Resonance Raman spectroscopy study of protonated porphyrin

: Resonance Raman microscopy was used to study the resonance Raman scattering of the diacid (diprotonated form) of free-base porphyrin ( 21 H ,23 H -porphine) in a crystal powder and KBr pellets. Intensive lines in the spectral range between 100  1000 cm -1 have been detected and assigned as spectral manifestation of out-of-plane modes. The Raman spectra were simulated by means of DFT methods and compared with the experimental data. It is evident from experimental and theoretical results that the activation of out-of-plane modes arises from saddle distortion of the porphyrin macrocycle upon formation of its diprotonated form.


INTRODUCTION
The molecule of porphyrin belongs to an interesting and important class of organic compounds. The porphyrins and their constitutional isomers have been used in the design of artificial photosynthetic systems [1,2], sensors [3], molecular optoelectronic devices [4], photoswitches [5], optical memories [6], and in photodynamic therapy [7]. Parallel to the application-oriented studies, the details of the chemical and physical properties of porphyrin still constitute a fruitful area of fundamental scientific research. The high importance of porphyrins for current science and technology has led to intensive studies of these compounds by different spectral methods. However, there still exist free-base porphyrin spectral features for which the detailed studies have not yet been carried out.
One of the most informative and sensitive tools for investigating of vibrational states of porphyrins is Resonance Raman Scattering (RRS) [8,9]. RRS methods have been used for detection, identification, quantification, and structural studies of porphyrins, porphyrazines and other compounds comprising the porphyrin chromophore in solvents, KBr pellets, and solid powder at ambient and low temperatures [10][11][12][13].
There is ample evidence that porphyrin moieties in their natural protein environment are highly distorted from planarity. Out-of-plane distortions of the porphyrin macrocycle lead to significant changes in their spectral properties and have been investigated because of their possible functional roles in heme-and chlorophyll-containing proteins. Because of the interest in the spectral consequences of non-planarity, numerous synthetic models of porphyrins have been prepared which mimic these distortions. The effect of distortion has been observed with the help of fine-line luminescence spectroscopy study of metallocomplexes with Mg(II), Zn(II) and Pd (II) ions [14,15]. The RRS aspects of non-planarity in the highly substituted metalloporphyrins and the effect of the central metal ions have also been previously discussed [16][17][18][19][20][21][22].
In general, the simplest distorted forms of porphyrin can be produced upon protonation of free-base, i.e. without metallic ions in the center of porphyrin macrocycle. From the early days of study of the acidic forms of porphyrin it is well known that free-base porphyrin is transformed into mono-and diprotonated forms in the acid medium [15,[23][24][25][26][27][28][29][30][31][32]. Protonation occurs on one or two available nitrogen atoms, resulting in the positively charged monoacid form or the dicationic diacid form. At ambient temperature [24,26,28,31] mono-and diprotonated forms are easily formed upon addition of a small amount of different kinds of commonly used acids. At low temperatures only the diprotonated forms of tetrapyrrolic compounds are observed [24]. The interest in the investigation of the protonated forms of porphyrins is attributed to the fact that the diprotonated species are considered as useful model compounds for studying the features of nonplanar distortion of tetrapyrrolic macrocycles and their molecular flexibility [30,[33][34][35].
X-ray crystallography studies revealed that the diacids generally undergo saddle-type distortions, although other geometrical shapes can also be produced [15].
The first fluorescence fine-line spectra of porphyrin (Н2P) diacids (Н4P 2+ ) have been obtained in a solid solution of trifluoroacetic acid matrix at 4.2 K [36] using fluorescence line narrowing (FLN) technique [37], but the data have not been interpreted. Several years ago the fineline spectrum of Н4P 2+ in inorganic tetraethoxysilane matrix at 4.2 K was detected [38]; these results reveal essential differences compared with the previous report [36] and the interpretation of the spectral data is also absent. Remeasured FLN spectra of Н4P 2+ have been recorded recently [39] and, on the basis of experimental data and theoretical treatment (DFT calculations) the manifestation of out-of-plane vibrations in the FLN spectra has been demonstrated. The relationship between the activity of out-of-plane modes and the geometry of the chromophore was discussed.
The RRS technique has also been used for investigating of structural issues in the acidic forms of porphyrin derivatives [40][41][42]. The photochemical formation of di-and monoacids of octaethylporphyrin in several solvents has been realized in [40] and the RRS data of the acidic forms (with the excitation wavelength of 441.6 nm) were recorded in the spectral range of 1100-1650 cm -1 . Later on, the Raman and IR spectra of H4OEP 2+ and its N-deuterated analogue (D4OEP 2+ ) were measured [41], but in the spectral range from 500 to 1700 cm -1 the recorded Raman spectrum was of very low quality. The set of free base tetraphenylporphyrin, psulfonatophenyl, and meso-phenyl-substituted porphyrins and their N-diprotonated derivatives have been studied by Raman and IR methods. The observed vibrational bands were assigned on the basis of the deuteration shifts and compared with those observed in the structural analogues of these compounds [42,43].
We have decided to study the RRS of the diacid form of free-base porphyrin in the solid state in a range from 100 to 1650 cm -1 , in order to compare the results of the experiment with the data obtained from FLN spectra and with the results of DFT calculations for the same compound.
The interest in the investigation was motivated by the possibility to examine in detail the relation between saddle conformation of the acidic form and activation of out-plane modes in the vibrational spectra.
In this work, the acidic form of porphyrin has been with the help of RRS in a crystal powder and KBr pellets coupled with theoretical modelling in order to elucidate spectral manifestation in Raman spectra of structural changes characteristic for the diacid form. In particular, the influence of the acidic form formation on the vibrational modes and the activity of out-of-plane and in-plane modes in the Raman spectra have been examined in detail.

EXPERIMENTAL Chemicals
Porphyrin (H2P) was synthesized according to well known procedures [44,45]. For purification and identification of the structure, standard procedures were used. The structural formulae of H2P and the diacid form are shown in Fig.1. Concentrated trifluoroacetic acid, HCl and methanol were used to produce the acidic form. These chemicals were purchased from Sigma-Aldrich Co. and used without further purification.

Preparation of the diacid form
The acidic form H4P 2+ was prepared using a methodology somewhat modified with respect to a previous reference [28]. MeOH was added to 100 mg of free-base porphyrin in 20 ml of concentrated trifluoroacetic or HCl acid until the free-base dissolved completely. The solution was concentrated to a small volume and deep purple crystals were obtained upon standing at room temperature, washed with diethyl ether and dried in air. The presence of the diacid form was confirmed by the appearance of the characteristic lines in IR absorption spectrum [46].

Raman setup
Renishaw InVia microscopic system was used for the measurements of Raman spectra. Ar + 514.5 nm (Stellar Pro Modu-Laser, LLC) laser line was used as the excitation source. The laser light was focused on a sample with a 100x microscope objective; the laser power at the sample was less than 5 mW to prevent the transformation of the diacid into the free-base porphyrin due to heating of the sample. The Raman-scattered light was collected by the same objective through a cut-off filter to block Rayleigh scattering. A grating of 1800 grooves/mm was used. The resolution was 5 cm -1 , with the wavenumber accuracy of 2 cm -1 , both calibrated with the Rayleigh line and the 520.6 cm -1 line of silicon. The Raman-scattered light was recorded by a 1024x256 pixel Peltiercooled RenCam CCD detector.

Theoretical treatment
Geometry optimizations, vibrational frequencies, and Raman activity calculations were performed using density functional theory (DFT). Becke three-parameter functional [47] with the Lee-Yang-Parr correlation functional (+) [48] was the model of choice. The basis set was cc-PVTZ, as implemented in Gaussian 09 [49]. In order to convert the calculated Raman activities into quantities comparable with the experimental results, they were multiplied by a factor of ((x -) 4 /(-exp(-hck))) [50], where x is the wavenumber corresponding to the laser wavelength used to record the spectra and equal to 19436.3 cm -1 for the 514.5 nm laser line, c is the speed of light, h and k are Planck's and Boltzmann's constants, T is the temperature,  (in cm -1 ) is the calculated transition frequency (after applying the scaling factor).

Raman spectra of H2P and H4P 2+
In order to explain the influence of diprotonation on spectral properties, the experimental data and the results of quantum-chemical calculations of H4P 2+ were compared with the data for H2P. Fig. 2 exhibits the RRS spectra of H2P and H4P 2+ obtained for a crystal powder. The vibrational frequencies of H2P and H4P 2+ observed in the Raman spectra are summarized in Table   1. The frequencies and the symmetry of the normal modes of H2P and H4P 2+ were previously assigned on the basis of quantum-chemical calculations and the results of fluorescence line narrowing (FLN) experiments [39] ( Table 1). The relatively high symmetry of H2P and H4P 2+ results in the similar set of vibrations observed in the FLN spectra [39] as well as in the RRS spectra. The comprehensive discussion of the experimental data and the results of quantum-chemical calculations of the normal modes for the studied compounds starts from the comparison of the Raman data for Н2P and Н4P 2+ molecules.  The formation of porphyrin diacid with four hydrogen atoms in the center of the porphyrin macrocycle leads to significant changes in the Raman spectra of Н2P (see Fig. 2 and the data in  [39].
According to the experimental results presented in Table 1  In the same spectral range of 100  1000 cm -1 one observes in the Raman spectrum of Н4P 2+ the lines corresponding to in-plane modes (111, 311, 357, 419, 717, 785, 806, 953, 997 cm -1 ). All these lines have analogues in the Raman spectrum of Н2P (see Fig. 2 and Table 1).
Evidently, the main reason of spectral manifestation and activation of the oop modes in the Raman and fluorescence spectra of the diacid of porphyrin (spectral range below 1000 cm -1 ) is the result of the saddle type distortion of the porphyrin macrocycle [15,36,39]. The distortion of the diacid macrocycle induces alterations in the ring kinematics as well as in the electronic structure of the absorption spectra (see, for example, [24,26,31]). As a result of distortion of the porphyrin macrocycle the oop vibrational modes become active in the resonance Raman spectra [51][52][53][54].
These modes are forbidden in the resonance Raman spectra of planar porphyrins because the resonant π-π* electronic transitions are polarized in the plane of the porphyrin macrocycle. The saddle distortion provides a nonzero z-component of the electronic transition moment, thereby inducing the RR intensity of the out-of-plane modes. Similar effects have been earlier investigated systematically for nickel porphyrins which are distorted to accommodate the short Ni-N (pyrrole) bonds and the experimental intensity pattern was predicted quite well [55,56].
In other words, the distortion of the saddle type should lead to realizing of 3D character of the absorption oscillator [39]. In this case, all electronic transition moments in Н4P 2+ can have three components (x, y, and z), contrary to the situation with only in-plane x and y in-plane components for the planar structure of Н2P. The 3D oscillator of the electronic (vibronic) transitions may be expressed as the superposition of the planar oscillator (x and y) and out-of-plane z oscillator with the orientation of the corresponding transition moment components.
As evidently follows from Fig. 2