Independent Reactions of Zn²⁺ and Cd²⁺ with Glycine Alone in Aqueous Solutions

Understanding more about the structure and function of the brain and its neurotransmitters is one of the biggest challenges for modern scientists. The brain is being studied by a broad range of investigators, collectively called neuroscientists. How can chemists aid in understanding the brain? The answer, as some scientists have proposed [1,2] is to analyze the small molecules found in the brain known as neurotransmitters. Thus far, there are thirtyone (31) known neurotransmitters that are categorized into five families namely, cholinergic agents, catechol amines, amino acids, peptides and gaseous neuro-transmitters [3]. Glycine (Gly) is one of these important neurotransmitters. This was one of the main reasons we studied Gly with zinc and cadmium. Gly is an inhibitory neurotransmitter. It has been reported that zinc has a very complex influence on Gly receptors, and it affects the efficacy of glycinergic transmission in mammalian brains [4,5]. It has been reported that Gly can reach a millimolar concentration during synaptic transmission [4]. Figure 1 shows the structure of free Gly in the form of glycine hydrochloride (Gly.HCl), or 2-amino acetic acid (chemical formula C2H5NO2. HCl) used in the current study.

Figure 1:Structural formula of glycine hydrochloride (Gly.HCl), or 2-amino acetic acid (chemical formula C₂H₅NO_2>.HCl) used in this study.

Zinc is only second to iron as the most abundant trace metal in the human body, with ~3.0g of zinc being present in a typical human adult [6]. It has been shown that it exists in more than 300 proteins and enzymes: carboxypeptidase, carbonic anhydrase and zinc finger proteins being the most famous [6-8]. Nearly 90% of zinc is found in muscles and bones. Considerable amounts of zinc are present in prostate, liver, kidneys, skin, lung, brain, heart and pancreas [6-13]. Absorption of zinc occurs throughout the small intestines. The absorption process is active, energy driven and mediated by specific binding proteins/ligands [6-8]. After absorption, distribution of zinc occurs via the serum, where it is bound predominately to proteins such as albumin, α-microglobulin, transferrin and to a limited extent is bound to low molecular mass ligands [6,13,14]. Individual compounds that have a positive effect on zinc absorption include glycine, lysine cysteine, and histidine [6].

Due to the complex nature of zinc regulations, the regular concentration of free zinc in circulatory fluids are in the femtomolar concentrations range, however, the hippocampus area of the human brain contains millimolar concentration ranges of zinc [7,9]. Cadmium has been considered to be a toxic metal ion for a century. It does not have a known essential role in biochemistry except in the replacement of the zinc metal ion at the catalytic site of a particular carbonic anhydrase enzyme in certain marine diatoms [15]. In addition, it is considered to be a human carcinogen [14,15].

Clearfield et al. [16] showed the reactions of zinc with glycine and/or glycine derivatives, have shown the crystal structures of both bis-Cu-Gly and bis-Zn-Gly [16]. The Zn-Gly crystal shows a tetra Zn-glycinate cluster creating coordination polyhedral. Other studies showed the reactions of zinc with Gly alongside other supporting ligand(s) [17,18]. As review of related literature indicated that not many reports were published for the reaction of free Zn²⁺ or free Cd²⁺ with glycine alone under ambient conditions. One report was for the cadmium ternary complexes with glycine and Vitamin D3 as auxiliary ligand [19], or a report at which Zn²⁺ was reacted with glycine along with Vitamin B9 by Ramadan and Fazary [20]. Further and more recent literature review showed the reactions of zinc ion and glycine in the presence of histidine [21]. In the present study, the millimolar independent reactions of glycine and Zn²⁺ alone (no other ternary or auxiliary ligands were added). Also, only glycine reactions with Cd²⁺ are carried out in separate experiments in aqueous solutions at 25 °C and 0.1M (I) NaNO₃. The presence of dimeric metal complexes is pervasive in the literature; reference number 22 is only an example [22].

Chemicals/solutions

All solutions were prepared using 99% Sigma reagent grade glycine hydrochloride. Zn²⁺ solutions were prepared using zinc nitrate hexa-hydrate, and Cd²⁺ solutions were prepared using cadmium nitrate tetra-hydrate. All solutions were prepared by using doubly deionized water (DI). The pH values of all solutions were adjusted using a standard sodium hydroxide (NaOH) solution that was standardized using dry primary standard potassium hydrogen phthalate (KHP) to the phenolphthalein endpoint. Both NaOH and KHP were purchased from Fisher Chemical Co. The pH values were measured using Orion Membrane pH meter (model 720) with a combination Orion-glass electrode in 0.1 M (I) NaNO₃ solution. Deuterium oxide (D₂O) was purchased from Cambridge Isotope Laboratory. High quality Norell 5 mm NMR tubes were a donation from the chemistry department at the University of Memphis.

Preparation of the potentiometric titration solutions

In all Zn(NO₃)₂, Cd(NO₃)₂, or Gly.HCl, or Zn(NO₃)₂-Gly.HCl, or Cd(NO₃)₂-Gly.HCl potentiometric titrations in 1:1, 1:2, 1:3, 1:4, and 1:5 ratios, NaOH solution was the titrant. NaOH solutions were prepared from NaOH laboratory grade, dry, pellets in carbonate free DI water. The detailed methods used to prepare and conduct potentiometric titrations have been described before in details [23]. In brief, the NaOH solutions were standardized using primary standard KHP. Before any KHP titration, the KHP was dried at 110 °C for 24 hours and stored in an airtight lid greased desiccator filled with Hammond brand anhydrous CaSO₄ (drierite) as a desiccant. A stock indicator solution of ~0.2% phenolphthalein in ~90% ethanol was prepared from reagent grade phenolphthalein. Typically, twelve runs were carried out to standardize the NaOH solution as shown in supplementary material. Microsoft Excel software program was used to carry out statistical treatment of all data.

Potentiometric titrations and speciation calculations

The detailed methods used to conduct potentiometric titrations has been described elsewhere [23]. In brief, we have followed the detailed studies of potentiometric titrations as well as the speciation calculations as protocols described previously [23- 26]. All speciation experiments were generated using Hyperquad simulation and speciation (Hyss) software program [24], pKa values were used from Martell and Smith [25], and the pKw value of 13.78 was taken from the literature [26,27]. In a typical titration, the Gly.HCl solutions were added first (in independent experiments) followed by the addition of Zn(NO₃)₂ or Cd(NO₃)₂ solutions. This sequence of addition allows for the metal ion to bind to Gly without forming zinc hydroxide or cadmium hydroxide. Then, the mixture was allowed to stand for a minimum of fifteen minutes to reach equilibrium. NaNO₃ solutions were added to adjust the final (I) to be equal to 0.10 M in the final solution. The total volume of the final titration solution was 100.00mL. The final concentration of the Zn²⁺ or Cd²⁺ ions titrated in the titration vesicle was in the range of 1mM. The exact concentrations of the two metal ions were: [Zn²⁺] = 8.46 x 10^-4 M and [Cd²⁺] = 9.02 x 10^-4 M. Before each titration, the final solution mixtures were allowed to stir for additional 25 minutes for a state of complete equilibrium.

UV-Vis spectroscopy

All UV-Vis spectroscopy measurements were gathered on either Thermo Scientific Evolution Array UV-Vis spectrophotometer connected to vision collect software version 1.4, or the T60 highperformance spectrophotometer in connection with UVWIN software version 5.0, purchased from Advanced ChemTech (Louisville, KY). UV-Vis Samples were prepared in DI water at 25 °C. The entire UV-Vis spectrum was scanned from 250 to 1000nm using quartz cuvettes with optical path length of 1cm. A reference cuvette filled with DI water was used with all measurements. The concentration of either Zn²⁺ or Cd²⁺ was in the range of 4.51×10^-2 M. The UV-Vis spectra were collected in duplicate at the pH value of 4.00

IR spectroscopy

All IR spectroscopy measurements were conducted using a Nicolet iS10 spectrophotometer in connection with OMINIC software version 8.1, both purchased from Thermo Fisher Scientific (Madison, WI). The entire IR spectrum was scanned from 400 to 4000cm-1 using the provided attenuated total reflectance (ATR) accessory cell compartment equipped with a diamond cell. We have used the following data parameters in a typical machine setup: number of sample and background scans was set at 32 with resolution of 4cm^-1, and laser frequency of 15798.7cm^-1. Typical IR spectra were generated in which the X-axis is wavenumbers incm^-1 and the Y- axis is set as percent transmittance.

¹H-NMR- spectroscopy

Room temperature ¹H-NMR spectra were collected on the Jeol 500MHz unit housed at the chemistry department of the University of Memphis. All NMR-solutions were prepared in D₂O to lock and shim the samples in high quality Norell NMR tubes. The preparation of the NMR sample solutions was carried out four hours prior to the data acquisition. In brief, 25mL of 0.1041M glycine.HCl solution was mixed with 25mL of 0.04998 M Zn(NO₃)₂ solution in a 100mL beaker. The pH-values were adjusted using ~11M NaOH using a micro-pipette to avoid the dilution effect. These ratios gave a final concentration of zinc of 25mM, which was sufficient for easy 1H-NMR data acquisition. 1H-NMR spectra were measured at the pH-values of 2.35, 3.02, 8.70 and 10.77. All peaks were referenced to the standard value of 4.80ppm for the D₂O solvent.

Potentiometric titrations of Gly.HCl, Zn²⁺ and Cd²⁺ ions

Most of the data gathered are presented in the supplementary material. Supplementary Tables 1-4 show the standardization of solutions used. Supplementary Figure 1 shows the potentiometric titrations of Gly.HCl, which shows plots of four independent titrations at which the acidity constants of both the carboxylic acid functional group and the ammonium groups are separated by a well-defined sharp inflection point. Supplementary Figure 2 is the mathematical treatment of supplementary Figure 1. In such figures, the Gly.HCl releases a net of two protons due to the fact that Gly.HCl has two titratable functional groups; the carboxylic acid (-COOH) group and the ammonium (NH³⁺) group as shown in supplementary Figures 1 & 2. Table 1 shows the data of this ligand alongside group 12 metal ions that had been reported in NIST standard reference database of critically selected stability constants of metal complexes [25].\

Supplementary Table 1:Standardization of NaOH using dry primary standard KHP to the phenolphthalein end point.

Supplementary Table 2:Standardization of Phosphoric acid (H₃PO₄) as a calibrating agent of the entire potentiometric titration system using standard NaOH as a titrant

Supplementary Table 3:Detailed potentiometric titration data of Zn²⁺ and Gly.HCl in different molar ratios in 0.1M NaNO₃ ionic strength solutions, 25 °C [Zn²⁺] = 8.46 x 10^-4 M

*Typical titration run contains 80 to 100 pH-data points

Supplementary Table 4:Detailed potentiometric titration data of Cd²⁺ and Gly.HCl in different molar ratios in 0.1 M NaNO₃ ionic strength solutions, 25 °C [C^d2+] = 9.02 x 10^-4 M

*Typical titration run contains 100 pH-data points

Supplementary Figure 1:Potentiometric titrations of the free Glycine hydrochloride solution (Gly.HCl, C₂H₅NO₂. HCl) in aqueous solutions, 25 oC, 0.10 M NaNO₃.

Supplementary Figure 2:First derivatives of the data shown in the potentiometric titrations of glycine hydrochloride in supplementary figure 1.

Table 1:Detailed potentiometric titration data of Zn²⁺ and Gly.HCl in different molar ratios in 0.1M NaNO₃ I solutions, 25 oC [Zn²⁺] = 8.46 x 10^-4M.

*Typical titration run contains 80 to 100 pH-data points

Data about the reaction of Zn²⁺ and Gly.HCl are catalogued in Table 2 (see below). As expected, the titrations of free Zn²⁺ solution and free Cd²⁺ solutions generated titration graphs with equivalence points that terminate at 2.0 equivalents of titrant. These data are shown in Supplementary Figures 3-6. The data presented herein are similar to the literature data presented previously for systems similar to the Zn²⁺:Gly and Cd²⁺:Gly systems [20-23].

Table 2:Detailed potentiometric titration data of Zn²⁺ and Gly.HCl in different molar ratios in 0.1M NaNO₃ I solutions, 25 oC [Zn²⁺] = 8.46 x 10^-4M.

*Typical titration run contains 80 to 100 pH-data points

Supplementary Figure 3:Potentiometric titrations of the free zinc nitrate solution (Zn(NO₃)₂) in aqueous solutions, 25 oC, 0.10 M NaNO₃.

Supplementary Figure 4:Potentiometric titrations of zinc nitrate in aqueous solutions, 25 oC, 0.10M NaNO₃.

Supplementary Figure 5:Potentiometric titrations of cadmium nitrate in aqueous solutions, 25 oC, 0.10M NaNO₃.

Supplementary Figure 6:Potentiometric titrations of cadmium nitrate in aqueous solutions, 25 oC, 0.10M NaNO₃.

¹H-NMR spectra

¹H-NMR experiment for the Zn²⁺-Gly reaction mixture indicated a linear correlation that as the pH-values increased from 2.35 to 10.77, they were high localized electron density concentrated on the glycine chelator. This in turn shifted the 1H-NMR chemical shift (δ) of the methylene (CH2) protons to lower values. This linear pH-correlation of the down shield shift of the 1H-NMR δ -values is depicted in Figure 2. To the best of our knowledge, this is the first study to present and combine 1H-NMR-data for the Zn²⁺- Gly reaction mixture in aqueous solution at room temperature alongside potentiometry, IR, speciation, and UV-vis spectroscopy. The solution pH-values were corrected according to eq. 1 [28].

Figure 2:Linear regression of the changes of the 1H-NMR peaks in parts per million () versus pH values of (2.35, 3.02, 8.70 and 10.77) for the Zn²⁺ and Gly.HCl reaction mixture in 1:2 molar ratio in D₂O, [Zn²⁺] = 2.50 x 10^-2M.

Independent potentiometric titrations of Zn²⁺ and Cd²⁺ with Gly.HCl in various molar ratios

Figure 3 is the potentiometric titration graphs for the Zn²⁺: Gly. HCl titration systems in 1:1 molar ratio. In the 1:1 titration graph, there is a plateau above pH-value of 8 which is due to the hydrolysis of the Zn²⁺ ion around this high pH-value. The literature value for the first hydrolysis constant of Zn²⁺ in 0.1M I is - 8.70 according to eq. (2) given below [25-27].

Figure 3:Potentiometric titrations of Zn²⁺ and Gly.HCl in 1:1 molar ratio, at 25 °C, in 0.10 M (I) NaNO₃, [Zn²⁺] = 8.46 x 10^-4 M.

The potentiometric titration graphs for the Cd²⁺: Gly.HCl titration systems in 1:3 molar ratios are shown in Figure 4. The plateau that was observed in the 1:1 titration system of Zn²⁺:Gly.HCl was missing from the 1:1 Cd²⁺:Gly titration system (shown in the supplementary figures for Cd²⁺), this is due to the difference in ionic radii of the two metal ions (88pm for Zn²⁺ versus 109pm for Cd²⁺) which makes Cd²⁺ have less hydrolysis properties compared to its smaller size family member Zn²⁺ ion [26,27,29].

Figure 4:Potentiometric titrations of Cd²⁺: Gly n 1:3.2 ratio at 25 °C, in 0.10 M I NaNO₃, [Cd²⁺] = 9.02 x 10^-4M. A mixture of monomer and dimers is formed.

Supplementary Figures 7-11 are the detailed potentiometric titration graphs of the Cd²⁺:Gly.HCl in 1:1, 1:2, 1:4 and 1:5 molar ratios respectively. The exact locations of the inflection points for both the Zn²⁺ and the Cd²⁺ titration systems with glycine are reported in Tables 2 & 3, respectively. The location of each inflection point gives the number of mols of protons released into the solution. For example, the titration plots of the Cd²⁺: Gly.HCl in 1:3.2 molar ratios indicated the release of four protons (Figure 4). By examining these plots in Figure 4 compared to that for the free Cd²⁺ graph (shown in the supplementary figures for Cd²⁺), clearly there has been an interaction between each of these individual metal ions Zn²⁺ or Cd²⁺ and Gly.HCl solutions due to the shift in the location of the inflection points to more than 2.0 equivalents compared to 2.0 equivalents as shown in the titration of the free Zn²⁺ and free Cd²⁺ ions (See Supplementary Figures 12-16). The location of the inflection point gives an idea on the identity of the metal complexes present; a mixture of monomer and dimers is formed.

Supplementary Figure 7:Potentiometric titrations of Zn²⁺: Gly n 1:3 ratio at 25 oC, in 0.10M ionic strength (I) NaNO₃, [Zn²⁺]=8.46 x 10^-4 M.

Supplementary Figure 8:Top: Potentiometric titrations of cadmium: Glycine in 1:2 molar ratio in aqueous solutions, 25 °C, 0.10M NaNO₃. Bottom: First derivatives of the data shown in the top graph.

Supplementary Figure 9:Top: Potentiometric titrations of cadmium: Glycine in 1:2 molar ratio in aqueous solutions, 25 °C, 0.10M NaNO₃. Bottom: First derivatives of the data shown in the top graph.

Supplementary Figure 10:Top: Potentiometric titrations of cadmium: Glycine in 1:4 molar ratio in aqueous solutions, 25 °C, 0.10M NaNO₃. Bottom: First derivatives of the data shown in the top graph.

Supplementary Figure 11:Top: Potentiometric titrations of cadmium: Glycine in 1:5 molar ratio in aqueous solutions, 25 °C, 0.10M NaNO₃. Bottom: First derivatives of the data shown in the top graph.

Table 3:Detailed potentiometric titration data of Cd²⁺ and Gly.HCl in different molar ratios in 0.1M NaNO₃ I solutions, 25 oC [Cd²⁺] =9.02 x 10^-4 M. *Typical titration run contains 100 pH-data points

Supplementary Figure 12:First derivatives of the potentiometric titrations of cadmium: Glycine in 1:3 molar ratio in aqueous solutions, 25 °C, 0.10M NaNO₃. A mixture of monomer and dimers is formed.

Supplementary Figure 13:IR of free Gly. HCl.

Supplementary Figure 14:IR of free Gly, free Cd(NO3)2, Cd²⁺:Gly Complex(es).

Supplementary Figure 15:IR of free Gly, free Zn(NO₃)₂, Zn²⁺:Gly Complex(es).

Supplementary Figure 16:UV-Vis spectra of DI H₂O, free Gly, free Cd(NO₃)

UV-Vis spectroscopy of Cd²⁺ with Gly.HCl

We have conducted novel UV-Vis absorption spectroscopy experiments. In these experiments, either Zn²⁺ or Cd²⁺ was reacted with the Gly.HCl that was potentiometrically titrated in the above graphs and figures. For example, the Cd²⁺ solution was mixed with Gly.HCl solution in 1:2 molar ratio. Figure 5 shows the UV-Vis absorption spectra for the control sample cuvette filled with DI H2O, free cadmium nitrate solution (Cd²⁺) and Cd²⁺: Gly.HCl solution in 1:2 ratio after 5 minutes equilibrium time. All samples were contained in 1cm quartz cuvettes. By repeating the experiment after 48 hours on the same set of cuvettes, there were no changes in the absorption pattern of the Cd²⁺: Gly reaction system. The UVVis absorption spectrum for the Cd²⁺: Gly solution in 1:2 ratio was collected at pH 4, in which no other salts were added to the reaction mixture is novel. It is noteworthy that from the literature review conducted to prepare the current revised study, it appeared that some other researchers showed some IR, some Raman, and some UV-Vis absorption spectra for similar, but not identical systems [30- 37]. In addition, there was a theoretical calculation study of glycine in the gas phase of a series of mono-valent and some di-valent metal ions including the Zn²⁺ ion [38]. So that, to the best of our knowledge, these UV-Vis absorption spectra presented in Figure 5 are new.

Figure 5:Ultraviolet-visible spectroscopy spectra of DI H2_Osub>, Gly.HCl, Cd(NO3)2 and a mixture of Cd:Gly.HCl solutions in 1:2 molar ratio at 25 °C. See supplementary figure 15 for absorbance. The ordinate is the molar absorptivity (ε) or molar extension coefficient graph. No other salts were added to adjust I, [Cd²⁺] = 4.51 x 10^-2 M.

It is clear from Figure 5 that the Gly.HCl had no absorption peaks within the UV-Vis range. Also, it is also not expected for a d10 metal ion configuration such as zinc and cadmium to possess absorption peaks in the same energy range. However, because cadmium was in the form of cadmium nitrate, the following peaks were observed. The absorption peaks that are shown had a maximum absorption value at λ_max=305nm, (Absorbance value of A=0.939, molar absorptivity ε_305nm=20.83M^-1cm^-1) for the free Cd(NO₃)₂ solution. The corresponding values for the Cd²⁺-Gly.HCl mixture of complex(es) are (Absorbance value of A=0.439, molar absorptivity ε_305nm=9.74 M^-1cm^-1) these values were due to the molar concentrations of [Cd²⁺]=0.04508M. also using the equilibrium constant for the 1:1 complex, Log Keq.1:1 = 4.25 from (Table 1) one can calculate the free energy change ΔG for the formation of the one-to-one Cd²⁺-Gly complex according to eq. (3).

At room temperature, T = 25 °C (298 K) and R=8.314 J. mol^-1. K^-1 and K_eq.1:1? = 104.25 accordingly, ΔG=-2.42 x 10⁴ J. mol^-1 which is indicative of a spontaneous and thermodynamically favored reaction. It is noteworthy to mention that there have been some kinetic studies of the zinc metal ion with glycine in aqueous solutions [36,37]. In these studies, they showed that the kinetic parameters (the formation and dissociation rate constants) are both ionic concentration and pH-dependent [36]. Also, these kinetic studies observed a relaxation time for the Zn²⁺-glycine reaction system in the millisecond time region [37].

IR Spectra of free Gly.HCl with Zn²⁺ and Cd²⁺

Figure 13-15 of the supplementary material show the overlaid IR-Spectra collected for free zinc nitrate, free Gly.HCl and the complex mixture for zinc nitrate plus Gly.HCl and free cadmium nitrate or cadmium chloride and cadmium nitrate or cadmium chloride plus Gly.HCl. Also, we are presenting the spectrum of air (showing the characteristic peaks for CO₂ at 2,360cm^-1) which was absent from the rest of the metal-Gly samples. The IR was an extra and further proof for the absence of the CO₂ from the current samples under study. The main peak that was changed due to the binding of Zn²⁺ or Cd²⁺ ions to Gly.HCl is the carbonyl peak of the carboxylate functional group that appeared at 1,734cm^-1. There were some changes in the locations of the peaks of the free Gly. HCl to that of the Cd²⁺-Gly.HCl mixture as well as the intensities of all observed peaks for free Gly.HCl were diminished due to the reaction of Gly.HCl with the given metal ion. 1734cm^-1 of free Gly. HCl versus 1731cm^-1 of the Cd²⁺-complex(es). It was encouraging to see that the peak at 1734cm-1 in the current study was observed in a previous IR study. The previous IR study was conducted in D₂O at in 1.0MKCl I solution at different pH-values [35]. These IR studies [35,36] were conducted many decades ago where instrumentations were not as accurate as the ATR-unit used in the current study.

Speciation calculations

Speciation diagrams shown in Supplementary Figures 17&18 were generated using the Hyss software program [24], we have used the pKa values from Martell and Smith [25] and the pKw value of 13.78 was used from Sweeton, Mesmer and Baes [26,27]. Supplementary figures 17&18 are showing the distribution of the total number of millimols of metal ions among the various metalglycinate complexes identified in the literature thus far [25]. The parameters set to generate the speciation diagrams shown in supplementary figures 17&18 were identical to the condition used for the potentiometric titrations. Zn²⁺ and Cd²⁺ concentrations were in the range of 1mM, and 0.10M NaOH titrant in 0.1M I [23]. In Supplementary Figure 18, we could not refine to a high degree of precision the formation of the tris- complex nor the Zn-glycinatehydroxo ternary complex that was proposed by others [25]. This is perhaps due to the fact that they did not form in appreciable quantities in our titration system. It is obvious that over a wide pHrange in our titration systems, in aqueous solutions in 0.10M I, under ambient conditions, the simple one-to-one and the bis-complexes are the dominant species and their presence over-shadowed the appearance of this tris- complex and other complexes.

Supplementary Figure 17:Speciation diagram of the total number of m moles Cd²⁺ among different Cd²⁺ - Glycomplexes.

Supplementary Figure 18:Speciation diagram of the total number of m moles Zn²⁺ among different Zn²⁺-Glycomplexes.

The available literature information thus far for the reactions of zinc and cadmium metal ions with the simplest amino acid glycine is in good agreement with the presented data. One problem we faced was that there were some studies that mixed auxiliary ligands with glycine to form what is known as the ternary reaction system. The fact that the crystal structure of Zn-glycinate complex presented in the literature in which the most stable complex is formulated into a tetra-zinc glycinate cluster indicate that the dimers we are seeing in the titrations is the precursor complex to the formation of the tetramer complex. From the values of the thermodynamic stability constants, the various speciation diagrams presented, and from the locations of the inflection points alongside the dramatic change in the UV-Vis absorbance value of the metal ion with glycine, we are very confident that the dominant species present in solutions are the simple one-to-one and the bis-complex. As the number of mols of Gly.HCl increased, the number of proton equivalents released into solution also increased forming the one-to-one, the bis- and the tris- metal complexes. We think that if the dimer complex proposed in this study is given extended time for crystallization, it will crystallize to the well-known tetramer glycine complex presented in the literature.

The fact that the detailed IR spectra of all peaks that appeared and changed its stretching frequencies due to binding to Zn²⁺ or Cd²⁺, in particular the stretching frequency of the carbonyl C=O group of the carboxylate at 1,734cm^-1, indicated a mono-dentate and not a bi-dentate binding of the glycine. We concluded that there was binding via this carboxylate which was supported by the similar experimental and theoretical calculation studies. In the future, we would like to carry out some two-dimensional NMR experiments that can show the presence of the tetramer precursor, the dimer complex.

This work was supported in part from NSF under Grant No. HRD-1332459. Acknowledgements go to the financial support of the ACS-SEED summer program. Special thanks go to Dr. T. C. Pham and Prof. T. Burkey of the University of Memphis for helping collecting the data on the JEOL 500 MHz NMR. The NMR material is based upon work supported by NSF under Grant No. CHE-1531466. We thank Prof. Delphia Harris for reading the manuscript.

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