protons of the ligand that can betitrated, and Ex is the e.m.f. from the acid/base titration. A second statistical parameterobtained is chi square (χ2) based on the Gaussian distribution of weighted residuals (a valueof 12.6 indicates 95% confidence). Additionally, Hyperquad allows simultaneous refinementof multiple data sets. HySS (Hyperquad Simulation and Speciation) program was used toobtain speciation diagrams.

Absorbance measurements were made using a HR2000(+) spectrometer and a DH-2000deuterium tungsten halogen source (Ocean Optics, Duiven, Netherlands) fitted with a fiberoptic cable. The titration was carried out in a quartz flow cell (HELMA UK Ltd., Southend-on-Sea) with a 10 mm path length. Background light (80% MEOH + 20% H2O with0.1 mol·dm−3 KNO3) and dark light were calibrated after the lamp had equilibrated for10 min. Absorbance titration data sets were analyzed using pHAB software to determine theformation constants for the system. This software is generally the same as Hyperquad andis designed to calculate formation constants from spectrophotometric data obtained fromsolutions for which the pH has also been measured.

3 Results and Discussions

3.1 Protonation Constants

The potentiometric titrations were carried out in 80% methanol + 20% water mixtures.Calibration of the electrodes was performed by pH titrations of 0.1 mol·dm−3 HNO3 in80% MeOH + 20% H2O with 0.1 mol·dm−3 NMe4NO3, background electrolyte. The pKw

value obtained was 14.49 ± 0.003 (σ = 0.67, ψ2 = 3.25), which compared reasonably wellwith previous reports (pKw = 14.42, Mussini et al. [14, 15]).

The stepwise protonation constants of the four sulfonamide ligands investigated (Table 1,Fig. 2) show the impact of the electron withdrawing effect of the highly electronegative−CF3 group compared to the tosyl group, on the pK1 values. The log10 K1 values for L1 andL4 were highly basic, 12.15 ± 0.004 and 12.58 ± 0.010 for L1 and L4, compared to 7.50 ±0.005 and 7.58±0.002 for L2 and L3. The electron withdrawing effect of the CF3 substitutedsulfonamide group results in an increase in the acidity of L2 and L3, which stabilizes theconjugate base of the sulfonamide. The values are similar to the literature values for simpletrifluoromethylsulfonamides [16]. An additional feature is the slight increase in basicity ofthe pyridyl nitrogen on L1 and L4 (compared to L2 and L3), following introduction of theα-methyl group (log10 K2 = 2.69 and 3.32 for L2 and L3 respectively).

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Table 1 Stepwise protonation constants for ligands L1, L2, L3 and L4 in 80% methanol + 20% watermixtures, containing 0.1 mol·dm−3 (CH3)4NNO3, pKw = 14.5, [L] = 10 mol·dm−3, 298 K

Ligand log10 K1 log10 K2 log10 β2 σ ψ2

L1 12.15 ± 0.004 3.27 15.42 ± 0.006 2.53 11.72

L2 7.50 ± 0.005 2.67 10.17 ± 0.016 7.31 7.45

L3 7.58 ± 0.002 3.33 10.91 ± 0.008 10.03 10.72

L4 12.58 ± 0.010 3.95 16.53 ± 0.012 13.19 11.63

Fig. 2 Potentiometric titrationcurves showing the impact ofligand acidity on the protonationconstants. L1/L4 are the titrationcurves for L1 and L4 tosylatedligands and L2/L3 the curves forthe −CF3 modified ligands

3.2 Formation Constants

The ligands L1–L4 were designed to either assist or inhibit square planar ML2 complexes.Cu(II) prefers square planar coordination geometry whereas Zn(II) prefers distorted tetrahe-dron geometry. The introduction of the α-methyl substituent (L3 and L4) sterically inhibitssquare planar geometry. It was also expected that the lower protonation constants of L2 andL3 compared to L1 and L4 would enable complex formation at physiological pH.

The co-ordination of the transition metals Cu(II) and Zn(II) was studied by pH titrations.The titration experiments were limited by the formation of precipitates of metal hydroxideswhen the formation constants with the ligands were weak compared to OH− as a competingligand (Table 2). A table of aqueous solubility products is included in the supplementarydata (Table 1).

3.3 Toluenesulfonamide Ligands (L1 and L4)

The refinement of the titration data (Table 2) indicated that the most stable complexis formed with Cu2+ and L1. The formation constants of the Cu2+/L1 were refined tolog10 βML = 11.35 and log10 βML2 = 20.50. This result is expected, as L1 was found toform a well-defined square planar complex with Cu(II), confirmed by X-ray crystallography(Fig. 3). Titrations in the pH range 3–10 were refined without the need to input any metalhydrolysis constants to improve the fitting parameters, presumably due to the strong com-plexation with of Cu2+ with L1. In contrast, Cu2+ complexation with the equivalent ligandL4, modified with the α-methyl substituent, forces the geometry into a distorted tetrahe-dron with a decrease in formation constants to log10 βML = 9.43 and log10 βML2 = 18.75(Table 2). Inclusion of the metal hydrolysis constant improved the data fitting parameters.The altered geometry (resulting in altered d-shell transitions) results in a change of colorfrom green with L1 to orange with L4. These observations were confirmed with UV/Visiblespectrophotometric titrations (vide infra).

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Table 2 Stepwise formation constants for ligands L1, L2, L3 and L4 in 80% methanol + 20% watermixtures, containing 0.1 mol·dm−3 (CH3)4NNO3 with Cu(II) and Zn(II), [L] = 10 mol·dm−3, [M] =5 mol·dm−3, at 298 K. Figure in italics in row 1(L1/Cu) and row 7(L4/Cu) show the binding constantsobtained from spectrophotometric titrations with the data fitted by pHAB software

Ligand M2+ log10 βML log10 βML2 log10 βMLOH log10 KM(OH)2 �2 σ

L1 Cu 11.35 ± 0.011 20.05 ± 0.014 9.39 0.48

11.58 ± 0.06 20.98 ± 0.06

Zn 7.12 ± 0.087 15.03 ± 0.23 10.67 1.33

L2 Cu 6.81 ± 0.015 13.28 ± 0.018 −0.34 20.51 8.85

Zn 5.11 ± 0.033 10.21 ± 0.023 11.07 4.45

L3 Cu 6.23 ± 0.008 12.46 ± 0.013 10.30 1.78

Zn 5.62 ± 0.074 11.50 ± 0.059 9.75 11.18

L4 Cu 9.43 ± 0.011 18.75 ± 0.011 −11.02 ± 0.015 7.49 1.86

9.98 ± 0.014 18.26 ± 0.03

Zn 7.80 ± 0.015 16.83 ± 0.002 −14.93 ± 0.023 3.50 0.77

(a) (b)

Fig. 3 Crystal structures of CuL12 and CuL4

2 showing the square planar and distorted tetrahedron geometries

Zn(II) forms complexes with a distorted tetrahedron geometry with both L1 and L4. Thetitration with Zn(II)/L1 (5 and 10 mmol·dm−3, respectively), was carried out in the pH range3–7 as Zn(OH)2 precipitation was evident beyond pH = 7.00. The formation constant withL1 refined at log10 βML = 7.12 and log10 βML2 = 15.03 with good ψ2 and σ values. In-terestingly with L4, the formation constants with Zn(II) were higher (log10 βML = 7.8 andlog10 βML2 = 16.83) resulting in the titration being possible in the pH range 3–11. Inclusionof the hydrolysis constant was necessary to optimize the fit. Both L1 and L4 complexes withZn(II) displayed βML values higher than the βML2 value, indicative of positive cooperativity.

3.4 Trifluoromethylsulfonamide Ligands (L2 and L3)

Introduction of a CF3 moiety leads to harder Lewis base character compared to the tosylligands. Therefore the formation constants of borderline Lewis acids such as zinc(II) and

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copper(II) to L2 and L3 is weaker. Additionally both Cu(II) and Zn (II) form distorted tetra-hedral structures with both ligands. Refinement of the Cu(II)/L2 titration required introduc-tion of an added species MLOH (log10 βMLOH = −0.34) for optimal fitting. The stability ofthe CuL2

2 complex is reduced to log10 βML = 6.81 and log10 βML2 = 13.28. The introduc-tion of an α-methyl moiety in the L3 complex further reduces the stability of the Cu(II)/L3

complexes to log10 βML = 6.23 and log10 βML2 = 12.46. Titration could not be carried outbeyond pH = 7.0 because of precipitation of a yellow ML complex and M(OH)2 formation.

Similar trends are observed with the Zn(II)/L2 and Zn(II)/L3 complexation. The Zn(II)/L2

titrations were refined with log10 βML = 5.11 and log10 βML2 = 10.21 in the pH range 3–7, before hydrolysis products were evident. With the L3 ligand, pH titration was possibleup to pH 6.0, beyond this pH precipitation was observed possibly due to the formation ofMLn(OH)m species.

3.5 Effect of Ligand/Metal Concentrations on Speciation

Ligand to metal ion concentrations can greatly affect the speciation of the complexes. Thisis illustrated using the Zn(II)/L2 complexes with log10 βML = 5.11 and log10 βML2 = 10.21.The species distribution plot indicates that, at 1:1 M :L ratios approximately 40%, bothZnL and ZnL2 are present in solution, with hydrolysis products appearing from pH = 6.5(Fig. 4a). At 1:2 M :L ratios, the ML2 species dominates as expected. Hydrolysis products

Fig. 4 Diagram illustrating theeffect of ligand: metalconcentration ratios on speciesdistribution.[M] = 0.001 mol·dm−3 in allthree plots; A: 0.001 mol·dm−3

L2; B: 0.1 mol·dm−3 L2;C: 1.0 mol·dm−3 L2

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are not apparent untill pH = 7.5 (Fig. 4b). However, an excess of ligand causes 100% com-plexation of the metal ion, with hydroxide complexes appearing at pH = 7.9 (Fig. 4c).

3.6 Spectrophotometric Titrations of Cu(II) with L1 and L4

Cu(II) complexation with L1 and L4 were further investigated with UV/VIS spectrophoto-metric titrations. The data were analyzed using PHAB. The refined β constants agree wellwith the values obtained using potentiometric titrations, with log10 βML = 11.58 ± 0.06 andlog10 βML2 = 20.98 ± 0.06.

The copper (II) complex gave rise to two main transitions, a ligand-to-metal charge trans-fer (LMCT) at 380 nm and a d-d transition in the 600–800 nm range. This indicated an in-crease in energy associated with the widening of HOMO/LUMO caused by greater ligandfield strength during formation of the square planar [CuL1

2] complex. The plot of λmax versuspH showed a plateau after pH = 7.0 that was in keeping with the formation of ML2 speciesaccording to species distribution models (Fig. 5a).

Absorbance measurement of the [CuL42] complex formation provided additional evidence

of steric hindrance inhibiting the complex from forming a square planar complex. A strongabsorbance with a ligand-to-metal charge transfer at 451 nm and another peak associatedwith d-d transition were observed. The absorbance data demonstrated that the absence ofsignificant change in the ligand field strength stabilization energy (LFSE) during complex-ation, with only small shift in λmax from 801 nm to 796 nm. Hydrolysis was noted at a pHof 11 by a slight shift to lower wavelength, and by a decrease in absorbance (Fig. 5b).

Fig. 5 Spectrophotometric titrations of Cu(II)/L1 and Cu(II)/L4 ([M] = 5 mmol·dm−3, [L] =10 mmol·dm−3). A: Plot of absorbance versus pH for Cu(II)/L1: Shift in λmax from 762 nm at pH = 3.06 to630 nm at pH = 7.15; and B: Plot of absorbance versus pH Cu(II)/L4: Shift in λmax from 801 nm at pH =3.06 to 796 nm at pH = 7.15

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Fig. 6 Species distribution diagrams illustrating the effect of ligand geometry and the magnitude of theformation constants. Ligands L2 and L3 form ML2 complexes at physiological pH with both Cu(II) andZn(II). [M] = 0.001 mmol·dm−3; [L] = 10 mmol·dm−3. A: (a) CuL4

2; (b) CuL12; (c) ZnL1

2; (d) ZnL42.

B: (a) CuL22; (b) CuL3

2; (c) ZnL22; (d) ZnL3

2

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Refinement was consistent with values previously reported in this study (Table 2) withlog10 βML = 9.98 ± 0.014 and log10 βML2 = 18.26 ± 0.0269.

4 Conclusions

This work demonstrates that by ligands can be carefully selected, modified and matchedwith a target analyte for the development of sensors at different pHs. Thus the ligands L2

and L3 form stable ML2 complexes with Cu(II) and Zn(II) at physiological pH (Fig. 6b) atless than micromolar levels of concentrations and are very suitable ligands for biological ap-plications. L1 forms a very stable square planar complex with Cu(II) at pH<7 (Fig. 6a) withno indication of hydrolysis products and is more suitable for sensing in acidic conditions.

Acknowledgement We would like to acknowledge Prof. David Parker and Dr. Aileen Congreeve for sup-plying the ligands and for helpful discussions.

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