Calix[4]arenes for the selective detection of Zn2+

Calix[4]arenes of Formula I are useful for selectively detecting Zn2+ ion.

FIELD

The present technology generally relates to ion detection.

BACKGROUND

Zinc is an essential nutrient and is necessary for the functioning of several metalloenzymes in humans and animals. Zinc deficiency is associated with anorexia, impaired immune, neural and reproductive functions. Zinc ions are present in neuronal cells. Due to its importance in human growth and development, human serum contains about 19 μM of this ion. Imbalanced homeostasis of Zn2+may cause a variety of diseases. However, an excess of zinc compounds such as oxides, sulfates, sulfides, and chlorides are known to cause problems in the respiratory tract and lead to bronchopneumonia and pneumonitis, developmental defects, inflammatory reactions, and even death. Prolonged oral exposure to zinc may also reduce copper absorption. Estimates of the minimal risk levels of zinc range from 77-600 mg/m3for inhalation, and is 0.3 mg/kg/day for oral exposure. There is a need for compounds that can detect Zn2+and for methods for detecting Zn2+, for example in blood serum. Provided herein are compounds and methods suitable for detecting Zn2+in a variety of samples.

SUMMARY

In one aspect, a compound of Formula I is provided:

or a salt thereof; wherein: each R1is a group of Formula:

In another aspect, a complex is provided including the compound of Formula I and a Zn2+ion. In another aspect, a method of determining the presence or absence of Zn2+in a solution is provided. This method includes contacting the compound of Formula I with a test sample to form a solution; and recording a fluorescence spectrum of the solution, wherein in the presence of Zn2+, the solution exhibits fluorescence intensity at about 450 nm that is greater than a fluorescence intensity of a solution that does not contain Zn2+. In one embodiment, the presence of Zn2+can be determined in the presence of other metal ions in the sample.

In another aspect, a method of synthesizing the compound of Formula I is provided which includes contacting a compound of Formula II:

with a compound of Formula III:

to provide a compound of Formula IV:

or a salt thereof, wherein R10is:

In one embodiment, the method also includes contacting the compound of Formula IV with R5NH2, or a salt thereof, to provide a compound of Formula I:

DETAILED DESCRIPTION

In the following detailed description, the illustrative embodiments described are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include plural reference.

Provided herein are calix[4]arene compounds, their complexes with Zn2+ion, methods of making these compound and complexes, and methods of determining the presence and absence of Zn2+ion in an analyte. Thus, in one aspect, a compound of Formula I is provided:

or a salt thereof; wherein: each R1is a group of Formula:

The above compounds may form salts with inorganic or organic acids or bases. In one embodiment, the salts include the phenoxide salts of compounds of Formula I. In another embodiment, the phenoxide moiety is part of the salicylaldimine or the R1moiety. In the phenoxide salts, the cation may be a variety of organic and inorganic cations. In one embodiment, the cation is a Zn2+cation. Salts may also include, without limitation, acid salts, formed with acids such of HClO4, HCl, H2SO4, and H3PO4, as well as acetic acid or trifluoroacetic acid.

In another aspect, a Zn2+salt or a Zn2+complex of the compound of Formula I and a Zn2+ion is provided. In certain embodiments, the Zn2+ion in the Zn2+complex is bonded to the salicylaldimine moiety via imino nitrogens and phenoxide oxygens.

In another aspect, a method of synthesis of the compound of Formula I is provided including contacting a compound of Formula II:

with a compound of Formula III:

to provide a compound of Formula IV:

or a salt thereof, wherein R10is:

R2is H, C1-C8alkyl or C3-C8cycloalkyl; and R4is H or C1-C8alkyl. In one embodiment, compounds of Formula II and Formula III are contacted in the presence of Cu2+. In a more specific embodiment, compounds of Formula II and Formula III are contacted in the presence of CuSO4.5H2O and sodium ascorbate in dichloromethane/water.

In one embodiment, the method also includes contacting the compound of Formula IV with R5—NH2, or a salt thereof, to provide a compound of Formula I:

In another embodiment, the method also includes contacting a compound of Formula I with a zinc salt to provide the zinc complexes. A variety of zinc salts may thus be employed including, without limitation, various zinc carboxylates. In another embodiment, the zinc carboxylate is zinc acetate.

In various other embodiments, R1, R2, R3, R4, and R5are defined as in any aspect or embodiment hereinabove. A skilled artisan will appreciate that the contacting may be performed in a variety of solvents including, without limitation, chlorinated solvents, dimethylformamide (DMF), ketones, alcohols, and water. After reacting, the product may be separated from the reaction mixture, for example, following an aqueous work-up. The product may be separated from other impurities by a variety of methods, including, without limitation, distillation, precipitation, crystallization, and chromatographic separation.

In another aspect, a method of determining the presence or absence of Zn2+in a solution is provided. The method may be qualitative (measuring the presence or absence of Zn2+) or quantitative (measuring the concentration of Zn2+). Such methods include contacting the compound of Formula I with a test sample to form a solution; and recording a fluorescence spectrum of the solution. The presence of Zn2+in the solution is confirmed by the exhibition of fluorescence intensity at about 450 nm that is greater than a fluorescence intensity of a solution or sample that does not contain Zn2+. The method may further comprise contacting the compound of Formula I with a blank sample lacking Zn2+to form a blank solution, and recording the fluorescence spectrum of the blank solution. In other words, any signal at 450 nm is enhanced in the presence of the Zn2+, or if no signal is present, the signal appears in the presence of Zn2+. For example, the enhancement at about 450 nm that is greater than a fluorescence intensity of a solution that does not contain Zn2+, may be from about 2 to 50, about 4 to 25, or about 8 to 10 fold greater.

In another embodiment, the test sample includes serum. In another embodiment, the test sample includes soil, water or food. In another embodiment, the test sample is one of biological origin. For example, samples of biological origin may include, but are not limited to blood, urine, cells, and/or tissue.

A wavelength of 450 nm may be used, or alternatively, a wavelength of about 450 nm may be used. As used herein, about 450 nm includes, from 400 nm to 525 nm, from 425 nm to 500 nm, from 450 nm to 475 nm, and 450 nm. The fluorescence spectrum is recorded using a an excitation wavelength (λex) from 360 nm to 400 nm, or from 370 nm to 390 nm, or which is about 380 nm.

The test sample may include aqueous methanolic (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Test samples may generally have any pH value. For example, Zn2+ions may be determined in test samples having a pH of 6, 7, 8, 9, 10, or 11, or from 6 to 11, 7 to 10, or from 8 to 9. Biological samples typically have pH values of about 7.

In some embodiments, the compound of Formula I and the test sample can both be dissolved in the same solvent or different solvents prior to the fluorescence testing. Alternatively, the compound of Formula I can be added without a solvent. The solvent can be a pure solvent or a mixture of solvents. If the solvents are different, they typically will be miscible with each other. For example, the compound of Formula I and the test sample can be dissolved in solvents which may include water, alcohol, or acetonitrile. In some embodiments, the test sample and/or the control sample includes an aqueous solution. In some embodiments, the test sample may be prepared by dissolving the sample to be analyzed in an aqueous solution including water in combination with water-miscible solvents. In some embodiments, the sample to be analyzed is dissolved in an aqueous solution that includes acetonitrile or methanol. In some embodiments, the sample to be analyzed is dissolved in a solution including aqueous methanol. In some embodiments, the aqueous methanol solution includes about 10% to about 90% methanol (by volume). In other embodiments, the aqueous sample solution includes about 40% to about 75% acetonitrile (by volume) or about 50% acetonitrile (by volume).

The methods provided herein can have very good sensitivity down to the part per billion (ppb) levels. In another embodiment, the presence of Zn2+ions may be detected, at a concentration of at least 20 ppb, at least 100 ppb, at least 200 ppb, at least 300 ppb, at least 500 ppb, or at least 1 part per million (ppm). In other embodiments, the Zn2+may be detected from about 20 ppb to about 10 ppm, from about 25 ppb to about 1 ppm, from about 30 ppb to about 500 ppb, or from about 30 ppb to about 100 ppb.

The methods provided are very sensitive for the presence of Zn2+, even in the presence of one or more other metal ions. Thus, in another embodiment, the compounds of Formula I may detect Zn2+in the presence of various other ions. Other metal ions may include, but are not limited to, divalent or trivalent metal ions. In another embodiment, the divalent metal ion is an alkaline earth metal ion, including without limitation, Mg2+, Ca2+Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Hg2+or Cd2+.

In another embodiment, the other metal ion is a monovalent metal ion. For example, monovalent metal ions may include, but are not limited to Li+, Na+, K+, Cs+, or Ag+. In another embodiment, the presence of Zn2+may be determined in the presence of Hg2+, Cd2+, Li+, Na+, K+, Cs+, Mg2+, Ca2+Sr2+, Ba2+, Mn2+, Fe2+, Co2+, Ni2+, Co2+, or Ag+ion. In one embodiment, a 100 ppb to 500 ppb range concentration of Zn2+ions can be detected in the presence of about 6.5 ppm concentration of Hg2+and about 6.5 ppm concentration of Cd2+.

The compounds of Formula I may detect Zn2+in the presence of blood serum and of various albumins that are known to form complexes with Zn2+. Thus, the presence of Zn2+may be determined in the presence of HSA, BSA, or LA. In another embodiment, the albumin is present in the test solution at a concentration of about 1 mg/mL of proteins. The Zn2+may also be detected in blood serum, at concentrations of about 300 ppb, 400 ppb, 500 ppb, 1 ppm, or from 100 ppb to 1 ppm.

As used herein, “alkyl” groups are monovalent hydrocarbon radicals and include straight chain and branched alkyl groups having from 1 to about 20 carbon atoms, and alternatively from 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include without limitation methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, without limitation, isopropyl, sec-butyl, t-butyl, neopentyl, and isopentyl groups. Alkyl groups may be unsubstituted or substituted. Representative substituted alkyl groups may be substituted one or more times with, for example, amino, carboxyl, thio, hydroxy, cyano, alkoxy, phenyl, and/or F, Cl, Br, and I groups.

As used herein, “alkoxy” refers to an —O-alkyl moiety. Examples of alkoxy groups include, without limitation, methoxy, ethoxy, isopropoxy, and benzyloxy.

As used herein, “cycloalkyl” groups are monovalent cyclic hydrocarbons. Examples of cyloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Cycloalkyl groups may be unsubstituted or substituted.

As used herein, “5-membered heteroaryl” refers to a cyclic aromatic ring containing 5 ring atoms and containing at least 1, and up to 4, heteroatoms selected from N, O, and S. Such 5 membered heteroaryl groups may be unsubstituted or substituted. Examples of 5 membered heteroaryls include, without limitation, diazoles, furan, imidazole, oxadiazole, pyrrole, thiadiazole, thiophene, triazoles, and the like.

As used herein, “substituted amino” refers to —NHRx or —N(Rx)2wherein each Rx independently is alkyl, —CO-alkyl, CO2-alkyl, SO2-alkyl, or two Rx groups together with the nitrogen atom to which they are bonded for a cyclic ring.

The present technology, thus generally described, will be understood more readily by reference to the following example, which is provided by way of illustration and is not intended to limit the present technology.

EXAMPLES

Overall Synthetic Scheme. L, a compound of Formula I, its precursors, and control compounds used to test the superior Zn2+detectability of L, were synthesized as shown in Scheme 1. To incorporate binding motifs and a fluorophore on the calix[4]arene platform, a triazole moiety was used as a linker. An aldehyde precursor, L3, was synthesized by reacting calix[4]arene based di-propargyl ether derivative (L1) with the substituted salicylaldehyde azide derivative (L2) through a click reaction as shown in Scheme 1. The receptor molecule (L) was synthesized in quantitative yield by the condensing L3with n-butyl amine in methanol (Scheme 1).

Synthesis of L2. L2, a compound of Formula III, was synthesized starting with p-t-butyl phenol, and via the intermediacy of compounds 1 and 2, as shown in Scheme 2.

Mass, NMR and UV-absorption spectroscopy confirming the zinc-complexation. The formation of a 1:1 complex between Zn2+and L was also confirmed by the m/z=1403.8 (100%, [L+Zn+K−H+]), 1388.9 (85-90%, [L+Zn+Na]), and 1365.9 (35-45%, [L+Zn]) peaks observed in (electrospray ionization mass spectroscopy) ESI MS, where the isotopic distribution demonstrated the characteristic signature of zinc in each of these peaks. The complex was also confirmed by comparing the1H NMR spectrum of L-Zn complex with that of L, where some resonances were shifted to down field and some to upfield, indicating a complex formation.

The absorption titration carried out between L and Zn2+in the same medium (FIG. 1A) exhibited three isosbestic points at 290, 335, and 405 nm indicating a transition between the unbound L and that of Zn2+bound L. L binds Zn2+via its two phenolic-oxygens and two imine nitrogens to form tetracordinated complex. The spectra also exhibited increase in absorbance in the about 375 nm bands and decrease in absorbance in case of about 320 and about 420 nm bands (FIG. 1B). The stoichiometry of the complex formed between L and Zn2+has been derived to be 1:1 based on Job's plot (FIG. 2).

Crystallization and structure determination of the zinc complex of L, Zn-L. X-ray diffraction quality crystals of the Zn-L complex were grown from a 1:1 methanol-acetonitrile mixture. The crystal structure exhibited a distorted tetrahedral Zn2+center where both the arms of L act as bidentate ligands through their imine nitrogen and phenoxide oxygen to give an N2O2core where the total complex is neutral. Crystal data for Zn-L is as follows. Empirical formula: C82H106N8O6Zn; formula. wt.: 1365.16; crystal system: triclinic, P1; unit cell dimension (Å): 15.5813(6), 17.3524(5), 18.4381(6), 79.959(3), 65.028(4), 76.432(3); V=4351.7(1) (Å3); Z=2; Dc=1.15 (g ml−1); unique reflections: 28611, R_obs: 0.068, wR2_obs: 0.205. In the primary coordination sphere about the zinc ion, bond lengths (Å) and bond angles) (° were Zn—O6=1.903(4), Zn—O5=1.911(3), Zn—N4=1.985(3), Zn—N8=1.996(2); N4-Zn—N8=122.1(1), N4-Zn—O6=118.4(1), N8-Zn—O6=97.6(1), N4-Zn—O5=96.4(1), O5-Zn—O6=106.7(1), O5-Zn—N8=115.9(1), demonstrating a substantially tetrahedral structure.

Fluorescence titrations. The receptor L exhibits very weak fluorescence emission at about 450 nm when excited at 380 nm in 10 mM methanolic HEPES buffer of pH=7.4 containing 4:1 (v/v) methanol and 50 mM HEPES buffer. Titrating such a solution of L with Zn2+, the fluorescence intensity enhances as a function of increasing Zn2+concentration (FIG. 3A). A plot of fluorescence intensity as a function of added [Zn2+]/[L] mole ratio (FIG. 3B) shows a stoichiometry of 1:1 between L and Zn2+and exhibits intensity saturation at >1 eq. An association constant of 148537±2930 M−1for the L-Zn complex was derived using Benesi-Hildebrand equation. When excited at 365 nm, the L+Zn2+complex is visibly fluorescent while L is not.

Competitive fluorescence titration in presence of other ions. To test L's ability to selectively detect Zn2+, fluorescence titrations were carried out in the same medium with the different metal ions, Li+, Na+, K+, Cs+, Mg2+, Ca2+, Sr2+, Ba2+, Nm2+, Co2+, Ni2+, Cd2+, Ag+and Hg2+. No significant fluorescence enhancement or quenching in presence of these ions was observed (FIG. 4). Concentration variation titration carried out between L and Zn2+, while maintaining their molar ratio at 1:1, resulted in a minimum detection limit of 36 ppb for Zn2+under these conditions. Since biological systems may possess large concentrations of alkali and alkaline earth ions, the selectivity of Zn2+has been studied by carrying out appropriate competitive metal ion titrations. No significant change in the fluorescence enhancement of L with Zn2+was observed. Hence the fluorescence of L with Zn2+does not exhibit changes in presence of other biologically important alkali and alkaline earth metal ions, however, shows strong complexation behavior of towards zinc (FIG. 5). As cadmium and mercury belong to the same period, the selectivity of Zn2+towards L was tested by carrying out the corresponding competitive ion titrations. Again, these ions did not appear to interfere in the detection of Zn2+(FIG. 5). Thus these tests further support L's use for selectively detecting Zn2+.

Testing the effect of pH variation on zinc detection. When titrations were carried out between L and Zn2+in the same medium but varying the pH from 6 to 9, no variation in the fluorescence intensity resulted (FIG. 6), suggesting that L can detect Zn2+in this pH range, which mostly covers the physiological systems. While the quantum yield of L is only Φ=0.028, binding of Zn2+to L enhances it to Φ=0.32. The observed ten-fold increase in the quantum yield of L in presence of Zn2+, simultaneously in the presence of water and buffer in the medium, and the observed low detection limit demonstrates the usefulness of L for detecting and quantifying Zn2+by switch on fluorescence spectroscopy, even in a biological medium.

Biological applicability of Zn2+detection by L. Biological applicability of L to sense Zn2+has been addressed by carrying out fluorescence titrations using blood serum, which includes albumin proteins, and albumins such as human serum albumin (HSA), bovine serum albumin (BSA) and an α-lactalbumin (LA), which are capable of complexing Zn2+. Fluorescence experiments were carried out by taking an in situ generated Zn-L complex and titrating this complex with varying concentrations of blood serum or the proteins (HSA, BSA and α-LA). Serum samples were obtained from a healthy volunteer after fasting. The blood sample was allowed to clot and serum was obtained via centrifugation. The serum samples were kept at −20° C. for storage. Serum (100 mL) was dissolved in HEPES buffer (3 mL) and used as stock solution. The bulk solution for proteins had a concentration of 1 mg/mL. Tests in this manner were not carried out beyond this concentration of the serum or the proteins due to precipitation.

Almost no change was observed in the fluorescence intensity of the about 450 nm band of L either in presence of these proteins individually or as a whole in presence of the serum (FIG. 7). Thus L can selectively detect Zn2+even in the blood serum milieu. The lower limit of Zn2+concentration at which L detected Zn2+was found to be 332 ppb in blood serum.

Titrations with the control molecules. The usefulness of the Schiff's base portion as well as the calix[4]arene platform in L for sensing Zn2+was addressed by employing L3(a precursor that possesses an aldehyde but not an imino group) and L4(a “single stranded” version of L) as control Zn2+detectors (see Scheme 1). Fluorescence titrations demonstrated that L detected Zn2+selectively, while the control detectors did not (FIG. 8). For L3and L4, the fluorescence enhancement was found to be very low, and that was only at very high molar equivalents of Zn2+, such as, greater than 30 molar equivalents. Thus the receptor molecule, L was much more sensitive toward Zn2+than the control molecules, L3and L4.

L has been demonstrated to detect Zn2+selectively by switch-on fluorescence. A 1:1 complex between L and Zn has been shown based on fluorescence, absorption, ESI MS and1H NMR. Further, L's selectivity to detect Zn2+has been demonstrated, in aqueous methanolic HEPES buffer, in the pH range of 6-11, in blood serum milieu, in presence, e.g., of albumins, of alkali and alkaline earth ions, and of cadmium and mercury. L has been demonstrated to be selective as compared to its precursor aldehyde derivative, L3, indicating the usefulness of the Schiff's base moiety. L has also been demonstrated to be a more efficient Zn2+detector than L4, a single stranded version of L, indicating the usefulness of the calix[4]arene scaffold for the detection.

EQUIVALENTS

The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms ‘comprising,’ ‘including,’ ‘containing,’ etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase ‘consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase ‘consisting of’ excludes any element not specified.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as ‘up to,’ ‘at least,’ ‘greater than,’ ‘less than,’ and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

Other embodiments are set forth in the following claims.