Synthesis and Characterization of Water-Soluble Star-Porphyrin Macromolecules and Their Interaction with Bovine Serum Albumin

The æ›°1 porphyrin compounds have unique macrocyclic conjugated structures, physicochemical properties and physiological functions, and have received increasing attention in recent years. In order to improve the solubility and reduce the aggregation of porphyrins, people connected porphyrin molecules to water-soluble polymers to obtain water-soluble porphyrin polymers. Heme, hematoporphyrin IX and protoporphyrin are linked to polyethylene glycol through ester bonds and amide bonds, and the resulting water-soluble porphyrin macromolecules have peroxidase activity, photosensitizing activity, and heme oxidase inhibitor action.

The polyethylene glycol derivative of hematoporphyrin is complexed with an active platinum compound to obtain an anticancer active substance having both cytotoxicity and phototoxicity. Protoporphyrin IX is copolymerized with other ethylenic monomers through double bonds and the resulting polymer has been used in biomimetic contact lines. There are many star-shaped water-soluble porphyrin polymers derived from the derivatization of tetraphenylporphyrins, which can be applied in the fields of biological enzyme simulation and oxygen-carrying drugs. Polyethylene glycol is covalently bonded to porphyrin via an ether bond, and the resulting polymer can be used as a catalyst for epoxidation, cyclopropanation, and aziridination of olefins, and can also be used as a catalyst for the recycling of photooxidation. Preparation of water-soluble porphyrin macromolecules from tetraphenylporphyrin derivatives includes polymerization method and functional group reaction method. Tetraphenylporphyrin derivative matrix is ​​mainly composed of four pairs of hydroxyphenyl porphyrin and four pairs of chlorophenyl groups. Porphyrins and four pairs of chloromethylphenylporphyrins.

After porphyrin compounds enter the body of humans and animals, they can bind to serum albumin, and their aggregation in vivo, as well as distribution, metabolism and other physiological processes in the body are affected by serum albumin. The interaction between small molecule porphyrin compounds and serum albumin has been studied. However, after the water-soluble porphyrin macromolecule enters the body, not only the interaction between porphyrin and serum albumin exists, but also the interaction between the macromolecular segment and serum albumin may be present. See the report. In order to investigate the biomedical properties of porphyrin macromolecules, it is necessary to understand the interaction between porphyrin macromolecules and serum albumin. In this study, tetra-b-aminophenyl) porphyrins were used as raw materials to prepare water-soluble star porphyrin polymers containing hydrophilic polymer chains. The structures of the water-soluble porphyrin macromolecules were characterized by IR, UV-Vis, 4 NMR and elemental analysis. The interaction with bovine serum albumin (BSA) was further studied.

2 Experimental 2.1 Instruments and reagents Polyethylene glycol monomethyl ether (MPEG, = 5000) was purchased from SIGMA. Bovine serum albumin (BSA) was provided by Institute of Hematology, Chinese Academy of Medical Sciences. N,N-dicyclohexylcarbodiimide was purchased from China Pharmaceutical (Group) Shanghai Chemical Reagent Company. N,N-dimethylaminopyridine was purchased from Tianjin Chemical Reagent Sixth Factory. Pyrrole was re-distilled before use, and other reagents and solvents were analytically pure domestic reagents. Bio-RadFTS135 Fourier infrared spectrometer, Varian UNITY Plus-400 U-570 UV-Vis spectrometer.

2.2 Preparation of terminal carboxyl tetraphenylporphyrin derivative TA4C Tetrakis(P-aminophenyl)porphyrin (P-TAPP) was synthesized according to the method and purified by column chromatography.

Under nitrogen protection, 0.4043 g (0.5991 mmol) of tetrakis(p-aminophenyl)porphyrin (p-TAPP) was dissolved in 5 ml of pyridine and 1.0226 g (10.22 mmol) of succinic anhydride was added. The oil bath was heated to 70° C. to 75° C. with stirring and reacted for 3 h. The reaction mixture was rotary evaporated at 60° C. to remove some of the solvent. After dilution with water, the mixture was slowly neutralized with 1 mol/L hydrochloric acid to keep the pH at 7.0. The precipitate was separated by centrifugation and the precipitate was washed three times with water. The resulting precipitate was dissolved in ethanol, further purified by silica gel column, and dried under vacuum to obtain tetrakis(p-phenyl)porphyrintetraamido succinic acid (TA4C) 0.3628 g, yield 2.3 The water-soluble star-shaped porphyrin macromolecule TA4G Preparation of TA4C (0.0829 g, 0.07711 mmol) was dissolved in 5 ml pyridine, 1.5469 g (0.3094 mmol) of polyethylene glycol monomethyl ether (Mw=5000) and 4A molecular sieves (0.5 g) were added. After the mixture was stirred for 3 h, 250 mg of N,N-dicyclohexylcarbodiimide (DCC) was added. The reaction mixture was stirred at a temperature of 25C for 10 days. After diluting with chloroform (100 ml), the insoluble by-product N,N-dicyclohexylurea (DCU) was removed by filtration. The filtrate was subjected to rotary evaporation to remove the solvent, and the residue was dissolved in water and centrifuged to remove insoluble matter. The supernatant was dialyzed against a dialysis bag with a molecular weight cut off of 14,000, protected from light, and re-evaporated with water every 6 hours. Rotary evaporation, drying to obtain the product TA4G0.2265g, yield 13.9/UV/Vis.0, H2OX427nm, 523nm, 561nm, 598nm, 653nm. Calculated from the integrated area of ​​the characteristic absorption peak of nuclear magnetic resonance spectra, TA4G number average molecular weight of 22000. 2.4 Preparation of water-soluble star-shaped porphyrin macromolecule zinc complex TA4G was dissolved in chloroform, saturated zinc acetate solution was added, and the mixture was stirred overnight. The excess ligand was removed by dialysis to obtain a spectral titration of the complex 2.5TA4G and its complex. A certain amount of TA4G or its complex was precisely weighed and dissolved in redistilled water. Quantitatively remove the solution and add it to 8 to 12 25 ml volumetric flasks. Each volumetric flask was respectively added with different volumes of BSA solution prepared with phosphate buffer (pH 7.4), then phosphate buffer solution and 0.01 mol/L NaCl solution were added to make the ionic strength of each solution the same. Protect from light and let stand overnight. The scanning absorption spectra of each solution were tested at 25C.

3 Results Discussion 3.1 Synthesis of TA4G The porphyrin TA4C obtained from the modification of tetra(P-aminophenyl)porphyrin with succinic anhydride has not been reported. The compound is soluble in 4% NaOH aqueous solution and some organic solvents such as tetrahydrofuran, acetone, pyridine, DMF, DMSO, etc., and is insoluble in solvents such as chloroform, dichloromethane, benzene, toluene, ethyl ether, and ethanol. Under mild conditions, DCC is used as a dehydrating agent to esterify carboxyl groups on TA4C molecules with hydroxy groups on MPEG molecules. TA4G.TA4G can be dissolved in water and some organic solvents such as benzene, chloroform, tetrahydrofuran and acetone. Wait. Since the four carboxyl groups in the TA4C molecule are incompletely reacted with hydroxyl groups in the MPEG molecule, the yield of the obtained target product is relatively low after the non-reacted substances and the incompletely reacted products are removed by dialysis. Under the experimental conditions, the obtained TA4G was tested by SEC with chloroform, tetrahydrofuran and water as the mobile phase. The obtained spectra were all single-peak, indicating that the product was quite pure and there was no unreacted MPEG and TA4C residues.

It has been reported that the correct molecular weight of star polymers cannot be obtained by GPC. The exact number average molecular weight can be calculated by calculating the integral area of ​​the corresponding proton absorption peak in the nuclear magnetic spectrum. The terminal methoxy (-OCH3) proton peak of the MPEG chain in the TA4G NMR spectrum appears at 51.8-2.2, the ethylene (-OCH2CH2-) proton peak at 53.3-3.6, and the phenyl group of the porphyrin unit. The peaks appear at 57.6 and 58.2, with the porphyrin ring and H's proton peak at 58.8.

By calculating the integral area of ​​the PH and ethylene proton peaks, the number average molecular weight of TA4G was found to be 22,000, which means that each porphyrin molecule has attached 4 PEG chains. Two Q absorption bands appear in the UV-visible absorption spectrum of Zn-TA4G, indicating that the zinc ion has undergone a coordination reaction with the porphyrin ring and a zinc complex has been formed.

3.2 Determination of Spectroscopic Properties of TA4G and Its Zinc Complex The spectral absorption of aqueous TA4G solution was titrated with BSA and the spectrum is shown in .

It can be seen that the Soret band shows a significant increase in color, but there is no change in the wavelength of the maximum absorption peak. The characteristics of TA4G absorption spectra showed that the degree of TA4G molecule association was reduced during the titration of BSA, while the porphyrin's environmental polarity did not change significantly. Takuzo Aida et al. believe that water-soluble porphyrin polymers form face-to-face aggregates in aqueous solution. Similarly, TA4G molecules also form face-to-face aggregates in aqueous solutions. After BSA solution was added, the porphyrins interacted with BSA and destroyed some of the porphyrin aggregates. The presence of porphyrins changed, so the Soret band showed a hyperchromic effect. In addition, there is no change in the Soret absorption peak position, indicating that the polarity of the porphyrin microenvironment has not changed substantially. Under the experimental conditions, although there was no special interaction between the polyethylene glycol polymer sidearm and bovine serum albumin, there was a mutual volumetric repulsion between the protein and the polymer chain segments. In addition, the polyethylene glycol segments adsorb to the non-polar surface in the aqueous electrolyte while rejecting the hydrophilic substance. The overall result is that the binding between TA4G and BSA is hindered and the polar environment around the porphyrin ring does not change.

In order to further study the effect of macromolecular porphyrin-complexed metal ions on the interaction between porphyrin and BSA, titration experiments of Zn-TA4G and BSA were performed. The spectrum obtained is shown in . Obviously, the combination of porphyrin macromolecules after complexing zinc ions with BSA also resulted in the increase of Soret band, and the chromogenic effect of Zn-TA4G reached 20.2%, which was significantly higher than that of TA4G (6.26%). It is generally believed that in addition to the hydrophobic interaction between the metalloporphyrin and BSA, there are also metal ions and BSA amino acid residues forming coordinate bonds. The experimental results show that due to the binding between zinc ions and histidine residues in BSA after zinc ion complexed with porphyrin ring, the binding ability of polymer porphyrin and BSA is enhanced. The experiment also found that the increase of polymer concentration will reduce the binding of porphyrin and BSA, because the volume of the side arm of the polymer interferes with the access of the BSA molecule to the porphyrin core. At the same time, the polymer concentration increases and the viscosity of the system increases. Affects the diffusion of molecules in solution.

The binding of macromolecule porphyrins to BSA is similar to that of small molecule porphyrins, but the binding behavior is obviously different. After the porphyrin ring enters metal ions, the coordination between metal and BSA enhances the interaction between porphyrin and BSA. Between the forces. Polymeric side chains have a very important influence on the interaction between porphyrin and BSA. The volume effect of the hydrophilic side chains often hinders the binding of BSA to the porphyrin ring, the microenvironment of the porphyrin ring changes little, and there is no change in the maximum absorption wavelength in the visible region.