Structural and Theoretical Evidence of the Depleted Proton Affinity of the N3-atom in Acyclovir

The hydronium salt (H 3 O) 2 [Cu(N7–acv) 2 (H 2 O) 2 (SO 4) 2 ]·2H 2 O (1, acv = acyclovir) has been synthesized and characterized by single-crystal X-ray diffraction and spectral methods. Solvated Cu(OH) 2 is a by-product of the synthesis. In the all-trans centrosymmetric complex anion, (a) the Cu(II) atom exhibits an elongated octahedral coordination; (b) the metal-binding pattern of acyclovir (acv) consists of a Cu–N7(acv) bond plus an (aqua)O–H···O6(acv) interligand interaction; and (c) trans-apical/distal sites are occupied by monodentate O–sulfate donor anions. Neutral acyclovir and aqua-proximal ligands occupy the basal positions, stabilizing the metal binding pattern of acv. Each hydronium(1+) ion builds three H-bonds with O-sulfate, O6(acv), and O–alcohol(acv) from three neighboring complex anions. No O atoms of solvent water molecules are involved as acceptors. Theoretical calculations of molecular electrostatic potential surfaces and atomic charges also support that the O-alcohol of the N9(acv) side chain is a better H-acceptor than the N3 or the O-ether atoms of acv.

Figure 1.Formula of acyclovir and the numbering used in this work (see also Figure A1).
In the Fourier transform infrared (FT-IR) spectrum of 1 (see also Figure A5 for acv•0.68H2O and Figure A6, Table A5), the monodentate sulfate ligands (~C3v symmetry) split the ν3 mode in two intense bands at 1122 and 1041 cm −1 , while only one ν3 band is observed for the free ion at about 1033-1440 cm −1 .Likewise, the sulfate ν4 mode consists of two medium intensity bands at 652 and 611 cm −1 , but only one

Results and Discussion
As part of our program expanding the frontiers of acv as a ligand, different reactions between acv and metal chelates were performed, using a large variety of tri-and tetra-dentate chelators.An attempt to obtain the ternary complex Cu(II)-DEA-acv (DEA = diethanolamine) yielded a DEA-free greenish powder with a few well-shaped single crystals corresponding to the formula (H3O)2[Cu(acv)2(H2O)2(SO4)2]•2H2O (1, 100 K, monoclinic system, space group P21/c, final R1 = 0.045.Table A1) along with bluish Cu(OH)2.The all-trans centrosymmetric anions (Figures 2 and A2 A2).It seems clear that the, shortest strongly-bound Cu-O(aqua) favor the cooperation of each Cu-N7(acv) bond with an intra-molecular interligand (aqua)O1-H1B•••O6(acv) interaction (2.615(3) Å, 157.3°) (Table A3), thus leading to the most common MBP of the acv ligand [2][3][4][5][6][7][8][9]12].This fact imposes the coordination of O-sulfate atoms towards the apical/distal sites of the copper(II) surrounding.In addition, three (hydronium)O-H•••O interactions stabilize the structure involving the O6, O(ol), and O(sulfate) atoms from three neighboring complex anions as acceptors, excluding the participation of O-water molecules within the intermolecular network (Table A4, Figures A3 and A4).The novel compound is closely related to the molecular compound all trans-[Cu(acv)2(H2O)2Cl2] [5] where chloride ligands are also moved to the trans-apical/distal coordination to favor the cooperation between Cu-N7(acv) bonds and (aqua)O-H•••O6(acv) interactions.In the Fourier transform infrared (FT-IR) spectrum of 1 (see also Figure A5 for acv•0.68H2O and Figure A6, Table A5), the monodentate sulfate ligands (~C3v symmetry) split the ν3 mode in two intense bands at 1122 and 1041 cm −1 , while only one ν3 band is observed for the free ion at about 1033-1440 cm −1 .Likewise, the sulfate ν4 mode consists of two medium intensity bands at 652 and 611 cm −1 , but only one In the Fourier transform infrared (FT-IR) spectrum of 1 (see also Figure A5 for acv•0.68H 2 O and Figure A6, Table A5), the monodentate sulfate ligands (~C 3v symmetry) split the ν 3 mode in two intense bands at 1122 and 1041 cm −1 , while only one ν 3 band is observed for the free ion at about 1033-1440 cm −1 .Likewise, the sulfate ν 4 mode consists of two medium intensity bands at 652 and 611 cm −1 , but only one at 613 cm −1 for the free ion [4].The identification of the hydronium ion by FT-IR spectroscopy is not an easy task.In compound 1, the H 3 O + ion seems responsible of the broad absorption (ν 1 and/or ν 3 ) at ~2743 cm −1 and the defined band (ν 4 ) at 1190 cm −1 [13].The electronic spectra of compound 1 (Figure A8) explain its greenish color (see Appendix A.4).
This structure, therefore, exhibits two uncommon features: (a) the apical/distal copper(II) coordination of the divalent sulfate anions versus the basal coordination of neutral aqua and acv ligands, and (b) the unexpected formation of hydronium(1+) cations instead of the protonation of the N3-acv atom.The molecular electrostatic potential surface (MEPS) was computed in the complex anion (Figure 3, Cartesian coordinates in Table A6) in order to better understand the basis of these features.As expected, the most negative region is located around the sulfate ligands, which are the best candidates to participate in H-bonding interactions with the H 3 O + ion.Indeed, this is observed in the crystal packing of compound 1.A comparison of MEPS values at the N3 and O(ol) atoms of the N9-acyclic chain reveals that the most negative electrostatic potential falls at the O(ol) atom, supporting the observed (H 3 O + )O-H•••O(ol) interaction, whereas no interaction with (H 3 O + )O-H•••N3(acv) is built.To further discuss the ability of the O(ol) atom and the N3(acv) atom, from the acv N9-side chain and the purine-like moiety, respectively, to participate in H-bonding interactions as acceptors, the atomic charges for [Cu(acv) 2 (H 2 O) 2 (SO 4 ) 2 ] 2− •2H 2 O were also computed.Results computed using two different methods for deriving atomic charges (see ESI for details) yield a more negative charge on the O(ol) atom than on the O(ether) and N3 atoms (Figure 4), in agreement with the experimental results.Therefore, the N3-acv atom is not protonated in the structure due to the significant depletion of its basicity.The steric hindrance on the N3(acv) atom imposed by the acv N9-side chain and ortho-2-amino group should also be considered.at 613 cm −1 for the free ion [4].The identification of the hydronium ion by FT-IR spectroscopy is not an easy task.In compound 1, the H3O + ion seems responsible of the broad absorption (ν1 and/or ν3) at ∼2743 cm −1 and the defined band (ν4) at 1190 cm −1 [13].The electronic spectra of compound 1 (Figure A8) explain its greenish color (see Appendix A4) This structure, therefore, exhibits two uncommon features: (a) the apical/distal copper(II) coordination of the divalent sulfate anions versus the basal coordination of neutral aqua and acv ligands, and (b) the unexpected formation of hydronium(1+) cations instead of the protonation of the N3-acv atom.The molecular electrostatic potential surface (MEPS) was computed in the complex anion (Figure 3, Cartesian coordinates in Table A6) in order to better understand the basis of these features.As expected, the most negative region is located around the sulfate ligands, which are the best candidates to participate in H-bonding interactions with the H3O + ion.Indeed, this is observed in the crystal packing of compound 1.A comparison of MEPS values at the N3 and O(ol) atoms of the N9-acyclic chain reveals that the most negative electrostatic potential falls at the O(ol) atom, supporting the observed (H3O + )O-H•••O(ol) interaction, whereas no interaction with (H3O + )O-H•••N3(acv) is built.To further discuss the ability of the O(ol) atom and the N3(acv) atom, from the acv N9-side chain and the purine-like moiety, respectively, to participate in H-bonding interactions as acceptors, the atomic charges for [Cu(acv)2(H2O)2(SO4)2] 2− •2H2O were also computed.Results computed using two different methods for deriving atomic charges (see ESI for details) yield a more negative charge on the O(ol) atom than on the O(ether) and N3 atoms (Figure 4), in agreement with the experimental results.Therefore, the N3-acv atom is not protonated in the structure due to the significant depletion of its basicity.The steric hindrance on the N3(acv) atom imposed by the acv N9-side chain and ortho-2-amino group should also be considered.We have also evaluated, energetically, the interaction energy of the H3O + ion with the O(ol) atom (observed experimentally) and the hypothetical complex with N3(acv), as indicated in Figure 4 (see Cartesian coordinates in Table A7).The interaction energies in both cases are very large (−88.8 and −88.1 kcal/mol, respectively) due to the strong electrostatic attraction between the counter ions.Interestingly, the complexation energy is slightly more favorable with the O(ol) atom than with N3(acv), in agreement with the experimental observation.We have also evaluated the complexation energy of the solid state assembly commented above in Figure 2 and the theoretical model is We have also evaluated, energetically, the interaction energy of the H 3 O + ion with the O(ol) atom (observed experimentally) and the hypothetical complex with N3(acv), as indicated in Figure 4 (see Cartesian coordinates in Table A7).The interaction energies in both cases are very large (−88.8 and −88.1 kcal/mol, respectively) due to the strong electrostatic attraction between the counter ions.Interestingly, the complexation energy is slightly more favorable with the O(ol) atom than with N3(acv), in agreement with the experimental observation.We have also evaluated the complexation energy of the solid state assembly commented above in Figure 2 and the theoretical model is depicted in Figure 4c.The interaction energy of this assembly is very large (−100.3kcal/mol) due to the contribution of both H-bonding interactions and also the pure electrostatic effects.depicted in Figure 4c.The interaction energy of this assembly is very large (−100.3kcal/mol) due to the contribution of both H-bonding interactions and also the pure electrostatic effects

Synthesis of Compound 1
Equimolar amounts (0.5 mmol) of CuSO4•5H2O and DEA were dissolved in 70 mL of methanol.Acyclovir (acv•0.66H2O,0.5 mmol) was added in small amounts to yield an apple-greenish solution that was filtered into a crystallizing dish.Slow evaporation yields compound 1 and bluish Cu(OH)2.Compound 1 can easily be collected by filtration and dried on a filter paper.Yield: 65%.

Crystal Structure Determination
A green plate crystal of (H3O)2[Cu(acv)2(H2O)2(SO4)2]•2H2O was mounted on a glass fiber and used for data collection.Crystal data were collected at 100(2) K, using a Bruker X8 KappaAPEXII diffractometer.Graphite monochromated MoK(α) radiation (λ = 0.71073 Å) was used throughout.The data were processed with APEX2 [14] and corrected for absorption using SADABS (transmissions factors: 1.000-0.907)[15].The structure was solved by direct methods using the program SHELXS-2013 [16] and refined by full-matrix least-squares techniques against F 2 using SHELXL-2013 [16].Positional and anisotropic atomic displacement parameters were refined for all non-hydrogen atoms.Hydrogen atoms were located in difference maps and included as fixed contributions riding on attached atoms with isotropic thermal parameters 1.2 times those of their carrier atoms.Criteria of a satisfactory complete analysis were the ratios of the RMS shift to standard deviation less than 0.001 and no significant features in final difference maps.Atomic scattering factors were taken from the International Tables for Crystallography [17].Molecular graphics were plotted from DIAMOND [18].

Theoretical Calculations
The energies and atomic charges of the compound included in this study were computed using the BP86-D3 functional [19,20] and def2-TZVP [21] basis set using the crystallographic coordinates within the TURBOMOLE 7.0 program [22].This level of theory, which includes the latest available dispersion correction (D3) [23], is adequate for studying non-covalent interactions, for which dispersion effects are important.The MEP surfaces were generated using Spartan'10 v. 1.1.0software [24] using the B3LYP [25][26][27] method and the 6-31+G* basis set.

Synthesis of Compound 1
Equimolar amounts (0.5 mmol) of CuSO 4 •5H 2 O and DEA were dissolved in 70 mL of methanol.Acyclovir (acv•0.66H 2 O, 0.5 mmol) was added in small amounts to yield an apple-greenish solution that was filtered into a crystallizing dish.Slow evaporation yields compound 1 and bluish Cu(OH) 2 .Compound 1 can easily be collected by filtration and dried on a filter paper.Yield: 65%.

Crystal Structure Determination
A green plate crystal of ( was mounted on a glass fiber and used for data collection.Crystal data were collected at 100(2) K, using a Bruker X8 KappaAPEXII diffractometer.Graphite monochromated MoK(α) radiation (λ = 0.71073 Å) was used throughout.The data were processed with APEX2 [14] and corrected for absorption using SADABS (transmissions factors: 1.000-0.907)[15].The structure was solved by direct methods using the program SHELXS-2013 [16] and refined by full-matrix least-squares techniques against F 2 using SHELXL-2013 [16].Positional and anisotropic atomic displacement parameters were refined for all non-hydrogen atoms.Hydrogen atoms were located in difference maps and included as fixed contributions riding on attached atoms with isotropic thermal parameters 1.2 times those of their carrier atoms.Criteria of a satisfactory complete analysis were the ratios of the RMS shift to standard deviation less than 0.001 and no significant features in final difference maps.Atomic scattering factors were taken from the International Tables for Crystallography [17].Molecular graphics were plotted from DIAMOND [18].

Theoretical Calculations
The energies and atomic charges of the compound included in this study were computed using the BP86-D3 functional [19,20] and def2-TZVP [21] basis set using the crystallographic coordinates within the TURBOMOLE 7.0 program [22].This level of theory, which includes the latest available dispersion correction (D3) [23], is adequate for studying non-covalent interactions, for which dispersion effects are important.The MEP surfaces were generated using Spartan'10 v. 1.1.0software [24] using the B3LYP [25][26][27] method and the 6-31+G* basis set.

Appendix A
Crystallographic data for 1 has been deposited with the Cambridge Crystallographic Data Centre, CCDC No. 1433120.Copies of this information may be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-336-033; email: deposit@ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk).

A2. FT-IR Spectrum
In the FT-IR spectra of copper(II) complexes having solvate and/or coordinated acv, this band is located very close to 1695 cm −1 .However, this is not the case of compound 1 (see Figure A6), where this ν(C=O) band appears at 1683 cm −1 because the exocyclic O6 atom of acv acts twice as an H-acceptor for an intra-molecular and an inter-molecular H-bonding interaction.
An additional band with good diagnostic value is that of the out-of-plane deformation mode δ(O-H) for the terminal alcohol functional group of the N9-side chain, -O(ol)-H, that appears as a more or less defined band near 1387(3) cm −1 (see band 33 at 1387 cm −1 ).
However, attention must be paid if the studied copper(II) complexes contain nitrate or carboxylate anions, which produce stretching bands near to 1385 cm −1 .The absorption band of the stretching mode ν(C=O) in various spectra recorded for commercial samples of acv⋅0.66H2Osplits into two partially-overlapped bands at 1720(3) and 1695(2) cm −1 .

A2. FT-IR Spectrum
In the FT-IR spectra of copper(II) complexes having solvate and/or coordinated acv, this band is located very close to 1695 cm −1 .However, this is not the case of compound 1 (see Figure A6), where this ν(C=O) band appears at 1683 cm −1 because the exocyclic O6 atom of acv acts twice as an H-acceptor for an intra-molecular and an inter-molecular H-bonding interaction.
An additional band with good diagnostic value is that of the out-of-plane deformation mode δ(O-H) for the terminal alcohol functional group of the N9-side chain, -O(ol)-H, that appears as a more or less defined band near 1387(3) cm −1 (see band 33 at 1387 cm −1 ).
However, attention must be paid if the studied copper(II) complexes contain nitrate or carboxylate anions, which produce stretching bands near to 1385 cm −1 .In the FT-IR spectra of copper(II) complexes having solvate and/or coordinated acv, this band is located very close to 1695 cm −1 .However, this is not the case of compound 1 (see Figure A6), where this ν(C=O) band appears at 1683 cm −1 because the exocyclic O6 atom of acv acts twice as an H-acceptor for an intra-molecular and an inter-molecular H-bonding interaction.
An additional band with good diagnostic value is that of the out-of-plane deformation mode δ(O-H) for the terminal alcohol functional group of the N9-side chain, -O(ol)-H, that appears as a more or less defined band near 1387(3) cm −1 (see band 33 at 1387 cm −1 ).
However, attention must be paid if the studied copper(II) complexes contain nitrate or carboxylate anions, which produce stretching bands near to 1385 cm −1 .Variable temperature (5-300 K) magnetic susceptibility measurements on polycrystalline samples were carried out with a Quantum Design MPMS-7 SQUID magnetometer under a magnetic field of 0.1 T. The experimental susceptibilities were corrected for the diamagnetism of the constituent atoms by using Pascal's tables.Magnetic susceptibility data show typical Curie-Weiss behavior.The calculated Curie constant (Cm = 0.44 cm 3 •K/mol) is in good agreement with the g-values obtained from ESR experiments (g = 2.170; Cm = 0.442).The Weiss temperature intercept is close to zero indicating that magnetic interactions between Cu(II) centers are very weak.Variable temperature (5-300 K) magnetic susceptibility measurements on polycrystalline samples were carried out with a Quantum Design MPMS-7 SQUID magnetometer under a magnetic field of 0.1 T. The experimental susceptibilities were corrected for the diamagnetism of the constituent atoms by using Pascal's tables.Magnetic susceptibility data show typical Curie-Weiss behavior.The calculated Curie constant (Cm = 0.44 cm 3 •K/mol) is in good agreement with the g-values obtained from ESR experiments (g = 2.170; Cm = 0.442).The Weiss temperature intercept is close to zero indicating that magnetic interactions between Cu(II) centers are very weak.Variable temperature (5-300 K) magnetic susceptibility measurements on polycrystalline samples were carried out with a Quantum Design MPMS-7 SQUID magnetometer under a magnetic field of 0.1 T. The experimental susceptibilities were corrected for the diamagnetism of the constituent atoms by using Pascal's tables.Magnetic susceptibility data show typical Curie-Weiss behavior.The calculated Curie constant (Cm = 0.44 cm 3 •K/mol) is in good agreement with the g-values obtained from ESR experiments (g = 2.170; Cm = 0.442).The Weiss temperature intercept is close to zero indicating that magnetic interactions between Cu(II) centers are very weak.

Figure 1 .
Figure1.Formula of acyclovir and the numbering used in this work (see also FigureA1).

19 Figure 1 .
Figure1.Formula of acyclovir and the numbering used in this work (see also FigureA1).

Figure 3 .
Figure 3. Compound 1: (a) molecular electrostatic potential surface (MEPS).The values at selected points of the surface are indicated.Color code: from red to blue, with red being the most negative and blue the most positive values.(b) Mulliken and Merz-Kollman charges obtained at the BP86-D3/def2-TZVP level of theory.

Figure 3 .
Figure 3. Compound 1: (a) molecular electrostatic potential surface (MEPS).The values at selected points of the surface are indicated.Color code: from red to blue, with red being the most negative and blue the most positive values; (b) Mulliken and Merz-Kollman charges obtained at the BP86-D3/def2-TZVP level of theory.

Figure 4 .
Figure 4. Theoretical models used to evaluate the electrostatic assisted H-bonding interactions in the solid state of compound 1. (a): Interaction of H3O + with O(ol) atom of acv; (b): Interaction of H3O + with N3 atom of acv; (c): Interaction of H3O + with O(ol) of acv and O-Sulfate atom.

Figure 4 .
Figure 4. Theoretical models used to evaluate the electrostatic assisted H-bonding interactions in the solid state of compound 1. (a): Interaction of H 3 O + with O(ol) atom of acv; (b): Interaction of H 3 O + with N3 atom of acv; (c): Interaction of H 3 O + with O(ol) of acv and O-Sulfate atom.

Figure A3 .
Figure A3.π,π-interactions between the six-membered rings of guanine moieties building 2D frameworks parallel to the bc plane of the crystal.

Figure A3 .Figure A4 .
Figure A3.π,π-interactions between the six-membered rings of guanine moieties building 2D frameworks parallel to the bc plane of the crystal.

Figure A4 . 19 Figure A4 .
Figure A4.Many H-bonds, some of them involving H 3 O+ ions, H 2 O molecules, and acv-O(ol)H groups as H-donors, linking the π,π-stacked 2D-layers in a 3D array in the crystal of compound 1.
lines contrasts with the structurally monomeric nature of the compound.The collapse of the hyperfine structure usually indicates the presence of long-range exchange coupling.The hydrogen bonding and/or the π,π-stacking of the acyclovir rings can provide the necessary exchange pathway.Crystals 2016, 6, 139 10 of 19 corresponds to an axially-elongated octahedral environment for Cu(II) ions.The absence of well-resolved hyperfine lines contrasts with the structurally monomeric nature of the compound.The collapse of the hyperfine structure usually indicates the presence of long-range exchange coupling.The hydrogen bonding and/or the π,π-stacking of the acyclovir rings can provide the necessary exchange pathway.

Figure A7 .
Figure A7.Q-band ESR powder spectrum of compound 1 registered at room temperature.Dotted line is the best fit; see text for the fitting parameters.

Figure A7 .
Figure A7.Q-band ESR powder spectrum of compound 1 registered at room temperature.Dotted line is the best fit; see text for the fitting parameters.

Appendix A. 4 .
Electronic Spectrum of Compound 1 Crystals 2016, 6, 139 10 of 19 corresponds to an axially-elongated octahedral environment for Cu(II) ions.The absence of well-resolved hyperfine lines contrasts with the structurally monomeric nature of the compound.The collapse of the hyperfine structure usually indicates the presence of long-range exchange coupling.The hydrogen bonding and/or the π,π-stacking of the acyclovir rings can provide the necessary exchange pathway.

Figure A7 .
Figure A7.Q-band ESR powder spectrum of compound 1 registered at room temperature.Dotted line is the best fit; see text for the fitting parameters.

Table A1 .
Crystal data, structure solution, and refinement of compound 1.

Table A1 .
Crystal data, structure solution, and refinement of compound 1.

Ligand or Solvent Chromophore Mode Wavenumber (cm −1 ) Band Number in the Read Spectrum
This band usually splits in two at 1720(3) and 1695(3) cm −1 in the spectra of acv⋅0.66H2Osamples, and appear at about 1695 cm −1 in the spectra of Cu(II)-acv complexes with monodentate acv ligands.Note that, in compound 1, the O6 atom of acv is involved as an acceptor in two H-bonds.

Table A6 .
Model in Figure3