Study on the Reactivity of Amino Acid Chemosensor , NPFNP , with Ethanol : Structural Elucidation through Single Crystal XRD and DFT Calculations

A novel ethoxy derivative of an amino acid chemosensor, 3-naphthyl-1-phenyl-5-(2ʹ-fluoro-5ʹnitrophenyl)-2-pyrazoline (NPFNP), has been synthesized and characterized by different spectroscopic methods. A single crystal of the ethoxy derivative, 3-naphthyl-1-phenyl-5-(2ʹ-ethoxy-5ʹ-nitrophenyl)-2-pyrazoline NPENP, has been obtained and characterized. The structure holds interest as it carries biologically active pyrazoline as a central ring attaching to electron donating and withdrawing substituents. The major motivation for this work was to gain detailed insight into the structural parameters of this compound for investigating the influence of crystal packing and geometrical dimensions on optical properties. Time-dependent DFT calculations have been employed for comparing the XRD data with theoretical parameters. The results show that the DFT method at B3LYP/6-31G level can well reproduce the structure of the title compound.


Introduction
he rising prevalence of heterocyclic compounds in bio-analytical chemistry has attracted the increasing attention of researchers to pyrazoline derivatives.Easily tunable properties suit these electron rich nitrogen heterocycles to be used extensively as useful synthons in organic synthesis [1][2] as well as for medicinal applications [3][4].These significant key motifs in heterocyclic chemistry are known not only from their pharmaceutical applications but also as materials showing excellent optical properties.Moreover, the synthesis of fluorophores with desirable properties based on a pyrazoline skeleton is of considerable interest in fluorescent materials research [5][6][7][8].A lot of work has been focused on the synthesis, crystal structure, optical properties and quantum chemical calculations of novel pyrazoline derivatives [9][10][11][12].
The hydroxyl group is a functional element of many drugs and naturally occurring compounds; however, its detection is still a challenging analytical subject [13].To date there have been relatively few compounds capable of tagging the hydroxyl groups directly.This inadequacy observed in the literature encouraged us to address this issue.Very recently, 3-naphthyl-1-phenyl-5-(5ʹ-fluoro-2ʹ-nitrophenyl)-2-pyrazoline was synthesized and characterized by our group and this probe showed a good affinity towards amino acid groups [14].As a continuation of the mentioned study, we inspected the preparation and usability of 3-naphthyl-1-phenyl-5-(2ʹfluoro-5ʹ-nitrophenyl)-2-pyrazoline NPFNP, a constitutional isomer of the reported probe in which the fluorine atom is situated at carbon-2ʹ and the nitro group at carbon-5ʹ (see Figure 1).In the present study we focus attention on the reactivity of NPFNP with a -OH group by reacting the probe with ethanol and aim to understand the structural details of the ethoxy product, 3-naphthyl-1-phenyl-5-(2ʹ-ethoxy-5ʹnitrophenyl)-2-pyrazoline NPENP in the solid state by means of X-ray crystallography (see Figure 2).Molecular geometries of NPENP in both ground and excited states have been calculated using the density functional theory, and the trend of calculated HOMO-LUMO gaps and geometrical parameters in excited state provides valuable information on the factors influencing the optical properties of NPENP.

Experimental
All reagents and solvents used in this study were obtained from Sigma Aldrich Chemical Company and were used without further purification.
Melting point was determined using GallenKamp MPA350 melting point apparatus.The purity of the synthesised compounds was checked by TLC and analyses were carried out on 0.25 mm thick pre coated silica plates (Merck Fertigplatten Kieselgel 60F254).Column chromatography was performed using Merck silica gel 60 (40-63 µm).IR spectra were determined on a Cary 630 FTIR spectrometer (Agilent Technologies, USA). 1 HNMR and 13 CNMR spectra were recorded using a 400 MHz Bruker spectrometer (Bruker Corp., UK).Chemical shifts (δc) are quoted in parts per million (ppm) to the nearest 0.1 and 0.01 and are referenced to the solvent peak (CDCl3).Mass spectra were obtained using a Quattro Ultima Pt tandem quadrupole mass spectrometer (Waters Corp. MA, USA).A Shimadzu (model multispec-1501) UV-Vis spectrophotometer (Shimadzu, Japan) was used to collect absorption spectra.All measurements were done repeatedly, and reproducible results were obtained.
The single crystal suitable for X-ray measurements was obtained by recrystallization from hot ethanol.The selected golden yellow crystal (0.350 x 0.300 x 0.250 mm 3 ) of the compound was mounted on a Bruker AXS KAPPA APEX-II diffractometer at 293 (2) K with a graphite monochromatic Mo-Kα radiation (k = 0.71073 Å).The corrections for LP factors and empirical absorption were applied to the data.The structures were solved by direct methods and refined by the full-matrix least-squares method   2 using the SHELXTL software package [15].All non-H atoms were anisotropically refined.The anisotropic displacement factor exponent takes the form of -2π 2  ].The hydrogen atom positions were fixed geometrically at calculated distances and allowed to ride on the parent C atoms.The final least-squares cycle gave R= 0.0975 and wR2 = 0.254 for 3547 independent reflections [R (int) = 0.0896].Atomic scattering factors and anomalous dispersion corrections were taken from International Table for X-ray crystallography [16].
The quantum chemical calculations were carried out with GAUSSIAN 09W programs and GaussView 05 was used for visualization of structures.DFT (Density Functional Theory) calculations were performed by using a combination of Becke ' s three parameter hybrid exchange potential [17] with the correlation functional of Lee et al. (B3LYP) [18].The basis set used was 6-31G.

Synthesis and 1 H NMR characterization of NPENP
The synthesis of NPENP commenced from an aldol reaction between 1-acetylnaphthalene 1 and 2ʹ-fluoro-5ʹnitro benzaldehyde 2 in acetonitrile using sodium carbonate as a base to give -unsaturated ketone 3 in 72 % yield.A cyclization reaction of compound 3 with phenylhydrazine in refluxing ethanol led to the formation of the pyrazoline derivative 4 which reacted in situ with ethanol to yield its ethoxy derivative 5 (Scheme 1).The synthetic route adopted was straightforward, and NPENP was obtained by following aromatic nucleophilic substitution, in which a NO2 group stabilises the anion intermediate through resonance, and the fluorine atom para to -NO2 can be easily replaced by the -OH group of the ethanol to yield the desired ethoxy derivative.
The 1 H NMR spectrum of the eight aliphatic hydrogens in NPENP displayed in Figure 3 exhibits three equally intense doublet of doublets, each accounting for one hydrogen atom on the pyrazoline ring; Ha, Hb and Hc.Among these, Ha, a stationary proton near to the electronegative nitrogen atom, appears in the mid field region of 5.52 ppm.It is clear from the structure that the geminal protons Hb and Hc are not in the same space of the pyrazoline ring, so Hb is shifted by the next benzene ring slightly to the left at a chemical shift of 4.03 ppm and Hc is located at a chemical shift of 3.20 ppm.The -CH2hydrogens show a quartet signal at 4.21 ppm and the methyl group appears as a triplet at 1.49 ppm.

Optical Spectroscopy
The NPENP is a chromophoric π-system composed of three aryl substituents in the 1-, 3-and 5-positions of pyrazoline ring atoms.As shown in Figure 4, this compound exhibits two prominent bands in all selected solvents, appearing at 298-312 nm and 373-391 nm, respectively, and the molar absorption coefficient values are in the same range of 6×10 4 M -1 cm -1 .The shorter wavelength is ascribed to a localized aromatic π-π * transition and the long wavelength is attributed to ICT transitions [24].However, in water there is a distinct bathochromic shift in the absorption maxima compared to those in non-aqueous solvents.The shift in magnitude of the peak position according to the polarity of the medium suggests that the ground state of the molecule is polar.Even though the molecule is designed in such a way that the aryl rings in this compound can communicate electronically through the pyrazoline π-system to produce an analytically useful signals, the question why the title compound is not fluorescent is crucial.In particular, the UV absorption spectra of NPENP in solvents of differing polarity clearly indicate that there is an intramolecular charge transfer throughout the system in ground state.

X-ray crystallography
To study the packing properties of NPENP, good-quality single crystals suitable for X-ray analysis were grown in ethanol solvent by slow evaporation at ambient conditions and were found to have a monoclinic crystal lattice with the P21/C space group.The molecular view of FNPFE is shown in Figure 5.A summary of crystallographic data collection parameters and refinement parameters are compiled in Table 1.One phenyl moiety, a naphthalene ring and a substituted phenyl ring are bonded to the pyrazoline ring at the atoms of N2, C9 and C7, respectively.The N2-N3 (1.398 Å) (6) and C9-N3 (1.283 Å) (7) bonds have pure singleand double-bond character, respectively.The torsion angle at (C9-C10-C15-C14) is -177.4(5) o , which shows that the molecular structure adopts a trans configuration about the C9N3 bond.The dihedral angle between the pyrazoline and substituted phenyl ring (C4-C5-C7-C8) is found to be 74.5 (6) o .The conformation of the naphthalene (C8-C9-C10-C11) and phenyl unit (C21-C20-N2-N3) with respect to the pyrazoline ring can be indicated by the torsion angles -1.3 (8) o and 23.1 (7) o , respectively.Even though the N2 atom is not involved in conjugation with the pyrazoline double bond, it is sp 2 hybridized with its lone-pair electrons delocalized through conjugation with the adjacent phenyl group, as shown by the N2-C20 bond length (1.387 Å) (7).This bond length is shorter than a C-N single bond (1.47 Å) and slightly longer than a CN bond (1.30Å).The sum of the three angles around N2 atom is 350 o , which clearly indicates the pyramidal disposition of the bonds at the atom N2.The C-C bond distances in its aromatic rings are in the normal range of 1.32-1.45Å, which is characteristic of delocalized aromatic rings [19].The selected bond length, bond angles and torsion angles are presented in Table 2.The 5-substituted phenyl ring on the asymmetric carbon of a pyrazoline moiety (C7) is oriented in such a manner that one of its hydrogen atoms (H27A) is located almost on top of the pyrazoline moiety at 2.6131 Å distance from the center of the pyrazoline ring.The angles around the asymmetric carbon are 111.7 o (4) (N2-C7-C5), 101.9 o (4) (N2-C7-C8), 113.0 o (4) (C5-C7-C8), 110.0 o (N2-C7-H7), 110.0 o (C5-C7-H7), 110.0 o (C8-C7-H7).The bond length of the C-C single bond bridging the naphthalene, the 5-substituted phenyl group and the pyrazoline ring (C9-C10, C5-C7) is calculated to be 1.49Å (9,7).This bond is significantly shorter than a typical C-C single bond of 1.54Å.This reflects an efficient charge delocalization over the pyrazoline π-system allowing electronic interaction between the attaching groups as they are electronically separated.Analogously it is clear that the presence of an electron attracting nitro group and electron donating ethoxy substituent distorts the interatomic distances so that the bond length of C4-O3 reduces to 1.339 (7) Å , in contrast to the normal C-O bond of 1.43 Å.
The bond lengths between these atoms are 2.6131 Å, 2.869 Å and 2.424 Å respectively.Similarly another four hydrogen bonds (O3H8, O3H7 and N3H16, N3H21) are bifurcated and their bond lengths are 2.996, 2.477, 2.223, and 2.446 Å, respectively, (Table 3).The establishment of CH-π interactions consequently result in the stacking of the NPENP molecules to be packed as a crystal along b axis like a chair as shown in Figure 6.The molecular packing diagram shows two layers of molecules, which are independently arranged in the unit cell.In the extended structure of NPENP it is clear that intermolecular CH-π interactions within each unit and ππ contact between the individual chain of a single molecule in a zig-zag manner to produce chains running parallel to each other.

Computational details
The DFT functional B3LYP coupled with the 6-31G basis set was used to perform geometry optimizations in vacuo, for comparing the XRD data with theoretical parameters.There are some differences between the experimental values and the calculated data as the geometry of the solid-state structure is subject to intermolecular forces, such as van der Waals interactions, crystal packing forces and hydrogen-bond forces [22], but the calculated data corresponds to the isolated molecule in gas phase.
As discussed previously for the crystal structure, the N2-N3 bond length (1.351 Å) is slightly lower than the experimental value; surprisingly C9-N3 (1.335 Å) shows deviation from the typical CN behavior of the pyrazoline ring (~1.28 Å) in solid state.The bond length of sp 2 hybridized N2 atom and the phenyl attaching carbon C20 is in good agreement with the experimental value, but the sum of the three angles around N2 atom is exactly 360 o indicating that the bonds meeting at N2 are almost coplanar in gaseous phase and this high degree of coplanarity invokes electronic transitions in ground state (see Figure 7).The calculated torsion angle at C9-C10-C15-C14 is found to be 178.597o and the difference of about 180 o from the experimental value corresponds to a rotation of 180 o about the C9-C10 axis of the naphthalene ring, which does not affect the molecular stability.The bond length of the C9-C10 single bond bridging the naphthalene ring (1.44 Å) is found to be lower than that of the experimental value (1.49Å) and also lower than the C5-C7 bond bridging the substituted phenyl group (1.51 Å).It was noticed that the distance between the pyrazolinic nitrogen connecting to the attaching carbon of the phenyl ring is shorter (1.388 Å) than a normal C-N single bond (1.47 Å).It implies that there is a chance for high degree electron density delocalization from the π-bridge towards the naphthalene and phenyl rings, which is crucial for the highly enhanced ICT character in ground state.The computational analysis of the frontier molecular orbitals (see Figure 8) provides more insightful information on the energies of occupied and virtual front orbitals and their influence on the excitation and emission properties of NPENP.

(i)
(ii)  The energies of HOMO, LUMO and their neighboring orbitals are all negative, which indicates the stability of NPENP in ground state, whereas the molecule is instable in excited state.In ground state the HOMOs are mainly localized on the naphthalene, pyrazoline and 1-phenyl rings and the LUMO is fully populated on the 5-substituted phenyl ring.By contrast, the contribution of naphthalene and pyrazoline is increased in LUMO+1 and the small energy gap (0.053 eV) underlines the highly delocalized nature of NPENP in ground state.From the figure it is observable that the bent -CH2-CH3 group has no charge density distribution, neither on the HOMO nor on the LUMO levels of NPENP in both ground and excited state.
On the other hand, there is a large distortion in the geometry of NPENP in excited state accompanied by unfavorable changes in the basic dimensions, particularly in the bond length and bond angles involving the pyrazoline ring.There is perceptible lengthening of N2-N3 (1.407 Å), N2-C20 (1.447 Å) and C9-C10 (1.463 Å) bond length in excited state geometry.The sum of the bond angles at N2 is found to be 347.28o and is considerably distant from the planarity.The dihedral angle C11-C10-C9-C8 (179.12 o ) is an indication of coplanar orientation of the pyrazoline and naphthalene rings in excited state while both the phenyl and substituted phenyl rings are folded slightly from their attached pyrazoline ring with torsion angles of 10.617 o (C21-C20-N2-N3) and -50.496 o (C4-C5-C7-C8), respectively (see Figure 9).As shown, for NPENP in excited state the electron densities of HOMO are mainly located on the naphthalene ring; there is an electron distribution from the substituted phenyl ring to the pyrazoline ring, except in case of the C8 carbon and a small node on the 1-phenyl ring.The increase in electron density on the left part including the pyrazoline and 1-phenyl rings was observed for HOMO-1 in excited state, and the LUMO is equally delocalized on the naphthalene and 5-substituted phenyl rings with a small contribution on C-N=N.The LUMO +1 looks like a flow of electrons from the 1-phenyl ring, including N2-C7, and as a separated cloud on the center of the 5-substituted phenyl ring.Compared to the MOs of the ground state (S0) the contribution of the 5-substituted phenyl ring is greater in excited (S1) state.This can increase the electron density on the conjugated back bone and its steric effects cause distortion of the conjugated back bone [23].On the other hand, it is clear that the phenyl group at N2 also destroys the coplanar character of the conjugated backbone in excited state.This ensures that a steric effect arises due to the distorted geometry of NPENP hampering the delocalization of a/ the lone pair of electrons of -N3 atom, and this may also disturb the conformational stability of CN-N which in turn will cause a decrease in conjugation, and thereby decrease in charge transfer throughout the system and make the LUMO-HOMO energy gap wider (0.134 eV).

Conclusion
Combining computational studies with single crystal X-ray diffraction facilitates a deeper understanding of the geometry of NPENP in solid and gaseous state.The analysis of the molecular geometry of NPENP in ground state exhibits a very good agreement with the experimental data.Considering the geometry of NPENP in excited state, one can clearly find that non-planarity and proximity of atoms play an important role in hindering the delocalization of π electrons throughout the system.The high molar absorptivity of NPENP underlines the importance of its mother dye NPFNP in the determination of alcohols and phenolic derivatives.

Figure 6 .
Figure 6.Packing diagram of NPENP viewed along b axis.

Figure 7 .
Figure 7.The optimized molecular geometry of NPENP in its ground state at B3LYP/6-31G level.

Table 1 .
Crystal data and parameters of X-ray diffraction experiment for NPENP.

Table 2 .
Selected geometrical parameters by X-ray and theoretical calculations at B3LYP/6-31G level of theory in ground state and excited state for NPENP.

Table 3 .
Parameters for the intra-and inter molecular hydrogen bonding.