**4. The equilibrium of porphyrins**

92 Macro to Nano Spectroscopy

absorption is between 380-500 nm depending on whether the porphyrin is - or *meso*substituted. The second region consists of a weak transition to the first excited state (S0 S1) in the range between 500-750 nm (the Q bands). These favourable spectroscopic features of porphyrins are due to the conjugation of 18 - electrons and provide the advantage of easy and precise monitoring of guest-binding processes by UV-visible spectroscopic methods (Yang et al. 2002; Gulino et al., 2005; Di Natale et al. 2000; Paolesse & D'Amico, 2007) CD, (Scolaro et al. 2004; Balaz et al. , 2005) fluorescence, (Zhang et al., 2004; Zhou et al.,

Fig. 7. Porphyrin HOMOs and LUMOs. (A) Representation of the four Gouterman orbitals in porphyrins. (B) Drawing of the energy levels of the four Gouterman orbitals upon symmetry lowering from *D*4*h* to *C*2V. The set of eg orbitals gives rise to Q and B bands.

The relative intensity of Q bands is due to the kind and the position of substituents on the macrocycle ring. Basing on this latter consideration, porphyrins could be classified as *etio*,

When the relative intensities of Q bands are such that IV > III > II > I, the spectrum is said *etio-type* and porphyrins called *etioporphyrins*. This kind of spectrum is found in all porphyrins in which six or more of the -positions are substituted with groups without electrons, *e.g.*, alkyl groups. Substituent with -electrons, as carbonyl or vinyl groups, attached directly to the -positions gave a change in the relative intensities of the Q bands, such that III > IV > II > I. This is called *rhodo-type* spectrum (*rhodoporphyrin*) because these groups have a "reddening" effect on the spectrum by shifting it to longer wavelengths. However, when these groups are on opposite pyrrole units, the reddening is intensified to give an *oxo-rhodo-type* spectrum in which III > II > IV > I. On the other hand, when *meso*positions are occupied, the *phyllo-type* spectrum is obtained, in which the intensity of Q

While variations of the peripheral substituents on the porphyrin ring often cause minor changes to the intensity and wavelength of the absorption features, protonation of two of the inner nitrogen atoms or the insertion/change of metal atoms into the macrocycle usually

2006) and NMR spectroscopy (Shundo et al., 2009; Tong et al., 1999).

*rhodo*, *oxo-rhodo* e *phyllo* (Prins et al 2001).

bands is IV > II > III > I (Milgron 1997).

strongly change the visible absorption spectrum.

Neglecting the overall charge of the macrocycle, a monomeric free-base porphyrin H2-P in aqueous solution can add protons to produce mono H3-P+ and dications H4-P2+ at very low pHs, or loose protons to form the centrally monoprotic H-P- at pH about 6 or aprotic P2 species at pH ≥ 10 (Fig. 8). These chemical forms of porphyrin may exist in equilibrium, depending upon the pH of the solution and can be characterized from the change of the electronic absorption spectrum. The change in spectra upon addition of acid or basic substances can generally be attributed to the attachment or the loss of protons to the two imino nitrogen atoms of the pyrrolenine-like ring in the free-base (Gouterman, 1979; Giovannetti et al, 2010). The N-protonation induced a red-shifts that are consistent with frontier molecular orbital calculations for protonated porphyrins (Daniel et al., 1996).

Fig. 8. Typical Uv-vis specrtum of dianion P2- (pH about 10) monoprotic H-P- (pH about 6) and dication H4-P2+ porphyrin (pH about 1).

Spectrophotometric titration was employed for determining the acid dissociation constants over the inter pH range and change in absorbance with pH can be attributed to the following acid dissociation reactions of porphyrins. Upon addition of acid the spectral pattern of porphyrins changes from the four Q-band spectrum, indicating *D*2h symmetry for free-base porphine, to a two Q-band spectrum for the formation of dications H4-P2+ (Fig. 8 c), indicating *D*4h symmetry, characteristic of porphyrin coordinated to a metal ion through the

The Use of Spectrophotometry UV-Vis for the Study of Porphyrins 95

metalloporphyrins that play key roles in several biochemical processes, due to their central role in photosynthesis, oxygen transport and in various redox reactions (Mathews et al., 2000; Garret & Grisham, 1999; Knör & Strasser, 2005; Lim et al., 2005; Martirosyan et al.,

Depending on their size, charge, and spin multiplicity, metal ions (*e.g.* Zn, Cu, Ni, Co, *etc.*) can fit into the center of the planar tetrapyrrolic ring system forming *regular* 

When divalent metal ions (e.g. Co(II), Ni(II), Cu(II)) are chelated, the resulting tetracoordinate chelate has no residual charge. While Cu(II) and Ni(II) in their porphyrin complexes have generally low affinity for additional ligands, the chelates with Mg(II), Cd(II) and Zn(II) readily combine with one more ligand to form pentacoordinated complexes with square-pyramidal structure (Fig. 11a). Some metalloporphyrins (Fe(II), Co(II), Mn(II)) are able to form distorted octahedral (Fig. 11b) with two extra ligand molecules (Biesaga et al.,

Most of the natural metalloporphyrins are of regular type, i.e. their metal centres are located within the plane of the macrocyclic ligand as a consequence of their fitting size. The cationic radii are in the range of 55–80 pm corresponding to the sphere in the porphyrin core surrounded by the four pyrrolic nitrogens. While the symmetry group of the free-base porphyrins is D2h due to the two hydrogen atoms on the diagonally located pyrrolic nitrogens, the coplanar (regular) metalloporphyrins (without these protons) are of higher

Fig. 11. Schematic pictures of square-pyramidal (a) and octahedral structures (b) (only

2004; Tovmasyan et al., 2008; Ren et al., 2010; Kawamura et al., 2011).

Fig. 10. Schematic representation of (a) regular and (b) SAT metalloporphyrins.

*metalloporphyrins* resulting in a kinetically inert complexes (Fig. 10a).

2000).

symmetry (Khan & Bruice, 2003).

enclose nitrogen N, metal M and extra ligands L).

four N-heteronuclei. In addition, in all cases, the intense Soret band is red-shifted (to an extent dependent on the particular meso-substituents).

### **5. The reaction of porphyrins with metal ions: Regular and sitting-atop complexes**

The metalloporphyrin formation reaction is one of the important processes from both analytical and bioinorganic points of view. The large molar absorption coefficient and the very high stability of porphyrins is valuable for the separation of various kinds of metal ions (Tabata et al.,1998). A variety of metalloporphyrin formation rates are also applicable for the kinetic analysis of metal ions (Tabata & Tanaka, 1991). Also, kinetic studies of metalloporphyrin formation are indispensable in order to understand in vivo metal incorporation processes leading to the natural metalloporphyrins. Generally porphyrins are synthesized in a metal-free form and metal ions are successively inserted.

When the metal ion Mn+ is incorporated into the porphyrin H2P to form MP(n-2)+, the two amine protons in H2P are dissociated from the two pyrrole groups as reported in equation (1):

$$M^{n+} + H\_2P \leftrightarrow MP^{(n-2)+} + 2H^+ \tag{1}$$

In the formation of metalloporphyrins an marked colour changes with trasformation of the Uv-Vis spectrum especially in the Q zone has been observed. The two Q band obtained are called and (Fig. 9). The relative intensities of these bands can be correlated with the stability of the metal complex; in fact when > , the metal forms a stable square-planar complex with the porphyrin, in the other case when > (e.g. Ni(II), Pd(II), Cd(II)), the metals are easily displaced by protons (Milgron, 1997).

Studies on water soluble and insoluble porphyrins have elucidated aspects of the mechanisms of metal ion incorporation into porphyrins to form metalloporphyrins (Bailey & Hambright, 2003; Hambright et al., 2001; Lavallee, 1987; Funahashi et al., 2001).

Fig. 9. Q band in the porphyrin metal complexes

The size of the porphyrin-macrocycle is perfectly suited to bind almost all metal ions and indeed a large number of metals can be inserted in the center of the macrocycle forming

four N-heteronuclei. In addition, in all cases, the intense Soret band is red-shifted (to an

The metalloporphyrin formation reaction is one of the important processes from both analytical and bioinorganic points of view. The large molar absorption coefficient and the very high stability of porphyrins is valuable for the separation of various kinds of metal ions (Tabata et al.,1998). A variety of metalloporphyrin formation rates are also applicable for the kinetic analysis of metal ions (Tabata & Tanaka, 1991). Also, kinetic studies of metalloporphyrin formation are indispensable in order to understand in vivo metal incorporation processes leading to the natural metalloporphyrins. Generally porphyrins are

When the metal ion Mn+ is incorporated into the porphyrin H2P to form MP(n-2)+, the two amine protons in H2P are dissociated from the two pyrrole groups as reported in equation

In the formation of metalloporphyrins an marked colour changes with trasformation of the Uv-Vis spectrum especially in the Q zone has been observed. The two Q band obtained are called and (Fig. 9). The relative intensities of these bands can be correlated with the stability of the metal complex; in fact when > , the metal forms a stable square-planar complex with the porphyrin, in the other case when > (e.g. Ni(II), Pd(II), Cd(II)), the

Studies on water soluble and insoluble porphyrins have elucidated aspects of the mechanisms of metal ion incorporation into porphyrins to form metalloporphyrins (Bailey &

The size of the porphyrin-macrocycle is perfectly suited to bind almost all metal ions and indeed a large number of metals can be inserted in the center of the macrocycle forming

Hambright, 2003; Hambright et al., 2001; Lavallee, 1987; Funahashi et al., 2001).

( 2)

<sup>2</sup> <sup>2</sup> *<sup>M</sup><sup>n</sup> <sup>n</sup> H P MP H* (1)

**5. The reaction of porphyrins with metal ions: Regular and sitting-atop** 

synthesized in a metal-free form and metal ions are successively inserted.

extent dependent on the particular meso-substituents).

metals are easily displaced by protons (Milgron, 1997).

Fig. 9. Q band in the porphyrin metal complexes

**complexes** 

(1):

metalloporphyrins that play key roles in several biochemical processes, due to their central role in photosynthesis, oxygen transport and in various redox reactions (Mathews et al., 2000; Garret & Grisham, 1999; Knör & Strasser, 2005; Lim et al., 2005; Martirosyan et al., 2004; Tovmasyan et al., 2008; Ren et al., 2010; Kawamura et al., 2011).

Fig. 10. Schematic representation of (a) regular and (b) SAT metalloporphyrins.

Depending on their size, charge, and spin multiplicity, metal ions (*e.g.* Zn, Cu, Ni, Co, *etc.*) can fit into the center of the planar tetrapyrrolic ring system forming *regular metalloporphyrins* resulting in a kinetically inert complexes (Fig. 10a).

When divalent metal ions (e.g. Co(II), Ni(II), Cu(II)) are chelated, the resulting tetracoordinate chelate has no residual charge. While Cu(II) and Ni(II) in their porphyrin complexes have generally low affinity for additional ligands, the chelates with Mg(II), Cd(II) and Zn(II) readily combine with one more ligand to form pentacoordinated complexes with square-pyramidal structure (Fig. 11a). Some metalloporphyrins (Fe(II), Co(II), Mn(II)) are able to form distorted octahedral (Fig. 11b) with two extra ligand molecules (Biesaga et al., 2000).

Most of the natural metalloporphyrins are of regular type, i.e. their metal centres are located within the plane of the macrocyclic ligand as a consequence of their fitting size. The cationic radii are in the range of 55–80 pm corresponding to the sphere in the porphyrin core surrounded by the four pyrrolic nitrogens. While the symmetry group of the free-base porphyrins is D2h due to the two hydrogen atoms on the diagonally located pyrrolic nitrogens, the coplanar (regular) metalloporphyrins (without these protons) are of higher symmetry (Khan & Bruice, 2003).

Fig. 11. Schematic pictures of square-pyramidal (a) and octahedral structures (b) (only enclose nitrogen N, metal M and extra ligands L).

The Use of Spectrophotometry UV-Vis for the Study of Porphyrins 97

The photoinduced behavior of normal metalloporphyrins have been thoroughly studied for several decades, while the investigation of SAT complexes started in this respect only in the past 8–10 years (Horváth et al., 2004; Valicsek et al., 2008; Valicsek et al., 2009; Valicsek et al.,

Interestingly, in the case of lanthanide ions as metal centers, triple decker porphyrin

While the natural porphyrin derivatives are exclusively hydrophobic, some artificial porphyrins having ionic substituents made it possible to prepare water-soluble metalloporphyrins of both regular and SAT type. Kinetically labile complexes are mostly

In the case of metalloporphyrins, however, metal ions are applied generally in excess, especially for spectrophotometric measurements, partly because of the extremely high molar absorbances (mainly at the Soret-bands) of the porphyrins. The formation of kinetically labile SAT complexes, deviating from the regular metalloporphyrins, is an equilibrium process. It can be spectrophotometrically monitored because the absorption and emission bands assigned to ligand-centered electron transitions undergo significant shift and

Special attention was devoted to the reaction of porphyrins with essential metal ions as manganese, iron and chromium show that the most important properties of manganese in complex biological systems is the highly variable oxidation states of the metal from +2 to +5 (Kadish et al. 1999). All these compounds can be easily spectrophotometrically distinguished among them; this is because they have different absorption spectra (Spasojevic & Batinic-Haberle, 2001, ) from which is possible to know the oxidation state. Manganese–porphyrin complexes have more extensively studied because were found to be similar to the biologically active compounds (Nakanishi et al. 2000; Meunier, 1992; Perie & Barbe, 1996; Balahura & Kirby, 1994; Haber et al., 2000; Cuzzocrea et al., 2001), and because were also used as catalysts for the oxygenation of alkanes, alkenes and compounds containing nitrogen and sulphur (Mansuy & Momenteau, 1982; Fontecave & Mansuy, 1984). The very important properties that influence the reactivity of the Mn(III)-porphyrin concerns the changes in the oxidation states of Mn in the complexes for its high reactivity with O2 (Cuzzocrea et al., 2001). Manganese, in the complexes obtained by the reaction of Mn(II) with the porphyrins, has oxidation number +3, so the complex of Mn(II) can be obtained only by reduction, while those of Mn(IV) and Mn(V) for the oxidation of Mn(III) complexes. Interesting is the reactions of a natural porphyrin, the acid 2,7,12,17 tetrapropionic of 3,8,13,18 tetramethyl-21H, 23H-porphyrin called Coproporphyrin- I (CPI), with manganese (III) that, with different pH and solvent compositions, show the formation of [MnIIICPI(H2O)2], [MnIIICPI(OH)2], [MnIV(O)CPI(OH)], [MnV(O)CPI(OH)], [MnIICPI(OH)] (Fig. 13) with specific Uv-Vis adsorptions as reported in Table 1. (Giovannetti et al., 2010).

Rates of the complexation of porphyrins with metal ions are very much slower by several orders of magnitude than those of acyclic ligands (Funahashi, S. et al., 1984). Such very slow rates have been discussed in terms of the rigidity of the planar porphyrin framework. The electronic nature of porphyrins, and also the steric accessibility of the bound metal center,

sandwich complexes were also synthesized and studied (Wittmer &. Holten, 1996).

2007; Huszánk et al., 2005; Huszánk et al., 2007; Valicsek et al., 2011).

examined in the excess of the ligand.

**5.1 Complexation kinetics** 

intensity change upon coordination of metal ions.

If, however, the ionic radius of the metal ions is too large (over ca. 80-90 pm) to fit into the hole in the centre of the macrocycle, they are located out of the ligand plane, distorting it forming *sitting-atop (SAT) metalloporphyrins* (Fig. 10b) that are characterized by special properties (Fleischer & Wang 1960; Barkigia et al., 1980; Liao et al., 2006; Walker et al., 2010) originating from the non-planar structure caused by, first of all, the size of the metal center.

These complexes are kinetically labile and display characteristic structural and photoinduced properties that strongly deviates from those of the regular metalloporphyrins. The latter kind of structure induces special photophysical and photochemical features that are characteristic for all SAT complexes. The symmetry of this structures is lower (generally C4v–C1) than that of both the free-base porphyrin (D2h) and the regular, coplanar metalloporphyrins (D4h), in which the metal center fits into the ligand cavity.

The rate of formation of in-plane (or normal) metalloporphyrins is much slower than that of the *SAT complexes* because of the inflexibility of porphyrins. In fact, in an SAT complex the distortion of the porphyrin caused by the out-of-plane location of the metal center makes two diagonal pyrrolic nitrogens more accessible on the other side of the ligand due to the increase of their sp3 hybridization (Tung & Chen, 2000).

Deviating from the regular metalloporphyrins, the SAT complexes, on account of their distorted structure and kinetic lability, display peculiar photochemical properties, such as photoinduced charge transfer from the porphyrin ligand to the metal center, leading to irreversible ring opening of the ligand and dissociation on excitation at both the Soret- and the Q-bands (Horváth et al., 2006). Moreover, the absorption and emission characteristics of these complexes are also significantly deviating from those of the normal (in-plane) metalloporphyrins (Horváth et al., 2006). Also the formation of bi and even trinuclear (bisporphyrin) complexes has been observed (Lehn, 2002).

In Figure 12 is shown a schematic Energy-level diagram of the frontier orbital of a porphyrin in free-base state (H2P), in a regular and in a SAT metalloporphyrin.

Fig. 12. Simplified energy-level diagram of the frontier orbital of a porphyrin in free-base state H2P, in a regular and in a SAT metalloporphyrin.

If, however, the ionic radius of the metal ions is too large (over ca. 80-90 pm) to fit into the hole in the centre of the macrocycle, they are located out of the ligand plane, distorting it forming *sitting-atop (SAT) metalloporphyrins* (Fig. 10b) that are characterized by special properties (Fleischer & Wang 1960; Barkigia et al., 1980; Liao et al., 2006; Walker et al., 2010) originating from the non-planar structure caused by, first of all, the size of the metal center. These complexes are kinetically labile and display characteristic structural and photoinduced properties that strongly deviates from those of the regular metalloporphyrins. The latter kind of structure induces special photophysical and photochemical features that are characteristic for all SAT complexes. The symmetry of this structures is lower (generally C4v–C1) than that of both the free-base porphyrin (D2h) and the regular, coplanar

The rate of formation of in-plane (or normal) metalloporphyrins is much slower than that of the *SAT complexes* because of the inflexibility of porphyrins. In fact, in an SAT complex the distortion of the porphyrin caused by the out-of-plane location of the metal center makes two diagonal pyrrolic nitrogens more accessible on the other side of the ligand due to the

Deviating from the regular metalloporphyrins, the SAT complexes, on account of their distorted structure and kinetic lability, display peculiar photochemical properties, such as photoinduced charge transfer from the porphyrin ligand to the metal center, leading to irreversible ring opening of the ligand and dissociation on excitation at both the Soret- and the Q-bands (Horváth et al., 2006). Moreover, the absorption and emission characteristics of these complexes are also significantly deviating from those of the normal (in-plane) metalloporphyrins (Horváth et al., 2006). Also the formation of bi and even trinuclear (bis-

In Figure 12 is shown a schematic Energy-level diagram of the frontier orbital of a porphyrin

MP regular H2P MP SAT

Fig. 12. Simplified energy-level diagram of the frontier orbital of a porphyrin in free-base

HOMO

LUMO

metalloporphyrins (D4h), in which the metal center fits into the ligand cavity.

increase of their sp3 hybridization (Tung & Chen, 2000).

porphyrin) complexes has been observed (Lehn, 2002).

state H2P, in a regular and in a SAT metalloporphyrin.

S

S1

S2

in free-base state (H2P), in a regular and in a SAT metalloporphyrin.

The photoinduced behavior of normal metalloporphyrins have been thoroughly studied for several decades, while the investigation of SAT complexes started in this respect only in the past 8–10 years (Horváth et al., 2004; Valicsek et al., 2008; Valicsek et al., 2009; Valicsek et al., 2007; Huszánk et al., 2005; Huszánk et al., 2007; Valicsek et al., 2011).

Interestingly, in the case of lanthanide ions as metal centers, triple decker porphyrin sandwich complexes were also synthesized and studied (Wittmer &. Holten, 1996).

While the natural porphyrin derivatives are exclusively hydrophobic, some artificial porphyrins having ionic substituents made it possible to prepare water-soluble metalloporphyrins of both regular and SAT type. Kinetically labile complexes are mostly examined in the excess of the ligand.

In the case of metalloporphyrins, however, metal ions are applied generally in excess, especially for spectrophotometric measurements, partly because of the extremely high molar absorbances (mainly at the Soret-bands) of the porphyrins. The formation of kinetically labile SAT complexes, deviating from the regular metalloporphyrins, is an equilibrium process. It can be spectrophotometrically monitored because the absorption and emission bands assigned to ligand-centered electron transitions undergo significant shift and intensity change upon coordination of metal ions.

Special attention was devoted to the reaction of porphyrins with essential metal ions as manganese, iron and chromium show that the most important properties of manganese in complex biological systems is the highly variable oxidation states of the metal from +2 to +5 (Kadish et al. 1999). All these compounds can be easily spectrophotometrically distinguished among them; this is because they have different absorption spectra (Spasojevic & Batinic-Haberle, 2001, ) from which is possible to know the oxidation state. Manganese–porphyrin complexes have more extensively studied because were found to be similar to the biologically active compounds (Nakanishi et al. 2000; Meunier, 1992; Perie & Barbe, 1996; Balahura & Kirby, 1994; Haber et al., 2000; Cuzzocrea et al., 2001), and because were also used as catalysts for the oxygenation of alkanes, alkenes and compounds containing nitrogen and sulphur (Mansuy & Momenteau, 1982; Fontecave & Mansuy, 1984). The very important properties that influence the reactivity of the Mn(III)-porphyrin concerns the changes in the oxidation states of Mn in the complexes for its high reactivity with O2 (Cuzzocrea et al., 2001). Manganese, in the complexes obtained by the reaction of Mn(II) with the porphyrins, has oxidation number +3, so the complex of Mn(II) can be obtained only by reduction, while those of Mn(IV) and Mn(V) for the oxidation of Mn(III) complexes. Interesting is the reactions of a natural porphyrin, the acid 2,7,12,17 tetrapropionic of 3,8,13,18 tetramethyl-21H, 23H-porphyrin called Coproporphyrin- I (CPI), with manganese (III) that, with different pH and solvent compositions, show the formation of [MnIIICPI(H2O)2], [MnIIICPI(OH)2], [MnIV(O)CPI(OH)], [MnV(O)CPI(OH)], [MnIICPI(OH)] (Fig. 13) with specific Uv-Vis adsorptions as reported in Table 1. (Giovannetti et al., 2010).
