Alkylation converts riboflavin into an efficient photosensitizer of phospholipid membranes.

A new decyl chain [-(CH2)9CH3] riboflavin conjugate has been synthesized and investigated. A nucleophilic substitution (SN2) reaction was used for coupling the alkyl chain to riboflavin (Rf), a model natural photosensitizer. As expected, the alkylated compound (decyl-Rf) is significantly more lipophilic than its precursor and efficiently intercalates within phospholipid bilayers, increasing its fluorescence quantum yield. The oxidative damage to lipid membranes photoinduced by decyl-Rf was investigated in large and giant unilamellar vesicles (LUVs and GUVs, respectively) composed of different phospholipids. Using a fluorogenic probe, fast radical formation and singlet oxygen generation was demonstrated upon UVA irradiation in vesicles containing decyl-Rf. Photosensitized formation of conjugated dienes and hydroperoxides, and membrane leakage in LUVs rich in poly-unsaturated fatty acids were also investigated. The overall assessment of the results shows that decyl-Rf is a significantly more efficient photosensitizer of lipids than its unsubstituted precursor and that the association to lipid membranes is key to trigger phospholipid oxidation. Alkylation of hydrophilic photosensitizers as a simple and general synthetic tool to obtain efficient photosensitizers of biomembranes, with potential applications, is discussed.

Antimicrobial-Loaded Polyacrylamide Hydrogels Supported on Titanium as Reservoir for Local Drug Delivery.

Arthroplasty is a highly successful treatment to restore the function of a joint. The contamination of the implant via bacterial adhesion is the first step toward the development of device-associated infections. The emerging concern about antimicrobial resistance resulted in a growing interest to develop alternative therapeutic strategies. Thus, the increment in the incidence of bacterial periprosthetic infections, the complexity of treating infections caused by organisms growing in biofilms, together with the rise in antibiotic resistant bacteria, expose the need to design novel surfaces that provide innovative solutions to these rising problems. The aim of this work is to develop a coating on titanium (Ti) suitable for inhibiting bacterial adhesion and proliferation, and hence, biofilm formation on the surface. We have successfully prepared polyacrylamide hydrogels containing the conventional antibiotic ampicillin (AMP), silver nanoparticles (AgNPs), and both, AMP and AgNPs. The release of the antibacterial agents from the gelled to aqueous media resulted in an excellent antibacterial action of the loaded hydrogels against sessile S. aureus. Moreover, a synergic effect was achieved with the incorporation of both AMP and AgNPs in the hydrogel, which highlights the importance of combining antimicrobial agents having different targets. The polyacrylamide hydrogel coating on the Ti surface was successfully achieved, as it was demonstrated by FTIR, contact angle, and AFM measurements. The modified Ti surfaces having the polyacrylamide hydrogel film containing AgNPs and AMP retained the highest antibacterial effect against S. aureus as it was found for the unsupported hydrogels. The modified surfaces exhibit an excellent cytocompatibility, since healthy, flattened MC3T3-E1 cells spread on the surfaces were observed. In addition, similar macrophage RAW 264.7 adhesion was found on all the surfaces, which could be related to a low macrophage activation. Our results indicate that AMP and AgNP-loaded polyacrylamide hydrogel films on Ti are a good alternative for designing efficient antibacterial surfaces having an excellent cytocompatibility without inducing an exacerbated immune response. The approach emerges as a superior alternative to the widely used direct adsorption of therapeutic agents on surfaces, since the antimicrobial-loaded hydrogel coatings open the possibility of modulating the concentration of the antimicrobial agents to enhance bacterial killing, and then, reducing the risk of infections in implantable materials.

Mono- and Bis-Alkylated Lumazine Sensitizers: Synthetic, Molecular Orbital Theory, Nucleophilic Index and Photochemical Studies.

Mono- and bis-decylated lumazines have been synthesized and characterized. Namely, mono-decyl chain [1-decylpteridine-2,4(1,3H)-dione] 6a and bis-decyl chain [1,3-didecylpteridine-2,4(1,3H)-dione] 7a conjugates were synthesized by nucleophilic substitution (SN 2) reactions of lumazine with 1-iododecane in N,N-dimethylformamide (DMF) solvent. Decyl chain coupling occurred at the N1 site and then the N3 site in a sequential manner, without DMF condensation. Molecular orbital (MO) calculations show a p-orbital at N1 but not N3 , which along with a nucleophilicity parameter (N) analysis predict alkylation at N1 in lumazine. Only after the alkylation at N1 in 6a, does a p-orbital on N3 emerge thereby reacting with a second equivalent of 1-iododecane to reach the dialkylated product 7a. Data from NMR (1 H, 13 C, HSQC, HMBC), HPLC, TLC, UV-vis, fluorescence and density functional theory (DFT) provide evidence for the existence of mono-decyl chain 6a and bis-decyl chain 7a. These results differ to pterin O-alkylations (kinetic control), where N-alkylation of lumazine is preferred and then to dialkylation (thermodynamic control), with an avoidance of DMF solvent condensation. These findings add to the list of alkylation strategies for increasing sensitizer lipophilicity for use in photodynamic therapy.

Immobilization of alkyl-pterin photosensitizer on silicon surfaces through in situ SN2 reaction as suitable approach for photodynamic inactivation of Staphylococcus aureus.

The tuning of surface properties through functionalization is an important field of research with a broad spectrum of applications. Self-assembled monolayers (SAMs) allow the surface tailoring through the adsorption of molecular layers having the appropriate functional group or precursor group enabling in situ chemical reactions and thus to the incorporation of new functionalities. The latter approach is particularly advantageous when the incorporation of huge groups is needed. In this study, we report the immobilization of pterin moieties on 11-bromoundecyltrichlorosilane-modified silicon substrates based on the in situ replacement of the bromine groups by pterin (Ptr), the parent derivative of pterins, by means of a nucleophilic substitution reaction. The modified surface was structurally characterized through a multi-technique approach, including high-resolution XPS analysis, contact angle measurements, and AFM. The designed synthesis method leads to the functionalization of the silicon surface with two compounds, O-undecyl-Ptr and N-undecyl-Ptr, with a higher proportion of the N-derivative (1:8 ratio). The alkyl-pterins immobilized via the proposed strategy, retain their photochemical properties, being able to inhibit Staphylococcus aureus growth under irradiation (84.3 ± 15.6 % reduction in viable cells). Our results open the possibility for the modification of several materials, such as glass and metal, through the formation of SAMs having the proper head group, thus allowing the design of photosensitive surfaces with potential microbiological self-cleaning properties.

Mechanistic Insight into the Photodynamic Effect Mediated by Neutral Red and a New Azine Compound in Staphylococcus aureus Cells.

The photodynamic activity of Neutral Red and the new monobrominated Neutral Red was studied in suspensions of Staphylococcus aureus. The effect of mannitol and sodium azide in the presence of 25 µm photosensitizer on lethal photosensitization were investigated. The results of the mechanistic evaluation of Neutral Red showed that both mannitol and sodium azide produced a completed protective effect after irradiation without significant differences between them. The evaluation of monobrominated Neutral Red also showed a protective effect of microorganisms with the addition of mannitol. Although sodium azide produced a protective effect of the photoinactivation, it was incomplete and less than that exhibited by mannitol. The results indicate that the starting reagent, Neutral Red, is a producer of radical species, acting through a type I mechanism, whereas the halogenated derivative of Neutral Red produced reactive oxygen species and a contribution of singlet molecular oxygen cannot be discarded in the photoinactivation of Staphylococcus aureus cells. These results, analyzed together with the previously evaluated properties of the dyes, allow us to explain the differences observed in the photoinactivation of Staphylococcus aureus mediated by both azine photosensitizers.

Alkane Chain-extended Pterin Through a Pendent Carboxylic Acid Acts as Triple Functioning Fluorophore, 1 O2 Sensitizer and Membrane Binder.

In order to develop a new long alkane chain pterin that leaves the pterin core largely unperturbed, we synthesized and photochemically characterized decyl pterin-6-carboxyl ester (CapC) that preserves the pterin amide group. CapC contains a decyl-chain at the carboxylic acid position and a condensed DMF molecule at the N2 position. Occupation of the long alkane chain on the pendent carboxylic acid group retains the acid-base equilibrium of the pterin headgroup due to its somewhat remote location. This new CapC compound has relatively high fluorescence emission and singlet oxygen quantum yields attributed to the lack of through-bond interaction between the long alkane chain and the pterin headgroup. The calculated lipophilicity is higher for CapC compared to parent pterin and pterin-6-carboxylic acid (Cap) and comparable to previously reported O- and N-decyl-pterin derivatives. CapC's binding constant Kb (8000 M-1 in L-α-phosphatidylcholine from egg yolk) and ΦF :Φ∆ ratio (0.26:0.40) point to a unique triple function compound, although the hydrolytic stability of CapC is modest due to its ester conjugation. CapC is capable of the general triple action not only as a membrane intercalator, but also fluorophore and 1 O2 sensitizer, leading to a "self-monitoring" membrane fluorescent probe and a membrane photodamaging agent.

Photo-Oxidation of Unilamellar Vesicles by a Lipophilic Pterin: Deciphering Biomembrane Photodamage.

Pterins are natural products that can photosensitize the oxidation of DNA, proteins, and phospholipids. Recently, a new series of decyl-chain (i.e., lipophilic) pterins were synthesized and their photophysical properties were investigated. These decyl-pterins led to efficient intercalation in large unilamellar vesicles and produced, under UVA irradiation, singlet molecular oxygen, a highly oxidative species that react with polyunsaturated fatty acids (PUFAs) to form hydroperoxides. Here, we demonstrate that the association of 4-(decyloxy)pteridin-2-amine ( O-decyl-Ptr) to lipid membranes is key to its ability to trigger phospholipid oxidation in unilamellar vesicles of phosphatidylcholine rich in PUFAs used as model biomembranes. Our results show that O-decyl-Ptr is at least 1 order of magnitude more efficient photosensitizer of lipids than pterin (Ptr), the unsubstituted derivative of the pterin family, which is more hydrophilic and freely passes across lipid membranes. Lipid peroxidation photosensitized by O-decyl-Ptr was detected by the formation of conjugated dienes and oxidized lipids, such as hydroxy and hydroperoxide derivatives. These primary products undergo a rapid conversion into short-chain secondary products by cleavage of the fatty-acid chains, some of which are due to subsequent photosensitized reactions. As a consequence, a fast increase in membrane permeability is observed. Therefore, lipid oxidation induced by O-decyl-Ptr could promote cell photodamage due to the biomembrane integrity loss, which in turn may trigger cell death.

Kinetic Control in the Regioselective Alkylation of Pterin Sensitizers: A Synthetic, Photochemical, and Theoretical Study.

Alkylation patterns and excited-state properties of pterins were examined both experimentally and theoretically. 2D NMR spectroscopy was used to characterize the pterin derivatives, revealing undoubtedly that the decyl chains were coupled to either the O4 or N3 sites on the pterin. At a temperature of 70°C, the pterin alkylation regioselectively favored the O4 over the N3. The O4 was also favored when using solvents, in which the reactants had increased solubility, namely N,N-dimethylformamide and N,N-dimethylacetamide, rather than solvents in which the reactants had very low solubility (tetrahydrofuran and dichloromethane). Density functional theory (DFT) computed enthalpies correlate to regioselectivity being kinetically driven because the less stable O-isomer forms in higher yield than the more stable N-isomer. Once formed these compounds did not interconvert thermally or undergo a unimolecular "walk" rearrangement. Mechanistic rationale for the factors underlying the regioselective alkylation of pterins is suggested, where kinetic rather than thermodynamic factors are key in the higher yield of the O-isomer. Computations also predicted greater solubility and reduced triplet state energetics thereby improving the properties of the alkylated pterins as 1 O2 sensitizers. Insight on thermal and photostability of the alkylated pterins is also provided.