Synthesis and Characterization of a Novel Series of Meso ( Nitrophenyl ) and Meso ( CarboxyPhenyl ) Substituted Porphyrins

Marco A. Schiavon, Lidia S. Iwamoto, Antônio G. Ferreira, Yassuko Iamamoto, Maria V. B. Zanoni and Marilda das D. Assis Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, 14040-901, Ribeirão Preto SP, Brazil. Departamento de Química, Universidade Federal de São Carlos, Rodovia Washington Luis Km 235, 13595-905, São Carlos SP, Brazil. Instituto de Química, Universidade Estadual Paulista, CP 355, 14801-970, Araraquara SP, Brazil


Introduction
The synthesis of porphyrins has gained special attention in recent years because of their importance in bioorganic and bioinorganic chemistry 1 and their applications in biomedical sciences.Porphyrins are used, for example, to mimic the function of hemeproteins such as cytochrome P-450 in oxidation catalysis 2 , as photosensitizers in photodynamic therapy of cancer (PDT) 3 , in electron transport chains 4 and as building blocks in molecular devices 5 .Each research area requires porphyrins with different and specific structural features, bearing a variety of different substituents.
Carboxy substituted porphyrins are attractive synthetic targets for three reasons: firstly, these substituents are present in natural porphyrins such as protoporphyrin IX, the prosthetic group of many important biological molecules like hemoglobin 6 .Secondly, the carboxy group confers an amphyphylic character to the porphyrins, and this is very important, for example, to improve the selectivity in tumour localization in PDT 3 .Lastly, carboxy groups can act as linkers to attach porphyrins to other materials, for example, they can be anchored to solid supports by amidation 7 .
We are interested in meso-para-carboxy and mesoortho-nitro phenyl substituted porphyrins.The carboxy groups when ionised provide the charges necessary to enable their intercalation into lamellar double hydroxides (LDH) 8 or to support them on cationic functionalized silicas through electrostatic binding.They can also be covalently linked to amino-functionalized silica to give heterogeneous porphyrin catalysts 7 .The ortho-nitro groups are important, since they remove electron density from the porphyrin ring and provide steric hindrance improving the stability of the metalloporphyrin catalysts in oxidation reactions 9 .The nitro group can improve the ability of porphyrin systems to act as radiosensitizers 10 and, like carboxy groups, they can act as linkers to attach porphyrins to other materials 7 .
The purpose the present study was to combine the advantages offered by both functional groups into one porphyrin ligand.The corresponding metalloporphyrins will be used as catalysts for hydrocarbon oxidations in homogeneous and supported systems.
In this paper, we report the synthesis and purification of anionic tetraarylporphyrins bearing carboxy-and nitrosubstituents on the phenyl rings (Figure 1), using the method of Gonsalves et al 11 .The characterisation of water-soluble porphyrins is much more difficult than the characterisation of porphyrin derivatives soluble in organic solvents because of their salt-like character.UV/Vis and 1 H NMR spectra are severely complicated by aggregation effects and a quantitative interpretation is rarely possible.The results of elemental analyses are not definitive because the substances are strongly hygroscopic 12 .Therefore, the synthesized porphyrins were characterised by combining thin layer chromato-graphy (TLC), mass spectrometry (FAB MS), 1 H NMR, UV/Vis and IR spectroscopies and electrochemistry and by comparison with the commercial tetra-(para-carboxyphenyl) and tetra-(ortho-nitrophenyl) porphyrins.
UV/Vis spectra were recorded on a Hewlett Packard 8452 Diode Array spectrophotometer.The spectra were recorded in 2 mm path length quartz cells (Hellma).The FTIR spectra were recorded on a FT spectrometer-BOMEN B-100 (4000 -400 cm -1 ) in KBr pellets; the FeP : KBr molar ratio was aproximately 1:2000.FAB mass spectra were recorded on a V.G.Analytical Autospec Instrument, using FAB + mode.4-Nitrobenzyl alcohol was used as the matrix and caesium ion bombardment to generate the ions.The 1 H NMR measurements were recorded on a BRUKER 400.13 MHz, DRX400 spectrometer operating at 303 K using a 5 mm BBO probe with temperature control.The 1 H NMR spectra of 2, 3 and 5 were obtained in CD 2 Cl 2 solution (2,5 x 10 -2 mol dm -3 ) and for 1 and 4 CD 3 OD solutions of the same concentration were used.All spectra were acquired with spectral widths of approximately 16000 Hz and 64 K data points for acquisition using TMS as internal reference.All electrochemical experiments were carried out with a Metrohm Polarecord E-506 coupled to a Metrohm 663 VA Stand which was connected to a computer for data storage and handling.A multimode electrode system was used in both the dropping mercury electrode for differential pulse polarographic measurements and the glassy carbon electrode for cyclic voltammetric measurements.The glassy carbon electrode was polished with alumina before each experiment, rinsed with water, cleaned in a ultrasound bath and dried at room temperature.The three-electrode system was completed with a glassy carbon auxiliary electrode and an Ag/AgCl (3 mol dm -3 KCl) reference electrode.A pulse amplitude of 50 mV was used for differential pulse polarography.Tetra-n-butylammonium tetraphenylborate (TBATFB) was obtained from Eastman Chemicals and was used as received.Porphyrin solutions were 1x10 -4 mol dm -3 , prepared by dissolving the purified substance in CH 2 Cl 2 .

Porphyrins syntheses
The greatest advance in the development of methods for porphyrin synthesis since the classic Rothemund process 14 has been the procedure of Lindsey et al 15 .
The gentle conditions of this synthesis provide means for converting a large range of pre-functionalized benzaldehydes into the corresponding porphyrins.For this reason, the method has been widely employed over recent years.However, Lindsey's method fails when the porphyrin synthesis involves benzaldehydes bearing ionic substituents which are insoluble in the reaction solvents dichloromethane or chloroform, unless the ionic group is masked.In this way, the synthesis of porphyrins bearing carboxy groups often requires the acid groups to be masked as esters.This consequently involves an extra step of group deprotection after the porphyrin has been formed 16 .We decided to use the method of Gonsalves et al 11 , which employs propionic acid and nitrobenzene medium, in the synthesis of the orthonitrophenyl and para-carboxyphenyl substituted porphyrin series.Whereas in Lindsey's method an expensive highly potent quinone is used as the oxidant, in the method of Gonsalves the oxidant is the nitrobenzene/propionic acid solvent.The latter also leads to the precipitation of the porphyrin from the reaction medium, thus facilitating its isolation and purification.
A mixed aldehyde condensation using stoichiometric amounts of ortho-substituted benzaldehyde (A), parasubstituted benzaldehyde (B) and pyrrole affords a mixture of six different porphyrins which can be separated chromatographically 5a .In order to decrease the number of porphyrins obtained in the synthesis and to avoid extremely tedious chromatographic purification processes the system was optimized with small scale reactions using different ratios of the two aldehydes.In this way each of the porphyrins A 3 B, A 2 B 2 (cis and trans) and AB 3 were prepared.
In the synthesis of 1 (AB 3 ) porphyrin, the initial ratio of pyrrole:ortho-nitrobenzaldehyde:para-carboxybenzaldehyde used was 4:0.8:3.2, rather than the stoichiometric 4:1:3 ratio.This synthesis gave a crude yield of 36% (estimated by the absorption of the Soret band in the UV/ Vis spectrum of the reaction mixture) of the two porphyrins 4 and 1 at a 13:1 ratio as determined through the mass of porphyrins after purification.In a second experiment, taking into consideration the higher reactivity of paracarboxybenzaldehyde, the relative amount of orthonitrobenzaldehyde was increased and the reagents ratio used was 4:1.6:2.4.This synthesis led to a high crude yield (60% estimated by UV/Vis) of the mixture of the three porphyrins 1, 2 and 3.In a third experiment, the relative amounts of the two benzaldehydes (4:1.3:2.7) were fitted to obtain a high crude porphyrin yield (60%, estimated by UV/Vis) corresponding to the mixture of 1 and 2. This was the optimized condition used to obtain 1.The final yield of 1 after purification was 5%, based on pyrrole.
To synthesize the (A 3 B) porphyrin 3, we used pyrrole : o-nitrobenzaldehyde : p-carboxybenzaldehyde at a molar ratio of 4 : 3.2 : 0.8.In this synthesis, the porphyrins 5, 3 and 2 were detected and the desired porphyrin 3 was obtained in 5% yield based on the starting pyrrole, after purification.Such yields are good, considering the losses in purification and if compared to other similar preparations involving mixed substituents 3b,3c, 17,18 .
For porphyrins purification, a careful study of the total polarity of the eluent to be employed was necessary.This study was carried out using TLC on silica gel and the ternary solvent mixtures initially used, such as benzene/ methanol/acetic acid, water/acetonitrile/p-dioxane or 2,6lutidine/water/ammonia (gas) described in the literature for natural and synthetic systems containing different numbers of carboxylic groups 19 .However, these water/ solvent mixtures were not effective in the present study.Winkelman's 20 quaternary solvent system, pyridine/ chloroform/water/acetic acid (2:1:1:1) employed for the separation of porphyrins containing different numbers of SO 3 H groups, obtained from the sulfonation of 4, was also ineffective for separating the o-nitro-and p-carboxysubstituted porphyrins.Finally, by using the ternary solvent mixture, chloroform/acetone/acetic acid (8 : 2 : 0,1) proposed by Harada et al 21 for the separation of the meso-mono-(p-carboxyphenyl)tri-phenylporphyrin from the mixture of this porphyrin and meso-tetraphenylporphyrin, good separations were obtained.By substituting chloroform for dichloromethane, we obtained the ideal solvent mixture for the purification of the porphyrins to be studied.Porphyrins 4 and 5 were also analysed in this solvent mixture as references.A difference of 0.10 -0.20 in the Rf values per added carboxy group was observed (see Table I), with the exception of 4, which did not elute from the origin.The fraction containing 2 was probably a mixture of the cis and trans isomers.However it was not possible to separate them under the conditions used.In conclusion the mixed ortho-nitrophenyl and paracarboxyphenyl substituted porphyrins can be obtained directly by the Gonsalves method.Furthermore, by controlling the molar ratio of the benzaldehydes, it is possible to optimize the synthesis and purification of the desired porphyrins.

Infrared spectroscopy
Infrared spectroscopy was very useful for the characterisation of the studied porphyrin series, due to the presence of the NO 2 and COOH substituents, whose absorption bands are defined and easy to assign.
Compounds containing the NO 2 group have strong absorption bands from symmetric and asymmetric deformations (1389 -1259 cm -1 and 1661 -1499 cm -1 , respectively) 22 .The exact position of these bands depends on the substitutions and unsaturations within the NO 2 group.Collman et al 23 assigned the 1542-1526 cm -1 and 1356-1340 cm -1 absorption ranges to the asymmetrical and symmetrical vibrations, respectively, for the NO 2 groups in 5,10,15,20-tetrakis-(o-nitrophenyl)porphyrin), 5. Goldberg et al 24 reported these absorptions at 1500 and 1330 cm -1 , respectively, for the same porphyrin.Table 2 shows the absorption frequencies observed for these groups on the porphyrins synthesized in this study.The values for 4 and 5 are also included for comparison purposes.
Porphyrins containing NO 2 groups as substituents on the meso-phenyl rings (Table 2) show the characteristic absorption bands of this group which agree with Collman's results 23 .The relative absorption intensities decrease in the order 5, 3, 2 and 1, as would be expected upon decreasing the number of NO 2 groups in these porphyrins (Figure 2). a reliable identification of these acids.The absorption near 1700 cm -1 is assigned to C=O stretching vibration.Carboxylic acids are capable of forming hydrogen bonds between the carbonyl and hydroxyl groups of the two molecules and this carbonyl frequency is accordingly reduced 25 .In this study, the porphyrins bearing carboxy substituents show carbonyl frequencies near 1690 cm -1 (Table 2 and Figure 2) indicating aggregation through hydrogen bonds.
The two other absorptions, near 1400 cm -1 (1440 -1395 cm -1 ) and near 1250 cm -1 (1320 -1210 cm -1 ) have been associated by many workers to the single bond C-O stretching vibration 25 .These bands appear in the spectrum of 4 as very intense and large bands centred at 1402 and 1268 cm -1 , which agree with data reported by Datta-Gupta and Bardos for this free-base porphyrin 16 .The intensity of these bands decreases in the order: 4 > 1 > 2 > 3, as expected, due to the decrease in the number of carboxylic acid groups in these porphyrins (Figure 2).
The others bands, not assigned here, are atributed to the vibration of the ring porphyrinic and phenyls group meso-substituents, and are well established in the literature 16,26 .

H NMR spectroscopy
The high frequency region of the 1 H NMR spectra shows the chemical shifts for pyrrole hydrogens (Hβ) and these data were used to characterise the porphyrins.Table 3 shows the chemical shifts and the multiplicities of the signals .
The chemical shifts (δ 8.69 br s) for Hβ hydrogens (close to the nitrophenyl groups) of 5, are at lower frequency than the Hβ hydrogens (close to the carboxyphenyl groups) of 4 (δ 8.88 br s).This effect is caused by the electron withdrawing effect of the ortho-nitro groups on the phenyl ring, which leads to less deprotection by the anisotropy effect on these β-pyrrole hydrogens.
For 1, one signal (multiplet, δ 8.78 -8.83) was observed for Hβ hydrogens between the carboxyphenyl substituents (Hc, Figure 3) and two signals (doublets) for the other Hβ hydrogens.The latter arise from hydrogens Ha, which are close to the nitrophenyl group (δ 8.69); and the former from Hb (δ 8.80) next to the carboxyphenyl group (Figure 3).The chemical shift differences for hydrogens Ha and Hb reflect the difference in the electron current ring, which induces different anisotropic effects on them.However, for hydrogens Hc, we expected to see a singlet and not a multiplet 27 .This is probably because the molecular aggregation leads to a non-equivalency between hydrogens Hc.Unfortunately, the N-H pyrrole hydrogens were not observed for this compound because the NMR  The carboxylic acids are well assigned in FTIR spectroscopy to regions near 1700, 1400 and 1250 cm -1 of the spectrum 22 and the study of all these regions provide spectrum was obtained in methanol d-4 since this porphyrin is insoluble in dichloromethane.Integration of the areas corresponding to Hβ (close to carboxy) and Hβ (close to nitro) hydrogens of 3, 2 and 1 gave the expected 3:1, 2:2 and 1:3 (H β -NO 2 /H β -COOH) ratios, respectively.For 2, 1 and 4 we can also observe a broad hump above the signal corresponding to hydrogens Hβ (close to nitro) and Hβ (close to carboxy), which we again suppose are due to intermolecular aggregation.It is well known that porphyrins bearing carboxylic groups can aggregate through hydrogen bonds and also the polar nitro substituent can act as a proton acceptor in hydrogen bonds 24 .The aggregation was indicated also by infrared spectra, which show broad signals corresponding to the carboxyl group.
The chemical shifts of the N-H hydrogens in the centre of the porphyrin are also affected by the number of nitrophenyl groups.Increasing the number of nitro groups leads to the expected high frequency shift of the N-H signal.In the region of the phenyl hydrogens the multiplet signals overlap makes the assignments difficult.

Electrochemical characterization
The electrochemical reduction of free base and iron ortho -nitro and para-carboxy substituted porphyrins was studied both on a glassy carbon electrode and a mercury electrode by cyclic voltammetry and differential pulse polarography, respectively, in CH 2 Cl 2 ; 0.1 mol dm -3 tetran-butylammonium tetraphenylborate (TBATFB).
The electroreduction of the free bases 5,10,15,20-(tetraphenyl)porphyrin ( 6) and tetrakis(o-nitrophenyl) porphyrin ( 5) and the corresponding iron porphyrins, 6-Fe, 5-Fe, were also investigated in order to understand the influence of substituents on the electrochemical behaviour of these compounds and for comparison purposes.The free base porphyrins 1, 4 and corresponding iron porphyrins were not investigated by this technique due to their insolubility in dichloromethane which was used as solvent in these studies.
The voltammetric reduction of 6 has two characteristic reversible one-electron transfer reactions (Figure 4, Table 4) as judged by wave analysis of ip/ν 1/2 = constant, Epa-Epc = 59 mV and ipa/ipc = 0.9-1.0 for ν (scan rate) = 10-2000 mV s -1 .These reductions correspond to the production of π-anion and di-anion radicals as well established in literature 28 .The cyclic voltammograms for nitro-substituted compounds, 2 (Figure 5), 3 and 5, exhibit two reduction processes at very close potentials (Table 4).Only one anodic peak associated with the reoxidation of product generated in the second reduction step is seen on the reverse scan, but the ipa/ipc ratio is always smaller than one.In addition, both peak potentials show cathodic shifting as a function of scan rate increasing, indicating that nitro-substituted compounds follow a reduction mechanism involving chemical reactions subsequent to the electron transfer 29 .In general, all nitro-substituted free base porphyrins are easier to reduce relative to the parent unsubstituted porphyrin (see Table 4).
A comparison of the electrochemical behaviour with other reported data 30,31 leads us to suppose that the redox reactions of the nitro-porphyrins reported here also involve the porphyrin π-ring system.Considering the predominant effect of the nitro groups on the electrochemical properties, all results were analysed as a function of the number of these groups.In order to confirm these results, differential pulse polarographic experiments were carried out with the aim to improve wave separation.The same trends are observed in the polarographic behaviour of these compounds, as shown in Table 5. Differential pulse polarograms show well defined reduction waves and the corresponding peak potentials indicate that the reductions are easier, increasing the number of nitro groups on the porphyrin ring.Although the electrochemical behaviour in both electrode systems was similar, a negative shifting in the reduction steps was noticed as compared to that obtained with a glassy carbon electrode, indicating that the nature of the electrode material affects the reduction process.A typical cyclic voltammogram obtained for nitro substituted iron(III) porphyrins is shown in Figure 6 and compared to that for 6-Fe obtained under the same conditions (Figure 7).All potential values obtained from voltammetric reduction of the iron porphyrins are shown in Table 4.As expected, the compounds containing the metal centre show two extra peaks at less negative potentials than that observed for reduction of the free base porphyrins.These peaks were assigned to the successive reduction of the iron (III)-iron(II) and iron (II)-iron (I) couples in the porphyrin complex 32,33 .However, the cyclic voltammograms are significantly changed in morphology and potential magnitude as the number of nitro groups is increased in the macrocycle.All investigated FeP undergo a reduction process involving a reversible one electron transfer of the metal reduction strongly depends on the number of nitro substituents in the porphyrin macrocyle.
In conclusion, our findings suggest that the electronwithdrawing NO 2 groups decrease the electron density in both metal centre and conjugated porphyrin π-ring system and this leads to easier reduction.On the other hand, the stability of the product electrochemically generated by the reduction of the Fe(II)-Fe(I) couple is decreased for the nitro-substituted porphyrins investigated in this study.Further work is now in progress in our laboratory in order to clarify the reduction mechanism of these compounds.

Conclusions
Despite great difficulties in working with ionic porphyrins due to aggregation, we have synthesized, isolated and characterised a new series of porphyrins containing mixed substituents, nitro and carboxy, in the meso-phenyl rings, with estimated overall porphyrin yield as high as 60%.
We have demonstrated that it is possible to optimise the benzaldehyde molar ratio in order to obtain fewer porphyrin isomers and higher concentrations of the desired porphyrins, which facilitates the purification process.These porphyrins are important as possible precursors of systems of the selfassembly type and they are potentially good catalysts due to the versatility of the carboxy and nitro groups which can be used to support them in different materials.Further studies are currently under way to explore the catalytic activities of these compounds in oxidation reactions.5).
The metal centre second reduction process in the 6-Fe also shows a characteristic reversible reduction involving one electron transfer.Nevertheless, the three nitro derivatives investigated exhibit cyclic voltammograms with the second reduction peak at less negative potential than that required for reduction of the iron(II)-iron(I) couple in 6-Fe.The difference between the first and second reduction potentials decreases as the number of nitro substituents increases.These data may indicate that the second reduction potential is markedly more sensitive to changes in the electronic nature of the porphyrin macrocycle.The cathodic shift of the peak potential could be interpreted as an increased interaction of the iron(II) with the porphyrin ring.The absence of an anodic peak on the reverse potential scan at a slow scan rate indicates that the second reduction process is complicated by fast chemical reactions which probably consume the generated product, assigned as the iron(I) porphyrin complex, under these conditions.This chemical reaction could be the loss or exchange of the axial ligand 34 or self protonation of the porphyrin 35 .
In addition, the reduction potential of the porphyrin ring system for all iron complexes is shifted up to 690 mV with the introduction of nitro groups, as shown in Tables 4  and 5.As previously observed, the reduction of free base nitro-porphyrins or corresponding iron porphyrins showed the same general trends, indicating that the ring system

Table 2 .
Main infrared absorption frequencies corresponding to the nitro and carboxy groups in the meso-substituted porphyrins.
Figure 2. FTIR spectra of porphyrins with NO 2 and COOH substituents in KBr pellets.Bands marked with * correspond to NO 2 vibration and bands marked with v correspond to the COOH group.

Table 3 .
Chemical shifts (δ in ppm) and coupling constants (J in Hz) for Hβ-pyrrole and N-H hydrogens for the nitro-and carboxy-phenyl substituted porphyrins.

Table 5 .
Substituent effect on the reduction potential of tetraarylporphyrins on a mercury electrode (differential pulse polarography) in CH 2 Cl 2 , 0.1 mol dm -3 TBATFB.