Synthesis , Structure and Molecular Modeling of a ZnII-Phenolate Complex as a Model for ZnII-Containing Tyrosinate Metalloenzymes

Apresentamos neste trabalho a síntese, estrutura cristalina e as propriedades espectrais de H NMR do complexo mononuclear [Zn(L-Br)].2H 2 O o qual contém em sua primeira esfera de coordenação, o ligante hexadentado H 2 L-Br (H 2 L-Br = N,N’-bis-(5bromo-2-hidróxibenzil)N,N’bis-(piridin-2-ilmetil)-1,2-etanodiamina). Cálculos teóricos utilizando DFT mostram boa correlação entre os parâmetros calculados e aqueles obtidos através de cristalografia de raios X em monocristais e revelam que somente os grupos fenolatos do ligante H 2 L-Br participam na formação do HOMO, enquanto somente os anéis piridínicos contribuem para a formação do LUMO.


Introduction
Many Zn II -containing enzymes have been discovered and their role in biological processes studied.Carbonic anhydrase, 1,2 carboxypeptidase, [3][4][5] β-lactamase II, 6 thermolysin, 7 alkaline phosphatase, 8 and astacin 9,10 are examples of such metalloenzymes.Of these, astacin, a digestive zinc-endopeptidase that is involved in hydrolytic processes should be highlighted since it represents the first example of a zinc enzyme that contains a tyrosine residue coordinated directly to the metal center in the active site. 11n fact, astacin, an endopeptidase isolated from crawfish Astacus astacus represents the prototype for the "astacin family", 12,13 which includes mammalian metalloendopeptidases 14 and developmentally regulated human, 15 fruitfly, 16 frog 17 and sea urchin 18 proteins.The X-ray crystal structure of astacin (R-value of 0.162) reveals that the Zn IIion lies in a trigonal bipyramidal coordination environment with three histidines, a water molecule and a more remote tyrosine as ligands. 11One histidine nitrogen and the tyrosine OH group, at distances of 2.3 and 2.6 Å to the zinc, respectively, are apically connected, whereas the other three ligands are coplanar and 2.1 Å apart from the zinc center.Following our interest in the search for new compounds as structural and functional models for the active site of zinccontaining metalloenzymes, [19][20][21] we report here the synthesis, X-ray structure and molecular modeling using Density Functional Theory (DFT) for the [Zn(L-Br)] complex, where L-Br 2-is the deprotonated form of the N,N'-bis-(5-bromo-2hydroxy-benzyl)-N,N'-bis-(pyridin-2-ylmethyl)-ethane-1,2diamine ligand.Importantly, theoretical calculations have been recently introduced in our group as a strategy for planning the synthesis of new ligands and model complexes. 22

Experimental
Abbreviations

Material and methods
All reagents and solvents were purchased from commercial sources and used as received. 1H NMR spectra were recorded on a Bruker 200 FT spectrometer, in CDCl 3 as a solvent.Infrared spectra were recorded with a Perkin Elmer FTIR 2000, in KBr pellets.Elemental analyses were performed with a Carlo Erba instrument model E-1110.

Synthesis of H 2 L-Br
The H 2 L-Br ligand was prepared according to the sequence of reactions depicted in Scheme 1, with slight modifications of the method described for the synthesis of the H 2 bbpen ligand. 23Ethylenediamine (10.0 mmol) was added dropwise to a THF/methanol -2:1 solution of 2hydroxy-5-bromobenzaldehyde (20.0 mmol) while stirring.After 30 min NaBH 4 (26.0 mmol) was added, and a few minutes later the deep yellow solution became colorless.Then, 4.0 mol L -1 HCl was added to adjust the pH to 7 and the solvent was removed under vacuum at 40 o C. Water was added to the precipitated product which was filtered off and washed with water followed by cold methanol.The white solid obtained (7.5 mmol) was added to a solvent mixture of THF and water 1:1 (100 mL) with 2-chloromethylpyridine hydrochloride (23 mmol) and sodium carbonate (38 mmol).This mixture was refluxed for 18 hours and the THF removed under vacuum at 40 o C. The residual water was decanted off and the product (yellowish oil) was solubilized in a mixture of 2-propanol and ethyl acetate.The H 2 L-Br ligand precipitated as a white powder after 24 hours (yield = 75%

Crystal structure determination
A colorless irregular block was prepared from a big crystal, which was selected from the crystalline sample of the [Zn(L-Br)] complex.The crystal data were measured on an Enraf-Nonius CAD4 diffractometer, using graphite monochromated Mo-K α radiation (λ=0.71069Å), at room temperature.Cell parameters were determined from 25 carefully centered reflections in the q range 8.76-15.30o and refined by the least-squares method.The collected intensities were corrected for Lorentz and polarization effects 24 and for Scheme 1.
absorption (face-indexed method; T mim 0.28 and T max 0.64).The structure was solved by direct methods and was refined by the full-matrix least-squares method using SHELXS97 25 and SHELXL97 26 computer programs, respectively.All nonhydrogen atoms were refined with anisotropic displacement parameters.H atoms bonded to C atoms were placed at idealized positions using standard geometric criteria, whereas the H atoms of the water molecule of crystallization were found from Fourier map and treated with a riding model.Further relevant crystallographic data are summarized in Table 1.The drawing of molecular structure was made with ORTEP3 program. 27

Computational details
All geometry optimizations were performed with B3LYP hybrid density functional theory in conjunction with the 6-31G (d,p) basis set and LACVP* basis set for the metal using the Spartan 04 program. 28The calculations were carried out on a 2.6 GHz Athlon PC, with 1 GB RAM and 40Gb HD under the operational system Windows 2000, using the Spartan 04 program.

Syntheses
The H 2 L-Br ligand was obtained in a good yield and pure enough to be fully characterized and used as a precursor for the synthesis of coordination compounds.The reaction between one equivalent of H 2 L-Br and one equivalent of Zn(OAc) 2 .2H 2 O produced the mononuclear complex [Zn(L-Br)].2H 2 O. Infrared spectral data reveal that upon coordination of H 2 L-Br to the zinc there is a general bathochromic shift of ~15 cm -1 and a decrease in intensity of the C=N and C=C stretching modes.Interestingly, dinuclear [ZnL].ZnCl 2 complexes were reported by Adams et al. 29 for the reaction between the similar ligands H 30 with ZnCl 2 , in a 1:1 stoichiometry.In these homodinuclear compounds, the coordinated phenolate oxygen atoms act as ligands toward a second zinc-containing entity yielding [ZnL].ZnCl 2 .The reaction between H 2 L 2 and Zn(OAc) 2 also produced a dinuclear species [Zn(L 2 )•Zn(OAc) 2 ], according to Adams's report.A mononuclear [ZnL 3 ] species was only obtained with the ligand H 2 L 3 (H 2 L 3 = N,N'-bis-(5-nitro-2-hydroxybenzyl)-N,N'-bis-(pyridin-2ylmethyl)-propane-1,3-diamine), due the electron withdrawing effect of the nitro group which makes the phenolic oxygen atoms weaker Lewis bases and consequently less able to coordinate a second zinc atom. 29,30The fact that the mononuclear species [Zn(L-Br)] described herein was obtained, instead of a dinuclear molecule, indicates that the electron withdrawing effect of the bromo groups in H 2 L-Br is working in the same way as the nitro groups in Fenton's H 2 L 3 , which suggests that the ligand substituent groups are playing an important role in the reaction stoichiometry.

Crystal structure of [Zn(L-Br)].2H 2 O
The molecular structure of the [Zn(L-Br)] molecule in [Zn(L-Br)].2H 2 O is depicted in Figure 1.Crystallographic data are shown in Table 1, and selected bond lengths and angles are listed in Table 2.The complex [Zn(L-Br)] consists of a distorted octahedral molecule, with the hexadentate N 4 O 2 -donor ligand binding the Zn IIion via two amine nitrogen atoms of the ethylenediamine backbone, two phenol oxygen atoms and two pyridine nitrogen atoms.Each half of the ligand provides a facial N 2 O-donor set with the phenolate oxygen atoms cis to each other and trans to the aliphatic nitrogen atoms.Completing the coordination sphere, the pyridine nitrogen atoms occupy apical sites and are trans to each other.This structural arrangement is essentially similar to that reported for [ZnL 3 ], 30     Zn II complexes already reported in the literature. 19,20,29,302][33] An exception is the [Ru(bbpen)] + cation complex which shows two amine nitrogens, two pyridine nitrogens and two phenolate oxygen atoms all as cis pairs. 34he coordination of phenolate moieties in cis positions to the metal center induces a intermolecular bifurcated H bond formation, where the water molecule of crystallization is the donor group (O1W-H1WA 0.88 Å) and the oxygen atoms O1 (H1WA…O1 2.29 Å; O1W…O1 3.00(1) Å; <O1W-H1WA…O1 138.

H NMR spectrum of [Zn(L-Br)].2H 2 O
Since the [Zn(L-Br)] complex is diamagnetic, 1 H NMR was used to investigate the species in CDCl 3 solution.The room-temperature 1 H NMR spectra, 200 MHz (Figure 2) clearly indicate the formation of the complex, and confirm that the symmetric solid-state structure is retained in solution.The assignments of all protons in the ligand and in the corresponding complex are based on the intensity of the signals and spin-pin splitting structure.The 1 H NMR spectra for the free ligand and the corresponding [Zn(L-Br)] complex, depicted in Figure 2, contain seven unique protons resonances in the aromatic region with some small differences, indicating that complexation has taken place.The pyridine H 6 hydrogen atoms are shifted downfield by 0.37 ppm for [Zn(L-Br)], relative to their positions in the free ligand spectrum.A 0.33 ppm upfield shift is also observed for the phenyl H 6' hydrogen atoms in the [Zn(L-Br)] spectrum.The most remarkable differences between the ligand and the complex are the resonances in the aliphatic region for the methylene groups.The free ligand, H 2 L-Br, contains three prochiral CH 2 groups with enantiotopic Hs isolated from the others.The Hs are observed as three singlet peaks shifted upfield to 3.72 (s, 4H, -CH 2 -py), 3.63 (s, 4H, -CH 2 -ph) and 2.68 ppm (s, 4H, -NCH 2 CH 2 N-).The [Zn(L-Br)] complex also contains three prochiral CH 2 groups, two groups with diastereotopic Hs (-CH a H b -py; -CH a H b -ph), and one with enantiotopic Hs (-NCH 2 CH 2 N-), all isolated from the other Hs.These Hs are observed as two pairs of doublets in the case of the diastereotopic Hs, with high geminal coupling constants

Theoretical calculations
The results for the principal calculated and experimental (for comparison) structural parameters of the [Zn(L-Br)] complex are shown in Table 2 and the optimized structure is shown in Figure S1 in the Suplementary Information.The maximum variation for the bond lengths is 0.16 Å and for angles is 9.1º.A comparison between the geometric parameters of the model and the experimental data shows that the results are in good agreement.The difference noted is due to the fact that the model complexes were considered in the gas phase while the experimental parameters were measured in crystalline form.The graphical representation of HOMO shows that only the phenolate rings participate in its formation (Figure 3 top).On the other hand, only one of the pyridinic rings contributes to the LUMO formation (Figure 3 bottom).The surface of electrostatic potential shows once again that the electronic density of the complex is localized around the phenolic rings.It can also be noted that the bromide substitutions pull the electronic density to the halogen atom (Figure S2 in the Suplementary Information).

Conclusions
Only recently mononuclear Zn II -phenolate containing complexes have been reported. 19,20,29,30In this paper we described the synthesis, crystal structure and 1 H NMR properties of such a complex.The good agreement between the theoretical and experimental data obtained for [Zn(L-Br)] indicates that the use of DFT is appropriate in the planning and synthesis of new structural and functional models for Zn II -containing phenolate enzymes.Based on this information, the synthesis of further multidentate ligands containing phenol as a coordinating group are under investigation, and will be the subject of further reports.
except for the fact that [Zn(L-Br)] contains an ethylenediamine backbone instead of a propane-1,3-diamine backbone in[ZnL 3  ].Consequently, the [Zn(L-Br)] complex presents a higher distorted geometry due to its five-membered ring in the equatorial plane compared to the six-membered ring in [ZnL 3 ].The higher distortion in [Zn(L-Br)] can be evidenced by the three trans angles which are 3.5 o (N1-Zn-O2), 4.8 o (N2-Zn-O1) and 11.1 o (N31-Zn-N41) smaller for [Zn(L-Br)] when compared to [ZnL 3 ].The average Zn-O bond lengths for [Zn(L-Br)] (1.997 Å) are 0.063 Å shorter than those for [ZnL 3 ].This is attributed to the higher distortion in the coordination sphere and the weaker electronwithdrawing effect of the bromo groups in [Zn(L-Br)].On the other hand, the average Zn-N py bond lengths are 0.058 Å longer in [Zn(L-Br)] when compared to [ZnL 3 ], whereas the Zn-N amine bonds are identical in both complexes.Since the [Zn(L-Br)] and the [Zn(bpa) 2 ] 19 complexes (Hbpa = N-(2-hydroxybenzyl)-N-(pyridin-2-ylmethyl) amine) possess identical coordination environments, a comparison of their structural parameters should also be of interest.Firstly, it should be noted that H 2 L-Br is a hexadentate N 4 O 2 ligand bound to the Zn II -ion in its deprotonated form, while in [Zn(bpa) 2 ] the Hbpa ligand corresponds to the half of H 2 L-Br without the ethylenediamine backbone and the bromo substitution in the para-position of the phenolate group (tridentate N 2 O-donor).Consequently, in [Zn(L-Br)] the tertiary amine nitrogen atoms must be coordinated in a cis-position to each other excluding the possibility of an inversion center at the zinc.Secondly, in both complexes the N-(2hydroxybenzyl)-N-(pyridin-2-ylmethyl)amine unity adopts a facial coordination arrangement.However, in [Zn(bpa) 2 ] the atoms of the same nature (two N amine , two N pyridine and two O phenolate ) are coordinated in trans positions