1. Katarzyna Polska, Stanisław Radzki Department of Inorganic Chemistry, Maria Curie-Skłodowska University Pl. M. C. Skłodowskiej 2, 20-031 Lublin, Poland Formation of the Porphyrin-Protein Complexes in Water Solution and Sol-Gel Materials

2. Studies of lectin-porphyrin interactions can be important from the point of view of the influence of lectins on porphyrin-containing biomolecules and the possible application of these conjugates in photodynamic therapy of cancer (PDT). PDT has attracted a great deal of attention in recent years as a new cancer treatment that utilizes porphyrins and metalloporphyrins as sensitizers. Porphyrins preferentially accumulate in tumour cells, when irradiated by light of appropriate wavelenght, they go into the excited state and cause irreparable damage of cancer cells. Concanavalin A, lectin of the jack bean (Canavalia ensiformis), was found in high concentration in growing tissues and have ability to interact preferentially with transformed (tumour) cells. Due to these properties this protein can be considered as a potential carrier for 3 rd generation photosensitizers to tumour tissues. Porphyrins have another potential application, they could be used as the peptide receptors which work in protic solvents. The goal of selective peptide complexation in aqueous solution was approached only recently, and still needs considerable progress until artificial receptors come close to the efficiency of biological systems. PURPOSE

3. Interactions of several free base porphyrins and their corresponding copper(II) complexes with lectin (concanavalin A) have been investigated by spectroscopic techniques. Experiments have been carried out in water solution and in monolithic silica gels. Porphyrin-protein systems immobilized in monolithic silica gels (obtained by polycondensation of tetraethoxysilane using sol-gel technique) have been also examined by atomic force microscopy (AFM). The present work was concerned on two water-soluble cationic porphyrins: tetrakis [4- (trimethylammonio)phenyl] porphyrin (H 2 TTMePP), tetrakis (1-methyl-4-pyridyl) porphyrin (H 2 TMePyP), their complexes with Cu(II) (CuTTMePP, CuTMePyP) and two water-soluble anionic porphyrins: tetrakis (4-carboxyphenyl) porphyrin (H 2 TCPP) and tetrakis (4-sulfonatophenyl) porphyrin (H 2 TPPS).

4. CONCANAVALIN A is a lectin of the jack bean (Canavalia Ensiformis), its conformation depends on pH, beetwen pH 4 and 5 it exists as a dimer and at pH above 7 it is predominantly tetrameric Fig.1. Structure of Concanavalin A protomer (a) and 1:1 H 2 TTMePP-Con A complex (b). a b

5. H 2 TTMePP H 2 TMePyP 5,10,15,20-tetrakis [4-trimethyl ammonio)phenyl] porphyrin 5,10,15,20-tetrakis [4-(1-methyl- 4-pyridyl)] porphyrin CATIONIC PORPHYRINS

6. Mixing of TEOS sol & ConA-H 2 P solution Gel formationAging TEOS sol ConA H 2 P SOL-GEL PREPARATION Con A–H 2 P

7. Con A (C M = 1·10 -4 ) H 2 TTMePP + Con A 1:1 (C M = 10 -4 /10 -4 )

8. H 2 TTMePP (C M = 10 -4 /10 -4 ) TEOS

9. Fig.2. H 2 TTMePP immobilized in monolithic silica gels after 7 days, 1 month and 6 months of drying (concentration = 7.5 x 10 -5 M).

10. SOLUTIONSOL-GEL Fig.3. Absorption and emission spectra of H 2 TTMePP and H 2 TTMePP/Con A systems measured in tris solution (pH 8.7) and in monolithic silica gels.

11. H 2 TTMePP (10 -3 M) H 2 TTMePP + Con A (1:1) H 2 TTMePP + Con A (2:1) H 2 TTMePP + Con A (1:2)Con A (10 -3 M) 1 H, 1 H COSY NMR

12. Both anionic and cationic porphyrins were found to interact with the lectin with comparable affinity, clearly indicating that the charge on the porphyrin does not play any role in the binding process and that most likely the interaction is mediated by hydrophobic forces. Upon binding to concanavalin A an increase in porphyrins fluorescence intensity and a red-shift in absorption and emission maxima have been observed. Each lectin subunit was found to bind one porphyrin molecule. The association constants estimated from absorption titrations for different porphyrins were comparable and were in the range 1 x 10 4 – 7.4 x 10 6 M -1 at room temperature. The UV-Vis titrations were carried out in the solution of TRIS buffer with different values of pH (2.8, 8.7 and 10). The strength of association increases with increasing pH and that observation could be explained by various degree of porphyrin protonation and by the conformation of concanavalin A, also depending on pH. Concanavalin A is a multimeric lectin, consisting of non-covalently associated two (below pH 6) or more (above pH 7) the same subunits. CONCLUSIONS

13. The sol-gel method allows to manufacture amorphous or crystalline materials from liquid phase at low temperatures and physiological pHs. Because of the low temperature growth procedure, dopands, such as fluorescent organic dye molecules, can be introduced in the solution phase of the sol-gel process to obtain optical materials with various interesting properties. Biologically important compounds encapsulated in silica gels have many unique features, as good mechanical durability, high resistance to chemical and biological degradation and, what is the most important, they retain their spectroscopic properties and biological activity. The advantages of biologicals captured in sol-gels might give them applications as biosensors, diagnostic devices and catalysts.