Metal ion complexation in acetonitrile by upper-rim benzyl-substituted, di-ionized calix[4]arenes bearing two dansyl fluorophores

The influence of Li + , Na + , K + , Rb + , Cs + , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Ag + , Cd 2+ , Co 2+ , Fe 2+ , Hg 2+ , Mn 2+ , Pb 2+ , Zn 2+ and Fe 3+ on the spectroscopic properties of two dansyl (1-dimethylaminonaphthalene-5-sulfonyl) groups linked to the lower rims of a series of three, structurally related, di-ionized calix[4]arenes is investigated by means of emission spectrophotometry. Di(tetramethylammonium) salts of the di-ionized ligands, L , L1 and L2 , which differ in having no, two and four benzyl groups, respectively, on the upper rim of the calix[4]arene scaffold, are utilized for the spectrofluorimetric titration experiments in MeCN. On complexation by alkaline earth metal cations, the emission spectra undergo marked red shifts.


Introduction
][9][10] Several photoinduced mechanisms have been proposed to explain the changes in fluorescent properties of the fluorophore group upon metal complexation, such as electron transfer (PET), charge transfer (PCT), energy transfer and excimer or exciplex formation or disappearance.
Calix [4]arenes possess a hydrophobic upper rim and a hydrophilic lower rim.They can exist in four different conformations: cone, partial cone, 1,2-alternate and 1,3-alternate. 11Among these, the cone conformation is used most widely for functionalization.3][14] The conformation of the calix [4]arene scaffold is important for metal ion complexation. 15Also, substituents on the upper and/or lower rims may influence the metal ion complexation process.The complex may form due to hydrogen bonding, hydrophobic bonding or electron donoracceptor interactions.
In an earlier study, we reported the interactions of didansyl-pendent, di-ionized calix [4]arenes with zero, two and four allyl groups on the upper rim (Figure 1) with a variety of metal cation species using spectrophotometric and spectrofluorimetric techniques. 16
In the present paper, we report the synthesis of two new calix [4]arene ligands 1 and 2 (Figure 2) with two pendent dansyl groups on the lower rim and two and four benzyl groups, respectively, on the upper rim and complexation properties of their di-ionized forms L1 and L2 with metal cations.Compared with the earlier investigated upper-rim allyl groups in the ligand series shown in Figure 1, upper-rim benzyl groups are more bulky and provide additional π bonds as potential interaction sites for metal ions.Responses of di(tetramethylammonium) salts of the di-ionized ligands L1 and L2 to a variety of metal cations are assessed by spectrofluorometric titration in MeCN.Changes in the fluorescence spectra of L1 and L2 in the presence of various metal cations are compared with those reported earlier for L, an analog with no benzyl groups on the upper rim.Stability constants and compositions of complexes of Hg 2+ , Pb 2+ and Fe 3+ with L1 and L2 are determined.A Stern-Volmer approach is utilized to probe the quenching mechanism.

Ligand synthesis
The syntheses of new di-dansylated, di-ionizable calix [4]arene ligands 1 and 2 are summarized in Schemes 1 and 2, respectively.For the preparation of ligand 1 with two benzyl groups on the upper rim of the calix [4]arene scaffold (Scheme 1), two methyl groups were attached to distal hydroxyl groups of calix [4]arene 3 to give known diether 4. Friedel-Crafts acylation of 4 with benzoyl chloride and AlCl3 in CH2Cl2 gave an 81% yield of the known disubstitution product 5. Attempted Wolff-Kishner reduction of the two benzoyl groups in 5 was unsuccessful.However, reduction of 5 to the upper-rim dibenzyl compound 6 in 90% yield was achieved with triethylsilane in trifluroacetic acid.Dialkylation of 6 with ethyl bromoacetate and NaH in THF gave a 71% yield of diester 7, which was hydrolyzed to diacid 8 in 97% yield with Me4NOH in aqueous THF.Diacid 8 was treated with oxalyl chloride in benzene to produce the corresponding di(acid chloride), which was added to the sodium salt of dansylamide in THF to give the di-dansylated calix [4]arene 1 in 30% yield.For the synthesis of ligand 2 with four benzyl groups attached to the upper rim of the calix [4]arene scaffold (Scheme 2), calix [4]arene 3 was reacted with benzoyl chloride and AlCl3 in CH2Cl2 to give the tetrabenzoylated tetrabenzoate ester, which was subjected to basic hydrolysis with NaOH in aqueous MeOH to remove the ester functions. 17An 89% yield of the tetrabenzoylated calix [4]arene 9 was realized.The benzoyl groups were converted into benzyl groups by reaction with hydrazine hydrate and K2CO3 in triethylene glycol to produce a 95% yield of known tetrabenzylated calix [4]arene 10.Reaction of 10 with K2CO3 and MeI in MeCN gave a 91% yield of diether 11, which was transformed into diester 12 in 90% yield by reaction with ethyl bromoacetate and NaH in THF.Treatment of diester 12 with Me4NOH in aqueous THF produced diacid 13 in 97% yield.Diacid 13 was treated with oxalyl chloride in benzene to produce the corresponding di(acid chloride), which was added to the sodium salt of dansylamide in THF to give the di-dansylated calix [4]  13 C NMR and IR spectra and by combustion analysis.The 1 H NMR spectra of new ionophores 1 and 2 showed broad, poorly defined absorptions revealing conformational mobility in CDCl3 solution.Ligands 1 and 2 were converted into their di(tetramethylammonium) salts L1 and L2, respectively, by a reported method. 18uorescence spectra When excited at 328 nm in MeCN, ligands L1 and L2 gave emission bands with maxima at 484 and 482 nm, respectively.Figure 3 shows the effects of 50 equivalents of metal cations on the fluorescence spectra of L1.As can be seen from Figure 3a, the emission band intensities increased somewhat in the presence of alkali metal cations with red shifts of the emission band from that of L1, except for Li + .Earlier, we observed quenching with alkali metal cations for analogous allyl-substituted ligands. 16As can be seen in Figure 3a, the largest red shift was produced by Li + .Similar results were obtained for alkali metal cations with L2 (Figure 4a).Thus it was found that increasing the number of benzyl groups on the upper rim from two to four did not change significantly the effect of alkali metal cations on the emission spectra.Previously, we obtained similar results with allyl groups on the upper rim. 16he emission band intensity for L1 was diminished substantially with red shifts for the alkaline earth metal cations (Figure 3b).The effects of Ba 2+ and Sr 2+ and of Ca 2+ and Mg 2+ on the fluorescence spectra are nearly the same for each pair of metal ions.There are larger red shifts and greater quenching for Mg 2+ and Ca 2+ .In this case, the responses to Mg 2+ and Ca 2+ were similar.For L2 in the presence of alkaline earth metal cations, the effect was also substantial quenching with red shifts (Figure 4b).The quenching effect of Mg 2+ was even greater.It is interesting that allyl and benzyl groups on the upper rim caused nearly same effect on the fluorescence spectra for interactions with alkali metal and alkaline earth metal cations. 16he effects of transition metal cations and Pb 2+ on the fluorescence spectra of L1 and L2 are presented in Figures 3c and 4c, respectively.Strong quenching of the fluorescence of L1 and L2 was observed in the presence of the transition metal cations and Pb 2+ .However, L2 caused greater quenching for all transition metal cations and Pb 2+ (Figure 4c).In particular, Fe 3+ , Hg 2+ and Pb 2+ cause greater than 99 % quenching of the dansyl fluorescence for both ligands L1 and L2.

Determination of stability constants
Stability constants and stoichiometries for complexation of Hg 2+ , Pb 2+ and Fe 3+ by ligands L1 and L2 in MeCN were determined by spectrofluorimetric titration.The ligand concentration was held constant at 2.58 × 10 -5 M. Stoichiometries of the complexes and their stability constants were determined from changes in the fluorescence intensity as a function of the metal ion concentration.Successive decreases of emission with increases of the metal ion concentration were observed in all of the fluorimetric titrations.
Figure 5 shows the fluorescence spectra of L2 in MeCN with increasing concentrations of Fe 3+ .The inserts in Figure 5   Io/(Io-I) Table 1 presents the stability constants and complex stochiometries for complexation of Fe 3+ , Hg 2+ and Pb 2+ by L 16 , L1 and L2.The log β values vary between 3.94 and 5.12 and show that the ionized ligands interact strongly with these metal ions in MeCN.For 1:1 complexation of Fe 3+ by the three ligands, the stability constants decrease in the order: L2 > L1 > L. Thus, the attachment of either two or four allyl groups to the upper rim of di-ionized ligand L increases the propensity for complexation of Fe 3+ in MeCN.A similar increase of stability constant for complexation of Fe 3+ was obtained with the upper-rim allyl-substituted analogues.As seen from the data in Table 1, all three ligands form 1:1 complexes with Fe 3+ , Hg 2+ and Pb 2+ .On the other hand, the upper-rim allyl-substituted analogues formed 1:2 complexes with Fe 3+ . 16It is clear that among the ligands L2 forms the most stable complexes with Fe 3+ , Hg 2+ and Pb 2+ .This result can be explained as arising from the effect of increased π electron density due to the four benzyl groups.

Stern-Volmer analysis
Stern-Volmer analysis was utilized to probe the nature of the quenching process in the complexation of Fe 3+ , Hg 2+ and Pb 2+ by L1 and L2.Stern-Volmer plots are a useful method of presenting data on emission quenching. 22,23Plotting relative emission intensities (Io/I) against quencher concentration [Q] yields a linear Stern-Volmer plot for a static quenching process.Expressed as Equation 1, the slope of this line is Ksv, the static quenching constant.I and Io are fluorescence intensities in the presence and in the absence of added metal cations.Io/I = 1+ Ksv [Q] (1) Figure 6 shows the steady-state emission Stern-Volmer analysis for complexation of Pb 2+ by L2.For both L1 and L2, linear behavior was observed for complexation of Fe 3+ , Hg 2+ and Pb 2+ .These results are consistent with static quenching.

Summary
This study investigates the influence of a systematic structural variation within calix [4]arene compounds with two ionized, dansyl group-containing side arms on the lower rim upon their spectroscopic responses to metal ions in MeCN.The upper rim possesses zero, two and four benzyl groups.In the presence of excess metal ions, fluorescence quenching increased in the order: alkali metal cations<alkaline earth metal cations<transition and heavy metal cations.The presence of Fe 3+ , Hg 2+ and Pb 2+ gave greater than 99% quenching of the dansyl fluorescence for the three ligands.For all three of the ionized ligands, only 1:1 (M/L) complexes are formed with these three metal ions.Information gained from this investigation aids in assessing the potential for such di-ionized, dansyl-containing ligands in fluorogenic metal ion sensors.

Experimental Section
General.The 1 H and 13 24 was prepared by a reported procedure.

Fluorescence measurements
Fluorescence spectra of the di-ionized ligands (2.58 × 10 -5 M) in MeCN solutions containing 50 molar equivalents of the appropriate metal perchlorate salt were measured using a 1-cm quartz cell.The excitation wavelength was 328 nm for all of the ionized ligands.Fluorescence emission spectra were recorded in the range 400-600 nm with a slit width of 1.0 nm.The stoichiometries of the complexes and their stability constants were determined according to a literature procedure. 19

Synthesis of 25,27-bis[N-(5-dimethylaminonaphthalene-1-sulfonyl)carbamoylmethoxy]-11,23-dibenzyl-26,28-dimethoxycalix[4]arene (1)
. Diacid 8 (0.50 g, 0.67 mmol) was dried by benzene-azeotropic distillation with a Dean-Stark trap.Oxalyl chloride (0.57 mL, 6.7 mmol) was added and the solution was refluxed for 5 h.The solvent was removed in vacuo to provide the corresponding di(acid chloride).A solution of the di(acid chloride) in THF (20 mL) was added to a mixture of dansylamide (0.37 g, 1.48 mmol) and NaH (0.16 g, 6.7 mmol) in THF (20 mL).The mixture was stirred overnight at room temperature.Water was added carefully dropwise to decompose the excess NaH.The THF was evaporated in vacuo and CH2Cl2 (100 mL) was added to the residue.The organic layer was washed with 1N HCl (50 mL) and then water (2 X 50 mL), dried over MgSO4 and evaporated in vacuo.The residue was chromatographed on silica gel with CH2Cl2-EtOAc (3:1) as eluent.Appropriate fractions were combined and evaporated in vacuo.The residue was dissolved in CH2Cl2.The solution was washed with 10% aq HCl and then water, dried over MgSO4 and evaporated in vacuo to give a 30% yield of 1 as a light yellow solid with mp 172-174 o C. IR 3453 and 3327 (N-H), 1729 (C=O) cm -1 . 1 H NMR δ 2.90-3.90(m, 34H), ARKAT USA, Inc.  (9).To a suspension of calix [4]arene 3 (5.00g, 11.8 mmol) and AlCl3 (12.60 g, 94.4 mmol) in CH2Cl2 (250 mL) was added benzoyl chloride (26.52g, 188.7 mmol) dropwise over a period of 30 min.The mixture was stirred at room temperature for 24 h and then poured into ice-water.The organic layer was separated, washed with 1N HCl and then with 10% aq NaOH (to remove the excess benzoyl chloride) and evaporated in vacuo.The yellow solid residue was mixed with MeOH (600 mL) and 10% aq NaOH (200 mL) and the mixture was refluxed for 2 days.The MeOH was evaporated in vacuo.The precipitate was filtered to give a yellow solid, which was added to CH2Cl2 (1500 mL) and 1N HCl (500 mL).Upon stirring the mixture overnight, the solid dissolved completely.The CH2Cl2 layer was separated, washed with water, dried over MgSO4 and evaporated in vacuo to give a red solid.Chromatography on silica gel with CH2Cl2-MeOH (80:1) as eluent gave an 89% yield of 9 as a white solid with mp 326-328 O C (lit. 25   (10).To a suspension of 9 (5.00 g, 5.95 mmol) and K2CO3 (16.43 g, 119.0 mmol) in triethylene glycol (300 mL) was added hydrazine hydrate (20 mL).The mixture was heated to reflux and water was removed with a Dean-Stark trip.When no additional amount of water was evolved, the Dean-Stark trap was removed and the mixture was refluxed for 20 h and then allowed to cool to room temperature.The mixture was diluted with 1N HCl (200 mL) and extracted with CH2Cl2 (2 X 200 mL).The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo.The yellow solid residue was chromatographed on silica gel with hexanes-CH2Cl2 (2:1) as eluent to give 10 as a white solid with mp 192-194 o C (lit. 26   (12).To a suspension of NaH (0.51 g, 21.4 mmol) in THF (100 mL), 11 (4.35 g, 5.36 mmol) was added and the mixture was stirred at room temperature until the evolution of hydrogen ceased.A solution of ethyl bromoacetate (3.58 g, 21.4 mmol) in THF (10 mL) was added over a period of 30 min and the mixture was stirred overnight at room temperature.Water was carefully added dropwise to destroy the excess NaH.The mixture was diluted with 1N HCl (200 mL) and extracted with CH2Cl2 (2 X 200 mL).The combined organic layers were washed with water, dried over MgSO4 and evaporated in vacuo.The residue was chromatographed on silica gel with hexanes-EtOAc (30:1) as eluent to give 12 in 90% yield as an oil.IR 1759 (C=O) cm -1 . 1 H NMR δ 1.27 (t, 6H), 2.60-4.60 (m, 30H), 6.17-7.41(m, 28H). 13  (13).A mixture of 12 (5.00g, 5.07 mmol), THF (150 mL) and 10% aq Me4NOH (100 mL) was refluxed overnight.The mixture was acidified to pH ~ 1 with 10% aq HCl.The solvent was evaporated in vacuo to give 13 as a yellowish solid with mp 96-100 o C in 97% yield.IR 3730-2520 (O-H), 1753 (C=O) cm -1 . 1   (2).Diacid 13 (0.30 g, 0.32 mmol) was dried by benzene-azeotropic distillation with a Dean-Stark trap.Oxalyl chloride (0.28 mL, 3.3 mmol) was added and the solution was refluxed for 5 h.The solvent was removed in vacuo to provide the corresponding di(acid chloride).A solution of the di(acid chloride) in THF (20 mL) was added to a mixture of dansylamide (0.18 g, 0.72 mmol) and NaH (0.080 g, 3.3 mmol) in THF (20 mL).The mixture was stirred overnight at room temperature.Water was added carefully dropwise to decompose the excess NaH.The THF was evaporated in vacuo and CH2Cl2 (100 mL) was added to the residue.The organic layer was washed with 1N HCl (50 mL) and then water (2 X 50 mL), dried over MgSO4 and evaporated in vacuo.The residue was chromatographed on silica gel with CH2Cl2-EtOAc (2:1) as eluent.Appropriate fractions were combined and evaporated in vacuo.The residue was dissolved in CH2Cl2.The solution was ARKAT USA, Inc.

Figure 3 .Figure 4 .
Figure 3.Effect of metal cations on the fluorescence spectra of L1 in MeCN: (a) for alkali metal cations; (b) for alkaline earth metal cations ; (c) for transition metal cations and Pb 2+ .
are a plot of Io-I vs. the ratio of [M]/[L] and a plot of the quantity Io/(Io-I) versus 1/[M].The break in the former at M]/[L] = 1.0 provides strong evidence for formation of a 1:1 complex.Similar plots were found with Pb 2+ and Hg 2+ for L1 and L2 in MeCN.The stability constant for a complex was obtained from a plot of the quantity Io/(Io-I) versus 1/[M].The ratio of intercept/slope gave the stability constant.19
point apparatus.Elemental analysis was performed by Desert Analytics Laboratory (now Columbia Analytical Services) of Tucson, Arizona.All reactions were conducted under nitrogen atmosphere.Reagents were obtained from commercial suppliers and used directly unless otherwise noted.Calix[4]arene 3 was obtained from Eburon Organics International of Lubbock, Texas.Tetrahydrofuran (THF) was dried over sodium with benzophenone as a indicator and distilled just before use.Spectrometric grade acetonitrile (MeCN) from EMD Chemicals was the solvent for the fluorescence measurements.All metal perchlorates purchased from Acros were of the highest available quality and vacuum dried over blue silica gel before use.The 25,27-dihydroxy-26-28-dimethoxycalix[4]arene 4 obtained with a SLM Aminco 800C photon counting spectrofluorimeter equipped with a 450-W ozone-free xenon lamp as the light source.Melting points were determined with a Mel-Temp ARKAT USA, Inc. melting