**3.2 Photosensitized protein damage by phosphorus(V) porphyrin through electron transfer**

Isolated amino acids, a water-soluble protein, and enzymes have been used as the targeting biomacromolecules to examine photosensitizer activity of phosphorus(V) porphyrins [42]. For example, human serum albumin (HSA), a water-soluble protein, is a convenient target. The crystal structure and amino acid sequence of HSA have been clarified [56]. In addition, HSA has major drug specific binding sites identified as Sudlow's site I and site II [57]. The mono-cationic phosphorus(V) porphyrins listed in **Table 1** are well-soluble in organic solvents (*e.g.*, alcohol) rather than water, indicating the hydrophobic character beside the hydrophilicity. Therefore, binding interaction between HSA and phosphorus(V)


*Ered: measured in acetonitrile (vs. saturated calomel electrode; SCE), PBS: 10 mM sodium phosphate buffer (pH 7.6) solution, EtOH: ethanol, PBSETOH2.5: PBS containing 2.5% ethanol, PBSETOH1.25: PBS containing 1.25% ethanol, PBSETOH1.0: PBS containing 1.0% ethanol,* Φ*f: Fluorescence quantum yield. ND: not detected.*

#### **Table 1.**

*Examples of phosphorus(V) porphyrin photosensitizers and their photochemical properties.*

#### *Electron Transfer-Supported Photodynamic Therapy DOI: http://dx.doi.org/10.5772/intechopen.94220*

porphyrins is expected and their binding site can be speculated. Because the electron transfer-mediated oxidation strongly depends on the distance between photosensitizer and the target molecule, a binding interaction is very important. HSA has one tryptophan, which is easily oxidized by oxidative stress, including 1 O2 and electron transfer reaction [42–47, 58]. Tryptophan can emit relatively strong fluorescence and its damage can be detected by fluorescence measurement [45, 58]. Using these characteristics of HSA, the oxidative damage of tryptophan residue by photosensitized reaction can be easily examined by a fluorometry [45–47, 58].

Qualitative study of HSA photodamage by phosphorus(V) porphyrins was reported using **Por2** [43]. **Por2** oxidized the tryptophan of HSA through 1 O2 generation and electron transfer. It has been considered that damaged tryptophan is changed to *N*-formylkynurenine and other decomposed products [59, 60]. 1 O2 can oxidize the tryptophan residue of HSA [61]. Using isolated amino acids, it has been demonstrated that tyrosine and tryptophan can be oxidized by photoexcited **Por2** [42].

Photosensitized HSA damage by **Por5** and **Por6** was quantitatively clarified [45]. **Por5** and **Por6** bound to HSA and damaged its tryptophan residue during photoirradiation. **Por5** and **Por6** photosensitized <sup>1</sup> O2 generation, and the contribution of 1 O2 was confirmed by the inhibitory effect of a 1 O2 quencher, sodium azide (NaN3, [62]). From the kinetic analysis, the contribution of electron transfer mechanism to HSA damage was demonstrated [45]. Fluorescence lifetime measurement and the calculation of Δ*G* supported the electron transfer mechanism.

To realize the effective PDT photosensitizer, response of photosensitizers to long wavelength visible light or near infrared region is important. To improve the abovementioned phosphorus(V) porphyrins, **Por5** and **Por6**, *meso*-phenyl substituted derivatives were designed and synthesized [46]. **Por8**, **Por9**, and **Por12** can be excited under the irradiation of long-wavelength visible light (> 630 nm). These phosphorus(V) porphyrins induced tryptophan oxidation in HSA under illumination with light-emitting diode (central wavelength: 659 nm), and this protein photodamage was barely inhibited by NaN3 [46]. Fluorescence lifetimes of phosphorus(V) porphyrins was decreased by HSA, suggesting the electron transfer quenching. The Δ*G* value of electron transfer from tryptophan to the S1 state of these porphyrins calculated from their redox potentials also supported the electron transfer-mediated oxidation.
