Metalation of Glycylglycine: An Experimental Study Performed in

Article pubs.acs.org/jced

Metalation of Glycylglycine: An Experimental Study Performed in Tandem with Theoretical Calculations Shilpi Mandal, Richard H. Duncan Lyngdoh, Hassan Askari, and Gunajyoti Das* Department of Chemistry, North-Eastern Hill University, Shillong, 793022, India S Supporting Information *

ABSTRACT: Interactions of the Ni2+, Cu2+, and Zn2+ ions with the simplest dipeptide glycylglycine (GlyGly) are explored using various experimental and computational techniques. Solid and aqueous phase syntheses of the metalated GlyGly complexes (by solid-state grinding and by coprecipitation respectively) lead to the same products, as confirmed by physicochemical and spectral properties which indicate metal-coordination through the −NH2 and −CO2− groups of the dipeptide. Phase-diagram and kinetic studies of the solid-phase reaction between GlyGly and copper acetate suggest that complexation occurs in 1:2 (metal/ligand) stoichiometry via a facile kinetic pathway (a barrier of only 22.22 kJ/mol). The right-handed α-helical conformer of GlyGly is considered in DFT modeling studies in gas and aqueous phases elucidating the effects of metalation and solvation upon structural, electronic, and vibrational properties of the complexes. The complexes are found to follow the stability order Cu2+ > Ni2+ > Zn2+ corroborating the Irving-Williams series. The Ni(GlyGly)2 complex is predicted to exist in its low-spin state. Hydration effects on structural aspects of the complexes are also investigated computationally. The BHandHLYP/6-311++G(d,p) level describes the Cu(GlyGly)2 complex more efficiently than the B3LYP/6-311++G(d,p) level (which, however, better predicts the vibrational spectra of the systems). Absorption titration experiments with calf thymus DNA together with in silico docking and molecular mechanical studies reveal that these metal−dipeptide complexes are DNA minor-groove binders primarily through Hbonding interactions, yielding a DNA-binding affinity order of Ni2+ > Zn2+ > Cu2+.

1. INTRODUCTION Metal complexes of small amino acid sequences are extensively used as biomimetic model systems in theoretical and experimental studies which explore the basic nature of the interactions occurring at metal binding sites of metalloproteins and metalloenzymes;1,2 an approach that has hugely benefited the field of proteomics. Much effort has also been expended on developing novel metallopeptide-based DNA-binders, taking advantage of their remarkable efficiency to recognize the DNA double helix in a way similar to transcription factors and recognition enzymes.3 The recent extensive literature on the biological/pharmacological activity of peptides and their metal complexes makes it very evident that the study of peptide− metal ion interactions holds much promise biologically and synthetically.4−11 Nickel, copper, and zinc ions are now widely used in inorganic pharmaceuticals.12,13 The biological occurrence and roles played by Ni2+, Cu2+, and Zn2+ ions in the intraand extracellular context of various life forms have been well illustrated.14,15 It is also believed that, before life emerged, divalent transition metal ions like Cu2+ may have played crucial roles in assembling simple amino acids to form large peptides.16,17 Owing to its structural resemblance to polyglycine18 with its broad spectrum of clinical activities,19,20 the simplest peptide glycylglycine (GlyGly) has been the subject of in-depth theoretical and experimental study of the metal− peptide coordination chemistry.21−25 However, there is yet no © 2015 American Chemical Society

combined experimental and quantum chemical study on the interactions of GlyGly with Ni2+, Cu2+, and Zn2+ ions in gaseous, aqueous, and solid phases. This paper attempts to investigate (a) the solid-phase metal-coordinating mode of GlyGly, (b) the kinetics of metal−dipeptide interactions in solid state, (c) the influence of metal ion identity on the backbone structural features of GlyGly and physicochemical properties of the complexes, and (d) the DNA-binding motifs and affinities of the Ni2+, Cu2+, and Zn2+ complexes of GlyGly. In line with the need for resource management and reduction of adverse environmental impact, solid-state synthesis has emerged as a viable alternative to traditional solution phase methods.26−30 On the other hand, high level computational chemistry ranks as the third most heavily explored field of current scientific research. Solvation and hydration effects upon the structure and properties of biomolecules are well discussed in the literature.30 State-of-the-art quantum chemical techniques can elucidate the theoretical basis of experimental observations,31 being also used to develop reliable force field parameters for molecular mechanics simulations of biomacromolecules.32 Received: August 28, 2014 Accepted: February 11, 2015 Published: February 24, 2015 659

DOI: 10.1021/je500799g J. Chem. Eng. Data 2015, 60, 659−673

Journal of Chemical & Engineering Data

Article

Figure 1. Pictorial illustration of free and metal-bound structures of GlyGly.

converted to anhydrous form by drying at 110 °C. The GlyGly ligand and metal acetates, ground separately at room temperature for 15 min in an agate mortar and pestle, were then mixed in 1:2 metal/ligand ratio and ground again at room temperature for 70 min. The color of the reactants changed immediately with the release of acetic acid fumes. The reaction was accelerated by heating the mixtures in an air-thermostat at 60 °C for 12 h. To ensure complete reaction, the process of grinding, pulverization, and heating was repeated several times. The products were then recrystallized with ethanol and ether and finally dried under reduced pressure over anhydrous CaCl2 in a desiccator. Progress of the reactions and purity of the products were monitored by TLC using silica gel G (yield, 84 % to 89 %). Solvent-Phase Synthesis. A 50 mL aliquot of an aqueous solution of metal acetate (10 mmol; Millipore water; pH ≈ 7) was mixed with 50 mL of aqueous solution of GlyGly (20 mmol; Millipore water; pH ≈ 7) in 1:2 ratio (metal/ligand), and refluxed on a water bath for 6 h to 8 h. Colored products appeared upon standing and cooling. The precipitated complexes were filtered, washed with ether, recrystallized with ethanol several times, dried under reduced pressure over anhydrous CaCl2 in a desiccator, then further dried in an electric oven at 50 °C to 70 °C (yield: 68 % to 73 %). 2.2. Phase-Diagram and Kinetic Studies. Phase-diagram studies for the Cu(GlyGly)2 complex followed the thaw-melt method.33 Accurately measured amounts of copper(II) acetate and GlyGly were taken in glass tubes to make mixtures of varying compositions. The tubes were sealed and heated on a water bath at a marginally higher temperature than the melting points of the components. The melts were shaken well and then cooled immediately in ice-cold water. The process of heating and chilling was repeated over and again to obtain homogeneous mixtures. Finally the tubes were broken and the solidified mass was crushed into fine powder. The melting points of the mixtures were determined by means of a precision mercury thermometer (which could read up to 0.1 °C) fitted on an IkonTM Instrument and plotted as a function of composition generating the phase diagram. The kinetics of the solid-phase reaction between GlyGly and copper(II) acetate were studied using the capillary technique.34 A glass capillary

This study investigates the properties of GlyGly as a potential metal-binding entity in the solid state in the context of the solid-phase synthesis and characterization of its complexes with Ni2+, Cu2+, and Zn2+ ions. The coprecipitation method is used to study the mode of interaction of GlyGly with these metal ions in aqueous environment as well. The solid-state kinetics of these interactions and the DNA-binding capacity of the synthesized metal−dipeptide complexes are also studied. Figure 1 shows the chemical structures and atom numbering schemes assigned here to GlyGly and its metal complexes. The B3LYP/6-311++G(d,p) strategy in gas and aqueous phases is used to examine metal-coordination effects on the backbone structure of GlyGly and geometry about the amide linkage, also yielding values of reaction enthalpies (ΔH) and Gibbs energies (ΔG), dipole moments, partial atomic charges, and bond strength indices for the species studied. Vibrational spectra and UV−vis spectra, along with highest occupied molecular orbital (HOMO)−lowest unoccupied molecular orbital (LUMO) energy gaps, are also calculated and analyzed. The polarizable continuum model (PCM) is used to simulate solvation effects for all species. An explicit solvation model is also used for the Ni(GlyGly)2 complex, incorporating three discrete water molecules around the peptide bonds of the complex, this hydrated complex being denoted as wNi(GlyGly)2. It is expected that this study would augment understanding of the physicochemical and biochemical properties of metal−GlyGly complexes, thereby yielding clues to such phenomena in a reallife macromolecular context.

2. EXPERIMENTAL SECTION 2.1. Materials and Synthetic Procedures. Copper(II) acetate monohydrate (Laboratory Reagent; 100 w = 99), nickel(II) acetate tetrahydrate (Laboratory Reagent; 100 w = 98), zinc(II) acetate dihydrate (Himedia; 100 w = 98), glycylglycine (Himedia; 100 w = 99), calf thymus DNA (Himedia) and all other reagents used were of analytical reagent grade, obtained from commercial sources, and used as supplied. Solid-State Synthesis. The complexes of GlyGly with the Ni2+, Cu2+, and Zn2+ ions were prepared according to a literature method.29,30 The hydrated metal acetates were 660



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tube sealed at one end was half filled with finely ground GlyGly and tapped for 5 min with a glass rod to achieve uniform packing density. On smoothening the surface, the remaining half of the tube was filled with copper acetate. The tube was sealed with sealing wax and kept horizontally in a thermostat at constant temperature. At the junction of the reactants, the reaction started with a color change to light blue. The kinetics was followed by measuring the thickness of the product layer at time intervals with a traveling microscope. The experiment was performed at 353 K, 363 K, and 373 K. Each set of reactions was studied thrice, and average values were reported (Figure 5). 2.3. Analytical and Spectral Measurements. The complexes were analyzed for C, H, and N content in a PerkinElmer 2400 series II CHN-OS analyzer. Molar conductance values were measured in DMSO (10−3 M) solution using a Coronation digital conductivity meter (cell constant = 1.0 cm−1). Infrared spectra of all species were recorded in KBr discs on a BOMEM DA-8 FTIR spectrophotometer at 4000 cm−1 to 400 cm−1. Electronic absorption spectra of GlyGly and its metal complexes were recorded in DMSO solution (10−5 M) on a PerkinElmer lambda 25 UV−vis spectrometer. The dehydration level of DMSO was 99 %. Fluorescence spectra of the products synthesized in solid and in solvent phases were measured using a PerkinElmer LS 55 fluorescence spectrometer. Thermogravimetry (TG) and differential thermal analysis (DTA) were carried out for the solid-phase reaction products in an inert N2 atmosphere on a PerkinElmer STA 6000 simultaneous thermal analyzer from 40 °C to 800 °C at a heating rate of 20 °C/min. EDAX-SEM micrographs for the Cu2+ complex of GlyGly were obtained in a Jeol JSM-6360 LV apparatus with an accelerating voltage between 15 kV and 20 kV at different magnifications. Transmission electron microscopy (TEM) and selected-area electron diffraction (SAED) images were obtained on a JEOLJEM-2100CX electron microscope operated at 200 kV without the addition of any contrast agent. Melting points of all species were determined on an IkonTM instrument. 2.4. Determination of DNA Binding Affinity. To evaluate the relative DNA recognition ability of the metallic complexes of GlyGly, absorption titration experiments were performed by adding homogeneous calf thymus DNA (CTDNA) solutions of varying concentrations (0 μM to 20 μM) dissolved in a tris-HCl buffer (pH 7) to the aqueous solutions of the metal−dipeptide complexes (10 μM dissolved in Millipore water). The concentration of CT-DNA was determined by UV absorbance at 260 nm. The solutions of CT-DNA gave a ratio of UV absorption at 260 nm and 280 nm A260/A280 of ∼1.8 to 1.9 (Figure S1 of the Supporting Information), indicating the DNA was sufficiently free of protein.35 Intrinsic binding constant (Kb) values for interaction of the complexes with CT-DNA were calculated from the spectroscopic titration data using36

were made at room temperature using the aforesaid UV−vis spectrometer.

3. COMPUTATIONAL METHODS Molecular geometries of the three conformers of GlyGly, the complexes with the Ni2+, Cu2+, and Zn2+ ions, and all species involved in eqs 2 and 3 were optimized in the gas phase and simulated aqueous phase using a polarizable continuum model (PCM)37 at the B3LYP/6-311++G(d,p) level38−41 by the Gaussian 09 (revision C.01) program.42 The adequacy of the 6311++G(d,p) basis set to treat Ni2+, Cu2+, and Zn2+ complexes with a host of biologically important chemical species has been well exemplified.43,44 Diffuse functions treat the presence of electron lone pairs,30 while polarization functions can treat stereoelectronic effects.30 The nonionic structure of GlyGly was considered in the gas phase, and the zwitterionic form was considered in the aqueous phase. Cu(GlyGly)2 was treated as an open-shell with a spin-unrestricted formalism, while Zn(GlyGly)2 was treated as a closed-shell. The BHandHLYP functional was used for the Cu(GlyGly)2 complex in the aqueous phase, since the higher percentage of exact exchange enables it to describe an open-shell system better than the commonly used B3LYP functional.45 Since the Ni2+ ion may exist in its complexes in both high- and low-spin states (triplet and singlet, respectively), both spin states were considered for Ni(GlyGly)2 in the aqueous phase. The explicitly hydrated complex, written as wNi(GlyGly)2, was treated at the B3LYP/6311++G(d,p) level. Frequency analyses confirmed all optimized geometries as true minima by the absence of negative Hessian eigenvalues. Zero-point energy (ZPE) corrections to the total energies of all species used a scaling factor of 0.9877.30 Vibrational frequencies obtained from the simulated IR spectra of GlyGly and its complexes with Ni2+, Cu2+, and Zn2+ were also scaled using appropriate correction factors,30 namely, 0.9679 for ν(C−H) and ν(N−H) stretching modes, and 1.01 for vibrational modes below 1800 cm−1. The gas and aqueous phase interaction enthalpies ΔH and Gibbs energies ΔG (energy changes for metal complex formation) were obtained as per the following equations: 2[+HNCH 2CONHCH 2CO−2 ] + M(CH3CO2 )2 solvent

⎯⎯⎯⎯⎯⎯→ M[H 2NCH 2CONHCH 2CO2 ]2 + 2CH3CO2 H phase

(2)

2[H 2NCH 2CONHCH 2CO2 H] + M(CH3CO2 )2 gaseous

⎯⎯⎯⎯⎯⎯→ M[H 2NCH 2CONHCH 2CONHCH 2CO2 ]2 phase

+ 2CH3CO2 H

(3)

Thus, ΔH (or ΔG) = [Etproducts] − [Etreactants], where Etproducts and Etreactants are the sum of the ZPE-corrected total electronic energies (or Gibbs energies) of the products and reactants, respectively. The theoretical electronic absorption λmax values for all representative reaction species were calculated in the gas and aqueous phase by performing single-point calculations employing time-dependent density functional theory (TDDFT)30,46 at the B3LYP/6-311++G(d,p) level. In silico rigid molecular docking analyses were carried out by the HEX 8.0.0 software47 using the B3LYP/6-311++G(d,p) aqueous phase optimized geometries of the metal−GlyGly complexes along with the d(CGCGAATTCGCG)2 B-DNA

[DNA]/(εa − εf ) = [DNA]/(εb − εf ) + 1/Kb(εb − εf ) (1)

The “apparent” extinction coefficient (εa) was obtained by calculating Aobsd/[DNA], where εf and εb correspond to extinction coefficients of the unbound and fully bound complexes, respectively. The Kb value for a particular metal− GlyGly complex was obtained from the ratio of the slope to the intercept, 1/(εa − εf) and 1/Kb(εb − εf), respectively, given by the plot of [DNA]/(εa − εf) versus [DNA]. All measurements 661



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Table 1. Physical, Conductivity, and Analytical Data of the Ni2+, Cu2+, and Zn2+ Complexes of GlyGly at Temperature (T) = 298.15 K and Pressure (p) = 0.1 MPaa F.W.

TF

ΛM

g/mol

(°C)

Ω−1 cm2 mol−1 (in DMSO)

132.118 320.913

262 to 264 237 to 245

4.9 5.8 (6.2)

blue

325.766

215 to 232

3.4 (3.5)

white

327.610

240 to 245

6.2 (6.2)

compound

empirical formula

color

GlyGly Ni(GlyGly)2

C4H8N2O3 C8H14N4O6Ni

white light green

Cu(GlyGly)2

C8H14N4O6Cu

Zn(GlyGly)2

C8H14N4O6Zn

found (calcd) (%) C

H

N

29.11 (29.94)b [29.73]c 29.56 (29.50) [29.51] 29.10 (29.33) [29.12]

4.52 (4.40) [4.32] 4.56 (4.33) [4.24] 4.29 (4.31) [3.99]

17.12 (17.46) [17.12] 17.19 (17.20) [17.12] 16.94 (17.12) [16.89]

F.W.= formula weight; TF = Melting point; ΛM = molar conductivity. aStandard uncertainties u are u(T) = 0.2 K, u(TF) = 1.0 °C, u(p) = 0.5 kPa, u(ΛM) = 0.05 Ω−1 cm2 mol−1. bFor reaction products obtained from coprecipitation technique. cCalculated values.

Figure 2. EDAX spectrum of Cu(GlyGly)2 synthesized via mechanochemistry.

Figure 3. TEM and SAED images of Cu(GlyGly)2 prepared in the solid phase.

sequence generated by AVOGADRO 1.1.1.48 The high ranking docked poses were visualized by the CHIMERA molecular graphics program (http://www.cgl.ucsf.edu/chimera/).

coprecipitation method had the same colors as the corresponding solid state products. Table 1 assembles the physical, molar conductivity, and analytical data of GlyGly and its metal complexes obtained by the grinding and coprecipitation methods. The elemental analyses for the complexes formed by the solid phase method are virtually identical to those for the complexes formed by the coprecipitation method and correspond to the empirical formulas and stoichiometries proposed. The conductivity values of the complexes in DMSO

4. RESULTS AND DISCUSSION Color changes after mixing the anhydrous metal acetates with GlyGly in 1:2 (metal:ligand) molar ratios were as follows− light-green for (GlyGly + Ni2+), blue for (GlyGly + Cu2+), and white for (GlyGly + Zn2+). The products formed by the 662



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indicate their neutral and nonelectrolytic nature.30,49 The energy-dispersive X-ray analysis (EDAX), the scanning electron microscopy (SEM), transmission electron microscopy (TEM), and selected-area electron diffraction (SAED) images of the Cu(GlyGly)2 complex prepared in the solid phase are shown in Figures 2 and 3, providing useful insights regarding chemical composition, surface morphology and particle size of this complex. The EDAX image of the gold coated sample confirms the presence of copper and purity of the complex, while the SEM photograph displays its agglomerated and homogeneous matrices. The TEM and SAED micrographs provide visual information regarding the crystalline nature of the sample. 4.1. Phase-Diagram and Reaction Kinetics. The phase diagram studies show the formation of the 2:1 (ligand/metal) complex from GlyGly and copper(II) acetate (Figure 4; Table

Figure 5. Plot of log x vs log t at a varying temperature range.

Figure 4. Solid−liquid equilibrium data of GlyGly−copper(II) acetate system. (A) GlyGly; (B) copper(II) acetate; (L) liquid.

S1 of the SI). The composite diagram has two simple eutectics joined at the position of the arrows in the figureone of the A−AB−liquid, and the other of the AB−B−liquid. The appearance of a flat maximum at ∼213 °C suggests that the Cu(GlyGly)2 complex is stable in the solid state, but dissociates in solution or the molten state. When the powdered solid copper acetate and GlyGly were kept in contact in a glass capillary, a light-blue product was formed at the copper acetate side, pointing to GlyGly as the diffusing species. The kinetics of the reaction was studied by measuring the thickness of the product layer as a function of time, obeying eq 4 below: x = kt n

Figure 6. Temperature dependence of rate constant for the GlyGly− copper(II) acetate system.

reaction as calculated from the Arrhenius equation is quite modest (22.22 kJ/mol). 4.2. Designing Metal−GlyGly Complexes. Earlier theoretical studies on the conformational propensities about the Ramachandran dihedrals50−52 ψ and Φ (Figure 1) for alanine dipeptide (AD) have revealed that the conformers C7eq (ψ = 90.1° and Φ = −86.3°) and C5 (ψ = 143.8° and Φ = −156.4°) are the predominant species in gas and nonpolar solvent phases.53 However, in a polar solvent the conformers C5, αR (ψ = −32.1° and Φ = −70.5°) and β (ψ = 142.1° and Φ = −64.0°) emerge as the dominant species with a relative stability order of C5 > αR > β; where the AD conformers differ in thermodynamic stability within a narrow range. The aqueous phase optimized geometries of the C5, αR, and β type conformers of GlyGly are shown in Figure 7. Values of their ZPE, total electronic energy (E), Gibbs free energies (G), and ZPE corrected energies Ecorr and Gcorr are listed in Supporting Information, Table S3. As for AD, these data suggest a stability order of C5 > αR > β for the three GlyGly conformers (energies of the αR-GlyGly and β-GlyGly conformers relative to C5GlyGly are 1.61 and 2.63 kcal/mol, respectively). Since there is no experimental evidence for C5 as the predominant conformer

(4)

where x is the thickness of the product layer at any time t, k is the apparent rate constant, and n is another constant. The plot of log x against log t gave straight lines (Figure 5), indicating the validity of eq 4. From the intercepts and the slopes of these plots, the values of k and n were calculated, as presented in Supporting Information, Table S2. The plot of log k versus 1/T gave a straight line (Figure 6), showing that k follows the Arrhenius equation (k = Ae−Ea/(RT)). During the solid-phase interaction between copper acetate and GlyGly, the GlyGly molecules may diffuse toward copper acetate via vapor phase, grain boundary defects, or bulk and surface migration. The activation energy for the complexation 663



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in both phases. The predicted coordination motifs of the three metal ions agree well with previous studies.54,55 It is known that the Zn2+ ion, owing to its fully filled d10 shell, does not lose ligand field stabilization energy (LFSE) in preferring a tetrahedral binding mode when interacting with proteins under physiological conditions.55 Gas and aqueous phase values of the interaction enthalpies (ΔH) and Gibbs energies (ΔG), dipole moments and HOMO−LUMO energy gaps for GlyGly and its metal complexes are presented in Table 2. The negative values of the interaction enthalpies (−22.10 kcal/mol to −45.46 kcal/mol in aqueous phase; −38.55 kcal/mol to −64.92 kcal/mol in gas phase) and Gibbs energies (−19.44 kcal/mol to −42.26 kcal/mol in aqueous phase; −31.38 kcal/ mol to −57.01 kcal/mol in gas phase) for all the complexes except triplet tNi(GlyGly)2 indicate that they are thermodynamically stable. The B3LYP aqueous phase metal binding affinity order of GlyGly emerges as Ni2+ > Cu2+ > Zn2+, which does not quite follow the Irving−Williams order of Cu2+ > Ni2+ > Zn2+. However, the BHandHLYP functional, which better describes spin-delocalization in open-shell systems, predicts an aqueous phase interaction enthalpy of −59.99 kcal/mol (ΔG = −60.17 kcal/mol) for Cu(GlyGly)2, thus yielding a binding affinity order of Cu2+ > Ni2+ > Zn2+ in precise agreement with the Irving−Williams series. This agrees with the fact that the Cu2+ ion is the strongest Lewis acid among the three metal ions considered here. The markedly higher stability predicted for the Ni(GlyGly)2 and Zn(GlyGly)2 complexes in the gas phase compared to that in aqueous phase can be attributed to the number and strength of the intramolecular H-bonds (Figure 8) present. The strength of an H-bond was assessed by following the geometrical criteria prescribed in the literature.56 The B3LYP stabilities of high- and low-spin states of square planar Ni(GlyGly)2 (Table 2) predict a singlet ground state. Thus, aqueous phase ΔH and ΔG values of triplet tNi(GlyGly)2 are 7.69 kcal/mol and 10.34 kcal/mol, respectively. Hence, the

Figure 7. Three most predominant conformers of GlyGly in the aqueous phase.

of a given dipeptide in the aqueous phase, we have used the biologically important right-handed α-helical conformer αRGlyGly to design the GlyGly complexes with the Ni2+, Cu2+, and Zn2+ ions. The conformer αR-GlyGly is simply referred to as GlyGly in the text, tables, and figures, with the metal complexes designated as Ni(GlyGly)2, Cu(GlyGly)2, and Zn(GlyGly)2 (Figures 7 and 8). Gas and aqueous phase values of the ZPE, total electronic energies (E), Gibbs free energies (G), and ZPE corrected energies Ecorr and Gcorr of the three metal complexes are presented in Supporting Information, Table S3. 4.3. Molecular and Structural Aspects. The gas and aqueous phase optimized geometries of the metal complexes (Figure 8) suggest that the Ni2+ and Cu2+ complexes of GlyGly are square-planar, while the Zn2+ complex is nearly tetrahedral

Figure 8. B3LYP/6-311++G(d,p) level optimized geometries of the metal−GlyGly complexes in gaseous and aqueous phases. 664



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Table 2. Calculated Interaction Enthalpies ΔH (kcal/mol) and Gibbs Energies ΔG (kcal/mol); Dipole Moments (Debye) and HOMO−LUMO Energy Gaps (eV) for All the Reaction Species in Gas and Aqueous Phase (Gas Phase Values Are Given in Parentheses) systems GlyGly Ni(GlyGly)2 t Ni(GlyGly)2 Cu(GlyGly)2 Cu(GlyGly)2a Zn(GlyGly)2 at

ΔH −45.46 7.69 −39.02 −59.99 −22.10

(−64.92) (−38.55) (−73.14) (−52.44)

ΔG −42.26 10.34 −32.39 −60.17 −19.44

dipole moments

HOMO−LUMO energy gaps

(−57.01)

15.721 (6.799) 9.192 (5.612)

5.764 (6.257) 4.257 (4.261)

(−31.38) (−69.23) (−43.89)

7.156 (4.228) 6.698 (3.505) 10.549 (1.263)

6.245 (5.974) 6.346 (6.008) 6.232 (5.997)

Triplet; BHandHLYP.

Figure 9. Depiction of electrostatic potentials mapped on the electron density surface for free and metal-bound GlyGly.

triplet tNi(GlyGly)2 complex is not discussed further. All three complexes exhibit smaller dipole moments (6.698 D to 10.549 D in the aqueous phase and 1.263 D to 5.612 D in the gas phase) than the free GlyGly molecule (15.721 D in the aqueous phase and 6.799 D in the gas phase). This is due to loss of the dipolar zwitterionic form of GlyGly molecule after metalation. Table 2 presents gas and aqueous values of energy gaps between the HOMO and the LUMO of GlyGly and its metal complexes in the aqueous phase, while Supporting Information Figure S2 depicts the 3D plots of these frontier orbitals. Fukui pointed out the important role played by the HOMO and LUMO in governing chemical reactions.57 The HOMO− LUMO energy gaps for GlyGly and its metal complexes are 4.257 eV to 6.346 eV in the aqueous phase and 4.261 eV to 6.257 eV in the gas phase. For the metal complexes, the HOMO−LUMO energy gaps increase upon solvation by a solvent (water) with its high dielectric constant. The decrease in this energy gap for free GlyGly upon hydration is due to the transformation from the nonionic form in the vacuum phase to its zwitterionic form in the aqueous phase.

The molecular electrostatic potential (MEP) maps for GlyGly and its metal complexes computed at the B3LYP/6311++G(d,p) level are depicted in Figure 9, while partial atomic charges derived from natural bond orbital (NBO) analysis are given in Supporting Information, Table S4, along with the partial charges at the BHandHLYP/6-311++G(d,p) level for the Cu(GlyGly)2 complex. Figure 9 highlights positively and negatively charged areas (blue and red, respectively) and possible sites for inter- and intramolecular H-bond interactions. The partial charges also reveal effects of the electronic configurations of the metal ions upon the charge distribution of the GlyGly moiety. For example, the Zn2+ ion with no vacant d orbital (d10 shell) cannot reduce charge density on the N6 and O7 atoms as efficiently as the other two metal ions do, being the weakest Lewis acid among the three studied here. 4.4. Experimental and Theoretical Vibrational Assignments. The experimental FTIR spectra of GlyGly and its metal complexes obtained by solid and solvent phase methods are shown in the Supporting Information, Figure S3, with some significant FTIR assignments listed in Table 3. A comparison 665


theor

exp

theor

exp

A C D E F A B C D A B C D G H A B C D

3332 3412 3467 3418 3421 3344 3336 3409 3409 3345 3342 3412 3419 3459 3538 3343 3342 3431 3384

(93) (61)

(57) (23) (204) (40)

(36) (25)

(113) (13) (146) (145)

νas(N−H) 3286 3357 3390 3369 3371 3287 3287 3324 3217 3287 3288 3295 3199 3352 3369 3288 3293 3330 3322 (266) (176)

(5) (454) (43) (449)

(165) (354)

(94) (67) (146) (182)

νs(N−H) 3418 3429 3490 3494 3376 3408 3409 3481 3474 3425 3419 3489 3485 3369 3620 3406 3411 3496 3493 (73) (34)

(74) (20) (325) (29)

(71) (15)

(58) (24) (89) (262)

ν(N3−H) 2926 3037 3027 3030 2985 2939 2937 3040 3045 2931 2922 3038 3043 3105 3071 2926 2931 3032 3049 (2) (2)

(6) (1) (3) (27)

(5) (0.6)

(4) (1) (6) (21)

ν(C2−H) 1671 1713 1789 1720 1713 1674 1673 1732 1807 1673 1672 1722 1779 1795 1781 1673 1672 1705 1748 (431) (222)

(611) (228) (465) (299)

(669) (263)

(549) (277) (474) (496)

ν(C4O)

1626 1634 1627 1628 1671 1719 1654 1654 1654 1700 1736 1732 1651 1642 1661 1721 (691) (611)

(31) (580) (1680) (831)

(891) (602)

(1164) (943)

1602 1621 (829)

νas(COO−) 1405 1361 1338 1374 1383 1389 1391 1338 1333 1390 1391 1347 1362 1441 1433 1394 1400 1373 1346 (55) (6)

(108) (72) (938) (460)

(266) (72)

(392) (258) (392) (440)

νs(COO−)

(36) (83)

(115) (120) (122) (4)

(143) (109)

(62) (34) (161) (17)

ν(C4−N3) 1259 1289 1289 1251 1244 1256 1255 1209 1226 1254 1253 1201 1199 1238 1252 1251 1253 1196 1183

1145 1078 (8) 1037 (9) 1005 (17) 1029 (9) 1134 1134 997 (7) 994 (3) 1134 1135 1000 (58) 999 (39) 1062 (9) 1063 (3) 1132 1135 1006 (19) 1027 (7)

ν(C5−N6)

(656) (15)

(919) (556) (36) (14)

(266) (609)

(66) (103) (161) (19)

ν(C−O) 1258 1323 1322 1301 1335 1283 1283 1338 1311 1310 1310 1328 1325 1361 1349 1281 1307 1352 1338 531s 531s 520 (77) 533 (29) 531s 531s 507 (101) 513 (45) 525 (13) 539 (53) 531s 531s 518 (62) 516 (51)

ν(M−O)

462w 464w 463 (41) 467 (16) 473w 471w 428 (10) 412 (44) 435 (1) 432 (47) 473w 468w 410 (25) 406 (1)

ν(M−N)

Δδ

252 251 238 237 333 386 264 263 307 338 295 299 257 242 288 375

197 260

a A = solid state; B = coprecipitation; C = solvent phase; D = gas phase; E = solvent phase; F = solvent phase; G and H = calculated at BHandHLYP/6-311++G(d,p) for Cu(GlyGly)2 in solvent and gas phases, respectively; w = weak; s = strong. Frequencies are expressed in cm−1 while the intensities are given in km/mol. The theoretical frequencies below 1800 cm−1 are scaled with 1.01 and for those above 1800 cm−1 a correction factor of 0.9679 is used.

Zn(GlyGly)2

Cu(GlyGly)2

theor theor exp

β-GlyGly C5-GlyGly Ni(GlyGly)2

theor

exp theor

GlyGly

cmpd

Table 3. Experimental (exp) and Theoretical (theor) IR Spectral Data, Calculated at B3LYP/6-311++G(d,p), of GlyGly and Its Metal Complexes Prepared in Solid State (Intensities Are Given in Parentheses)a

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Table 4. Experimental and Computed Absorption Maxima (λmax) for All the Reaction Species λmax (experimental) in aqueous phase (nm) λmax (calculated) TD-B3LYP (nm) systems GlyGly Ni(GlyGly)2 Cu(GlyGly)2 Zn(GlyGly)2

solid state 220, 218, 211, 217,

327 339, 379, 554 375, 566 335, 384

gas phase

aqueous phase

coprecipitation

wavelength

oscillator strength

wavelength

oscillator strength

255, 375, 552 254, 369, 560 252, 357

236.81 638.87 700.6 243.52

0.0012 0.0002 0.0012 0.0030

242.45 673.47 669.63 229.39

0.054 0.0002 0.0013 0.0003

and amino groups of GlyGly, not the central amide moiety. These binding modes agree with previous studies on copper(II)−tryptophan−tryptophan complex.62 The gas and aqueous phase theoretically simulated vibrational spectra of GlyGly and its metal complexes were calculated at the B3LYP/6-311++G(d,p) and BHandHLYP/6311++G(d,p) levels. Computed harmonic vibrational frequencies are usually larger than their corresponding experimental values63 due to neglect of anharmonicity effects in theoretical treatments, incomplete incorporation of electron correlation and the use of finite basis sets. Even then, the predicted vibrational modes of GlyGly and its complexes provide valuable information on metal binding and solvation effects. It is known that the B3LYP level predicts vibrational frequencies better than the MP2 level.64 The GlyGly molecule, in both zwitterionic and nonionic forms, has 45 normal modes of vibration while each metal−GlyGly complex possesses 93 modes. The theoretical IR spectra of each species (with a scaling factor of 0.955) are reported in Supporting Information, Figure S4 while some important frequencies along with their intensity values (in parentheses) are collected in Table 3. Six out of ten of the predicted harmonic modes are closer to experimental FTIR assignments for the right-handed α-helical GlyGly conformer than for the C5-GlyGly or β-GlyGly conformers. The theoretically predicted frequency shifts of the νas(N−H), νs(N−H), νas(COO−), and νs(COO−) modes of GlyGly as a result of metal coordination are in good agreement with experimentally observed values. Calculated frequencies of the ν(M-O) and ν(M-N) modes also agree well with the experiment. On the other hand, the aqueous phase frequency values of the polar exposed bonds of GlyGly and its complexes, namely N3−H3 and C4O4, are consistently lower than the corresponding gas phase values by up to 75 cm−1 due to elongation of these bonds in the aqueous phase (see later). Similarly, an increase in the aqueous phase frequency values of ν(C4−N3) modes can be attributed to shortening of the C4−N3 bonds in the aqueous environment. It is also apparent that the commonly used B3LYP functional performs far better than the BHandHLYP level in reproducing the experimental vibrational frequencies of the Cu(GlyGly)2 complex. 4.5. Electronic Absorption and Fluorescence Properties. Optical properties of free GlyGly and its metal complexes, prepared in both solid and aqueous phases, were studied using electronic absorption and fluorescence spectroscopy. Table 4 gathers the observed and calculated λmax values for each species while their theoretical absorption spectra are shown in Supporting Information, Figure S5. The experimental UV−vis spectra of the reacting species show intense absorption bands in the region 211 nm to 339 nm which are assigned to n → π* and π → π* transitions. Appearing only in the metal−GlyGly complexes, the relatively less intense peaks at 357 nm to 384 nm can be attributed to ligand-to-metal charge transfer (LMCT) transitions affirming the formation of the metal−

here between the free zwitterionic GlyGly molecule and the metal complexes confirms that no free ligand remains after the grinding or coprecipitation procedures. Appearance of the ν(N3−H), ν(C4O), and ν(C4−N3) vibrational modes of the amide moieties of GlyGly and its metal complexes in the ranges of 3406 cm−1 to 3425 cm−1, 1671 cm−1 to 1674 cm−1, and 1251 cm−1 to 1259 cm−1, respectively, suggests that GlyGly does not interact with the metal ions via its CONH group.58 On the other hand, the asymmetric and symmetric ν(N−H) stretching frequencies of GlyGly (observed at 3332 cm−1 and 3286 cm−1, respectively) are shifted to higher wave numbers (3336 cm−1 to 3345 cm−1, and 3287 cm−1 to 3293 cm−1, respectively) in the metal complexes. This implies that the NH3+ group of free GlyGly gets deprotonated and binds to the metal ions through its nitrogen atom.30,59 Metal coordination via the amino N6 atom of GlyGly is also indicated by the red shift of the ν(C5− N6) stretching mode of GlyGly (1145 cm−1) to 1132 cm−1 to 1135 cm−1 in the complexes The appearance of new strong bands in the range of 462 cm−1 to 473 cm−1 in all three metal complexes30,59 (assigned to asymmetric ν(M-N) stretching frequencies60) also confirms involvement of the N6 atom of GlyGly in metal coordination. Note that the two coordinating −NH2 groups are positioned trans to each other in the complexes since the symmetric ν(M-N) stretching mode of the N-M-N group (M = Ni2+, Cu2+, and Zn2+) which usually appears around 400 cm−1 to 450 cm−1 is IR inactive.60 Regarding the carboxylate groups of the ligand and its metal complexes, the νas(COO−) mode of GlyGly at 1602 cm−1 increases to higher wave numbers (up to 1654 cm−1) while the νs(COO−) stretch at 1405 cm−1 shifts to the lower frequencies in the complexes (1389 cm−1 to 1400 cm−1). These indicate that GlyGly binds to the metal ions via its carboxylate group.30,59 Table 3 also lists the differences (Δδ values) between the νas(COO−) and νs(COO−) stretches for GlyGly and its metal complexes which are indicative of the M-O bond strengths and provide evidence that the carboxylate groups bind to the metal ions in monodentate fashion.30,59 The Δδ values of the metal complexes (237 cm−1 to 264 cm−1) indicate that the M-O bonds possess significant covalent character.30,59 The new bands at 531 cm−1 in all three metal complexes, assignable to ν(M-O) stretching modes,30,59 also confirm that GlyGly interacts with metal ions via its carboxylate group. The peaks at 2922 cm−1 to 2939 cm−1 can be assigned to ν(C2−H) stretching modes. Thus, the experimental IR spectra of this study furnish noticeable signatures indicating that GlyGly binds to the metal ions through its amino and carboxylate groups in both the solid and aqueous phases. It is worth mentioning that Angkawijaya et al.61 have shown that in alkaline media, it is the deprotonated amide moiety which bind to metal ions, as seen in a set of glycine-containing metal−peptide complexes. In our study the metal−GlyGly complexes were synthesized at neutral pH with the amide nitrogen remaining undeprotonated, resulting in metal-coordination via the terminal carboxylate 667



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reasonably conclude that the reaction products formed by following both the methods (solid state grinding or coprecipitation) are the same and that it is the predominant right-handed α-helical conformer of GlyGly that forms the metal complexes involving its −NH2 and −CO2− groups. 4.6. Mass Spectral Analysis and Thermal Behavior. Mass spectra of the solid state products recorded at room temperature (Supporting Information, Figure S6) were used to compare the stoichiometric compositions of the metal complexes of GlyGly. The molecular ion peaks for the Ni2+, Cu2+, and Zn2+ complexes of GlyGly, observed at 320, 325, and 327 m/z respectively, confirm the [ML2] type stoichiometry. The mass spectra of the three complexes exhibit similar disintegration patterns; the peaks at 303, 309, and 311 m/z for the Ni2+, Cu2+, and Zn2+ complexes, respectively, may be due to the loss of amino groups. Thermal stability of the complexes synthesized in solid-state was studied using thermogravimetry (TG) and differential thermal analysis (DTA). Table 6 gives values of the maximum decomposition temperature along with corresponding weight loss, while the TG/DTA curves for all the complexes are reported in Supporting Information, Figure S7. The TGA curves indicate that the metal complexes are thermally stable up to about 202 °C to 227 °C above which, that is, between 202 °C and 700 °C, the actual mass loss steps begin and most of the organic parts of the metal complexes are lost. This sharp decomposition period brings about a 74 % to 77 % weight loss for the complexes; and leads to complete formation of the respective metal oxides. The DTA curves of the metal complexes also show similar behavior that corresponds to the TGA curves. The small endothermic peaks in the range 231 °C to 245 °C can be attributed to melting of the complexes. 4.7. Effects of Metalation and Explicit Solvation. Quantum chemical calculations can gauge effects of solvation and metal coordination on the backbone structural features of dipeptides, which are crucial for supporting or refuting existing theories predicting protein structure. Values of some significant dihedral angles of GlyGly and its complexes calculated at the B3LYP/6-311++G(d,p) level in gas and aqueous phases are presented in Table 7. The values of those dihedrals involving ligand atoms bound to the metal ion change appreciably from the free GlyGly ligand to the metal complex, predicting that coordination to the metal ion in each case would exert profound effects upon the conformational shape of the ligand upon complexation. It is noteworthy that, in general, such deviations in dihedral values are maximum for Zn(GlyGly)2, while the deviations for Ni(GlyGly)2, Cu(GlyGly)2, and wNi(GlyGly)2 are less appreciable. This is because the Zn2+ complex is tetrahedral in geometry around the metal ion,

dipeptide complexes. The weaker and less well-defined broad bands in the absorption spectra of Ni(GlyGly)2 (∼552 nm to 554 nm) and Cu(GlyGly)2 (∼560 nm to 566 nm) may be ascribed to 1A1g → 1B1g and 2B1g → 2A1g transitions, indicating square planar-type geometries for the Ni 2+ and Cu 2+ complexes.65 Such d−d transitions are not exhibited by the Zn(GlyGly)2 complex due to the completely filled d-orbital of the Zn2+ ion.30 The efficacy of the TD-DFT method in predicting theoretical UV−vis spectra is well exemplified.29,30 The gas and aqueous phase theoretical electronic absorption spectra of GlyGly and its metal complexes calculated using TDDFT furnish characteristic λmax values around 236 nm to 700 nm in the gas phase and 229 nm to 669 nm in the aqueous phase. Occurrence of d−d transitions for the Cu2+ and Ni2+ complexes but not for the Zn2+ complex is correctly predicted by our theoretical calculations. The photoluminescence properties of GlyGly and its metal complexes were recorded at room temperature in DMSO, and the observed spectra are shown in Figure 10. In general,

Figure 10. Emission spectra of GlyGly and its complexes (Cu*, Ni*, and Zn*) in Millipore water; a plot of fluorescence intensity (Y-axis) versus wavelength (X-axis).

transition metal ions are effective fluorescent quenchers66 and the often reported fluorescence quenching of a ligand by transition metal ions during complexation can be attributed to processes like magnetic perturbation, redox activity, and electronic energy transfer. As listed in Table 5, the emission spectrum of the GlyGly molecule excited at 356 nm shows a peak at 402 nm while those of the complexes, excited at 357 nm to 384 nm, are observed in the range of 415 nm to 439 nm. These results agree well with previous experimental studies30,67 which establish that the extent of the fluorescence quenching increases with decreasing number of d-orbital electrons in the coordinating transition metal ions. Thus, by considering the experimental and theoretical results of Tables 1 to 5, we may

Table 5. Fluorescence Parameters of GlyGly and Its Complexes in Aqueous Phase (Intensities Are in Parentheses)a excitation wavelength λmax systems

methods

nm

GlyGly Ni(GlyGly)2

solid state solid state coprecipitation solid state coprecipitation solid state coprecipitation

356 379 375 375 369 384 357

Cu(GlyGly)2 Zn(GlyGly)2

a

εmax −1

L mol

emission wavelength λmax cm

52 572 54717 3599 49 001 81 018 57 916 15 832

−1

nm 402 438 429 439 422 437 415

(228) (107) (102) (115) (132) (137) (149)

quantum yield

Stokes’ shift

φf

nm

0.0401 0.0205 0.0221 0.0218 0.0301 0.0313 0.0345

46 52 54 54 53 52 58

εmax = extinction coefficient; Quantum yield determined by using quinine sulfate as a standard (φfs = 0.546). 668



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Table 6. Maximum Temperaturea Values for Decomposition along with Corresponding Weight Loss Values for Solid State Reaction Products

a

cmpd

decomposition range (°C)

peak temperature (°C)

percentage weight loss (%)

product expected

residue state/ color

mass changes calcd (found)

Ni(GlyGly)2 Cu(GlyGly)2 Zn(GlyGly)2

227 to 700 202 to 656 215 to 689

243 231 245

77 74 75

NiO CuO ZnO

black powder black powder white powder

2.89 (2.76) 2.58 (2.66) 2.95 (2.93)

Standard uncertainties u are u(T) = 2.0 °C.

Table 7. Calculated Dihedral Angles (in deg) for Free GlyGly and Its Metal Complexes Using the B3LYP Level of Theory in the Gas and Aqueous Phase (Gas Phase Values Are Given in Parentheses)a Dihedrals C2−N3−C4−O4 C2−N3−C4−C5 H3−N3−C4−O4 H3−N3−C4−C5 N3−C4−C5−N6 C1−C2−N3−C4 O7−C1−C2−N3 O8−C1−C2−N3 O4−C4−C5−N6 C4−C5−N6−Ha C4−C5−C6−Hb

GlyGly −33.6 138.7 −178.1 −5.7 −82.1 −73.8 4.8 −175.3 90.6 150.6 −89.8

(−24.7) (148.1) (−178.4) (−5.6) (−73.1) (−89.4) (9.9) (−170.1) (99.7) (158.4) (−82.7)

Ni(GlyGly)2 −34.1 141.3 −173.2 2.2 −77.4 −57.7 −21.4 160.1 98.1 92.4 −151.3

Cu(GlyGly)2 −28.0 145.8 −171.4 2.3 −86.5 −56.0 −24.0 156.5 87.3 100.7 −142.6

(−35.5) (140.7) (−168.4) (7.7) (−82.8) (−57.3) (−20.9) (159.0) (93.5) (99.4) (−142.8)

(−28.7) (145.3) (−166.4) (7.7) (−90.6) (−54.1) (−25.2) (153.8) (83.6) (103.2) (−138.4)

Zn(GlyGly)2 −13.7 159.3 −178.0 −5.0 −86.6 −89.8 12.1 −167.8 86.7 133.0 −110.6

(−26.4) (146.4) (−165.5) (7.3) (−117.2) (−56.4) (−30.0) (148.2) (55.9) (160.2) (−81.9)

w

Ni(GlyGly)2 −33.8 141.8 179.5 −4.9 −56.6 −86.4 8.1 −168.6 119.1 81.1 −162.2

aw

Ni(GlyGly)2 = water complex.

Figure 11. Interactions of Ni(GlyGly)2 with three explicitly considered water molecules.

while the Ni2+ and Cu2+ complexes are square planar. Values of the dihedrals C2−N3-C4−O4 and C2−N3-C4−N5 which describe the peptide bond of the ligand are also given, and show that the central amide moiety is not completely planar in either the free ligand or its complexes. Predicted values of the ψ(N3−C4-C5−N6) and Φ(C1−C2-N3−C4) Ramachandran dihedrals suggest that backbone structural features of GlyGly do not get modified dramatically in the aqueous environment after metal coordination. For the explicitly hydrated wNi(GlyGly)2, significant differences with Ni(GlyGly)2 in the simulated aqueous phase are seen only for the O4−C4−C5−N6 and N3−C4−N5−N6 dihedral angles.

Supporting Information, Table S5 lists relevant bond lengths on the GlyGly ligand and its metal complexes as computed at B3LYP/6-311++G(d,p) level in gas and aqueous phases. Bond orders for the metal−ligand atom bonds are also given. The C1−O7 bond which involves the O7 atom coordinated to the metal ion undergoes the maximum deviation (decrease in length) on going from the free ligand to the metal complex, while the uncoordinated C1−O8 bond undergoes less change. However, the C5−N6 bond which involves the N6 atom coordinated to the metal ion undergoes increase in length upon complexation. Lengths of the M-O7 and M-N5 metal−ligand atom bonds follow the order Zn2+ > Cu2+ > Ni2+ with respect 669



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Figure 12. Absorption spectra of metal complexes in the absence (black line) and presence of increasing addition of CT-DNA (tris-HCl buffer, pH 7); [[M(GluGly)2] = 10 μM; M = Ni2+, Cu2+, Zn2+; [CT-DNA] = 0 μM to 20 μM; Inset: Linear plot shows the binding isotherms with CT-DNA].

Figure 13. Most favorable docked poses of (a) Ni(GlyGly)2, (b) Cu(GlyGly)2 and (c) Zn(GlyGly)2 complexes with B-DNA.

S6, show minimal variations (only up to 2.2°): however, with a deviation of 5° in the case of the C2−C1−O7 angle the geometry about the carboxylate group seems to be sensitive to the effects of explicit solvation. Of the six dihedral angles (listed in Table 7), considered to examine the planarity of the amide planes as well as the backbone structural features of GlyGly and its complexes, the Φ dihedral value shows a maximum deviation of 28.7°. 4.8. Interactions with DNA. The DNA-binding properties of a host of Ni2+, Cu2+, and Zn2+ biochelates have been recently reviewed by Barone et al.69 Low molecular weight metallopeptides are efficient DNA binders, and have attracted much interest owing to their ability to serve as biomimetic models for metalloprotein−DNA interactions as well as for applications in biotechnology and medicine.70,71 The absorption spectra of the complexes at constant concentration (10 μM) in the absence and presence of different concentrations of CT-DNA (0−20 μM) are portrayed in Figure 12. In the presence of increasing

to metal ion, while the order followed by the corresponding bond orders is Ni2+ > Cu2+ > Zn2+. These suggest that strength of metal−ligand atom binding would follow the order Ni2+ > Cu2+ > Zn2+, which, however, conflicts with the Irving− Williams series Cu2+ > Ni2+ > Zn2+. Interactions of the water molecules with proteins via intermolecular H-bonds are crucial in affording the biologically active three-dimensional structures of proteins, and study of the interactions of water molecules with peptide moieties has been the subject of intense investigation.50,68 The intermolecular Hbonds present in wNi(GlyGly)2 are depicted in Figure 11 along with their intermolecular H-bond distances. As presented in Supporting Information, Table S5, interactions of explicit water molecules with the amide planes of Ni(GlyGly)2 result in elongation of the exposed polar C4O4 and N3−H3 bonds up to 0.006 Å and shortening of the embedded N3−C4 bond by a maximum value of 0.013 Å. The bond angles related to the amide plane and α-carbon atom geometries, collected in Table 670



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Gas and aqueous phase DFT calculations carried out using the biologically relevant αR conformer of GlyGly at the B3LYP/ 6-311++G(d,p) and BHandHLYP/6-311++G(d,p) levels furnish a cation-binding affinity order of Cu2+ > Ni2+ > Zn2+ in accordance with the Irving−Williams series, and suggest a lowspin state for Ni(GlyGly)2. Theoretically obtained square planar type geometries for Ni(GlyGly)2 and Cu(GlyGly)2 and a tetrahedral geometry for Zn(GlyGly)2 agree with conclusions from experimental absorption spectra. The calculated absorption spectral peaks correctly predict d−d transitions only for Ni(GlyGly)2 and Cu(GlyGly)2, not for Zn(GlyGly)2. Theoretical vibrational spectra give good general agreement with the experimental FTIR spectra and confirm the assignments of the various vibrational modes. Values predicted for relevant dihedrals in the ligand moiety of the complexes distinguish the square planar Ni(GlyGly)2 and Cu(GlyGly)2 from the tetrahedral Zn(GlyGly)2. The computed charge distribution of the complexes shed light on charge transfers between ligand and metal ion as per the metal electronic configurations. UV− visible titration experiments in tandem with in silico docking and molecular mechanical studies suggest that the metal complexes bind to the minor-groove of DNA via H-bonding interactions using their −CO2−, −NH2, and the −CONH groups, giving a DNA-binding affinity order of Ni2+ > Zn2+ > Cu2+.

amounts of CT-DNA, the UV−visible titration spectra of the metal−GlyGly complexes exhibit amplifications in the LMCT peak intensities at ∼375 nm to 384 nm (hyperchromicity), while their absorption band-positions remain basically unaltered (i.e., no batho- or hypsochromic shifts are observed). These results provide strong evidence regarding the possibilities of groove binding for the metal−dipeptide complexes to DNA.69,70 Groove-binding, mainly facilitated by the highly negative electrostatic potential of the DNA-grooves, has now been realized as an important noncovalent ligand−DNA binding motif in the field of drug development.69 The Kb values for the interactions of the Ni(GlyGly)2, Zn(GlyGly)2, and Cu(GlyGly)2 complexes with CT-DNA, calculated according to eq 1, are found to be 2.27·106, 2.5·105 and 2.38· 105 M−1, respectively. Thus, the metallic GlyGly complexes show a DNA binding-affinity order of Ni2+ > Zn2+ > Cu2+, which is consistent with the results of several previous studies reviewed by Barone et al.69 Docking procedures are now routinely used for in silico screening of the drug-receptor interactions because of their remarkable efficiency to describe the “‘best-fit’” orientation of a ligand to a particular receptor; both energetically and geometrically. It is known that the minor grooves of a double helical DNA molecule are the main binding sites for most antibiotic and anticancer drugs.72,73 The highest ranking docked poses of the Ni(GlyGly)2, Zn(GlyGly)2, and Cu(GlyGly)2 complexes with the classical d(CGCGAATTCGCG)2 B-DNA sequence are depicted in Figure 13. In general, the square planar Ni(GlyGly)2 and Cu(GlyGly)2 complexes are found to prefer the AT rich segment of the minor-groove of DNA, while the tetrahedral Zn(GlyGly)2 complex favors the portion that consists of a mixture of both AT and GC base pairs. The H-bond interactions (whose intermolecular H-bond distances range from 2.41 to 2.63 Å) established by the Zn(GlyGly)2 complex with DNA include the interactions of COO− groups of the complex with the NH2 groups belonging to the G9 and G10 residues of DNA. In addition, the amide-NH group of Zn(GlyGly)2 also participates in H-bond interactions with the furanose ring O4′ atom of the A8 residue. On other hand, the Ni(GlyGly)2 and Cu(GlyGly)2 complexes interact with DNA primarily via their NH2 groups with the O2 atom of the T5 residue of DNA. The predicted DNA binding-affinity order of the three metal−GlyGly complexes (listed in Supporting Information, Table S7), determined by performing single point energy calculations using the all atom potential UFF level (details are given in Table S7), is also in agreement with the results of our absorption titration experiments.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental and calculated data; bond lengths, angles, and indices; 3D HOMO and LUMO plots; experimental spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

Financial assistance from the Special Assistance Program of the University Grants Commission, New Delhi, India, to the Department of Chemistry, NEHU, is gratefully acknowledged. S.M. is also grateful to the University Grants Commission, Government of India, New Delhi, for financial assistance through a research fellowship. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The analytical services provided by SAIF, NEHU, are highly appreciated. G.D. is thankful to the Council of Scientific and Industrial Research, New Delhi, India, for generous allocation of computational facilities through Research Project No. 37(1481)/11/EMR-II.

5. CONCLUSIONS Solid and aqueous phase syntheses of the complexes of Ni2+, Cu2+, and Zn2+ with GlyGly lead to the same products, where GlyGly interacts with the metal ions via its −NH2 and −CO2− groups, while the amide group does not bind to the metal. Kinetic studies of the solid-state reaction between GlyGly and copper acetate suggest that GlyGly diffuses toward the metal acetate, forming the complex in 1:2 (metal/ligand) stoichiometry with an activation energy of 22.22 kJ/mol. Electronic absorption spectra distinguish Zn(GlyGly)2 from Cu(GlyGly)2 and Ni(GlyGly)2 in good accord with the coordination modes of the metal ions. The FTIR spectra cast light on which ligand atoms are involved in binding to the metal ions.

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REFERENCES

(1) Hambley, T. W. Why Does Cisplatin Bind to ApG but Not GpA Sequences of DNA? A Molecular Mechanics Analysis. J. Chem. Soc., Chem. Commun. 1988, 221−223. (2) Herr, U.; Spahl, W.; Trojandt, G.; Steglich, W.; Thaler, F.; Eldik, R. v. Zinc(II) Complexes of Tripodal Peptides Mimicking the Zinc(II)-Coordination Structure of Carbonic Anhydrase. Bioorg. Med. Chem. 1999, 7, 699−707.

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