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.

Singlet Oxygen Flux, Associated Lipid Photooxidation, and Membrane Expansion Dynamics Visualized on Giant Unilamellar Vesicles.

The physical properties of lipid membranes depend on their lipid composition. Photosensitized singlet oxygen (1O2) provides a handle to spatiotemporally control the generation of lipid hydroperoxides via the ene reaction, enabling fundamental studies on membrane dynamics in response to chemical composition changes. Critical to relating the physical properties of the lipid membrane to hydroperoxide formation is the availability of a sensitive reporter to quantify the arrival of 1O2. Here, we show that a fluorogenic α-tocopherol analogue, H4BPMHC, undergoes a >360-fold emission intensity enhancement in liposomes following a reaction with 1O2. Rapid quenching of 1O2 by the probe (kq = 4.9 × 108 M-1 s-1) ensures zero-order kinetics of probe consumption. The remarkable intensity enhancement of H4BPMHC upon 1O2 trapping, its linear temporal behavior, and its protective role in outcompeting membrane damage provide a sensitive and reliable method to quantify the 1O2 flux on lipid membranes. Armed with this probe, fluorescence microscopy studies were devised to enable (i) monitoring the flux of photosensitized 1O2 into giant unilamellar vesicles (GUVs), (ii) establishing the onset of the ene reaction with the double bonds of monounsaturated lipids, and (iii) visualizing the ensuing collective membrane expansion dynamics associated with molecular changes in the lipid structure upon hydroperoxide formation. A correlation was observed between the time for antioxidant H4BPMHC consumption by 1O2 and the onset of membrane fluctuations and surface expansion. Together, our imaging studies with H4BPMHC in GUVs provide a methodology to explore the intimate relationship between photosensitizer activity, chemical insult, membrane morphology, and its collective dynamics.

Synthesis, Characterization and Photocleavage of Bis-decyl Pteroic Acid: A Folate Derivative with Affinity to Biomembranes†.

Here, we provide mechanistic insight to the photocleavage of a compound in the folate family, namely pteroic acid. A bis-decyl chain derivative of pteroic acid was synthesized, structurally characterized and photochemically investigated. We showed that, like folic acid, pteroic acid and the decylated derivative undergo a photocleavage reaction in the presence of H2 O, while no reaction was observed in methanol solution. Furthermore, density functional theory calculations were carried out to predict relative stabilities of hypothetical mono-, bis- and tris-decylated pteroic acid derivatives to help rationalize the regioselectivity of the bis-decyl pteroic acid product. Additionally, the lipophilicity of the bis-decyl pteroic acid appears to confer a hydrophobic property enabling an interaction with biomembranes.

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.

Photochemistry of tyrosine dimer: when an oxidative lesion of proteins is able to photoinduce further damage.

The tyrosine dimer (Tyr2), a covalent bond between two tyrosines (Tyr), is one of the most important modifications of the oxidative damage of proteins. This compound is increasingly used as a marker of aging, stress and pathogenesis. At physiological pH, Tyr2 is able to absorb radiation at wavelengths significantly present in the solar radiation and artificial sources of light. As a result, when Tyr2 is formed in vivo, a new chromophore appears in the proteins. Despite the biomedical importance of Tyr2, the information of its photochemical properties is limited due to the drawbacks of its synthesis. Therefore, in this work we demonstrate that at physiological pH, Tyr2 undergoes oxidation upon UV excitation yielding different products which conserve the dimeric structure. During its photodegradation different reactive oxygen species, like hydrogen peroxide, superoxide anion and singlet oxygen, are produced. Otherwise, we demonstrated that Tyr2 is able to sensitize the photodegradation of tyrosine. The results presented in this work confirm that Tyr2 can act as a potential photosensitizer, contributing to the harmful effects of UV-A radiation on biological systems.

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.

S,S-Chiral Linker Induced U Shape with a Syn-facial Sensitizer and Photocleavable Ethene Group.

There is a major need for light-activated materials for the release of sensitizers and drugs. Considering the success of chiral columns for the separation of enantiomer drugs, we synthesized an S,S-chiral linker system covalently attached to silica with a sensitizer ethene near the silica surface. First, the silica surface was modified to be aromatic rich, by replacing 70% of the surface groups with (3-phenoxypropyl)silane. We then synthesized a 3-component conjugate [chlorin sensitizer, S,S-chiral cyclohexane and ethene building blocks] in 5 steps with a 13% yield, and covalently bound the conjugate to the (3-phenoxypropyl)silane-coated silica surface. We hypothesized that the chiral linker would increase exposure of the ethene site for enhanced 1 O2 -based sensitizer release. However, the chiral linker caused the sensitizer conjugate to adopt a U shape due to favored 1,2-diaxial substituent orientation; resulting in a reduced efficiency of surface loading. Further accentuating the U shape was π-π stacking between the (3-phenoxypropyl)silane and sensitizer. Semiempirical calculations and singlet oxygen luminescence data provided deeper insight into the sensitizer's orientation and release. This study has lead to insight on modifications of surfaces for drug photorelease and can help lead to the development of miniaturized photodynamic devices.

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.

Lipophilic Decyl Chain-Pterin Conjugates with Sensitizer Properties.

A new series of decyl chain [-(CH2)9CH3] pterin conjugates have been investigated by photochemical and photophysical methods, and with theoretical solubility calculations. To synthesize the pterins, a nucleophilic substitution (SN2) reaction was used for the regioselective coupling of the alkyl chain to the O site over the N3 site. However, the O-alkylated pterin converts to N3-alkylated pterin under basic conditions, pointing to a kinetic product in the former and a thermodynamic product in the latter. Two additional adducts were also obtained from an N-amine condensation of DMF solvent molecule as byproducts. In comparison to the natural product pterin, the alkyl chain pterins possess reduced fluorescence quantum yields (ΦF) and increased singlet oxygen quantum yields (ΦΔ). It is shown that the DMF-condensed pterins were more photostable compared to the N3- and O-alkylated pterins bearing a free amine group. The alkyl chain pterins efficiently intercalate in large unilamellar vesicles, which is a good indicator of their potential use as photosensitizers in biomembranes. Our study serves as a starting point where the synthesis can be expanded to produce a wider series of lipophilic, photooxidatively active pterins.

Type I and Type II Photosensitized Oxidation Reactions: Guidelines and Mechanistic Pathways.

Here, 10 guidelines are presented for a standardized definition of type I and type II photosensitized oxidation reactions. Because of varied notions of reactions mediated by photosensitizers, a checklist of recommendations is provided for their definitions. Type I and type II photoreactions are oxygen-dependent and involve unstable species such as the initial formation of radical cation or neutral radicals from the substrates and/or singlet oxygen (1 O21 ∆g ) by energy transfer to molecular oxygen. In addition, superoxide anion radical (O2·-) can be generated by a charge-transfer reaction involving O2 or more likely indirectly as the result of O2 -mediated oxidation of the radical anion of type I photosensitizers. In subsequent reactions, O2·- may add and/or reduce a few highly oxidizing radicals that arise from the deprotonation of the radical cations of key biological targets. O2·- can also undergo dismutation into H2 O2 , the precursor of the highly reactive hydroxyl radical (·OH) that may induce delayed oxidation reactions in cells. In the second part, several examples of type I and type II photosensitized oxidation reactions are provided to illustrate the complexity and the diversity of the degradation pathways of mostly relevant biomolecules upon one-electron oxidation and singlet oxygen reactions.

Degradation of tyrosine and tryptophan residues of peptides by type I photosensitized oxidation.

Pterin derivatives are involved in various biological functions, including enzymatic processes that take place in human skin. Unconjugated oxidized pterins are efficient photosensitizers under UV-A irradiation and accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder. These compounds are able to photoinduce the oxidation of the peptide α-melanocyte-stimulating hormone (α-MSH), which stimulates the production and release of melanin by melanocytes in skin and hair. In the present work we have used two peptides in which the amino acid sequence of α-MSH was mutated to specifically investigate the reactivity of tryptophan (Trp) and tyrosine residues (Tyr). The parent compound of oxidized pterins (Ptr) was used as a model photosensitizer in aqueous solution at pH5.5 and was exposed to UV-A radiation, a wavelength range where the peptides do not absorb. Trp residue yields N-formylkynurenine and hydroxytryptophan as oxidized products, whereas the Tyr undergoes dimerization and incorporation of oxygen atoms. In both cases, the first step of the mechanism involves an electron transfer from the amino acid to the photosensitizer triplet excited state, Ptr is not consumed and hydrogen peroxide (H2O2) is released. The role of singlet oxygen produced by energy transfer from 3Ptr⁎ to dissolved O2 was negligible or minor. Other amino acid residues, such as histidine, might be also affected.

Thymidine radical formation via one-electron transfer oxidation photoinduced by pterin: Mechanism and products characterization.

UV-A radiation (320-400nm), recognized as a class I carcinogen, induces damage to the DNA molecule and its components through different mechanisms. Pterin derivatives are involved in various biological functions, including enzymatic processes, and it has been demonstrated that oxidized pterins may act as photosensitizers. In particular, they accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder. We have investigated the ability of pterin (Ptr), the parent compound of oxidized pterins, to photosensitize the degradation of the pyrimidine nucleotide thymidine 5'-monophosphate (dTMP) in aqueous solutions under UV-A irradiation. Although thymine is less reactive than purine nucleobases, our results showed that Ptr is able to photoinduce the degradation of dTMP and that the process is initiated by an electron transfer from the nucleotide to the triplet excited state of Ptr. In the presence of molecular oxygen, the photochemical process leads to the oxidation of dTMP, whereas Ptr is not consumed. In the absence of oxygen, both compounds are consumed to yield a product in which the pterin moiety is covalently linked to the thymine. This compound retains some of the spectroscopic properties of Ptr, such as absorbance in the UV-A region and fluorescence properties.

Soybean phosphatidylcholine liposomes as model membranes to study lipid peroxidation photoinduced by pterin.

Oxidized pterins, efficient photosensitizers under UVA irradiation, accumulate in the skin of patients suffering from vitiligo, a chronic depigmentation disorder. Soybean phosphatidylcholine (SoyPC) liposomes were employed as model membranes to investigate if pterin (Ptr), the parent compound of oxidized pterins, is able to photoinduced lipid peroxidation. Size exclusion chromatography and dialysis experiments showed that Ptr is not encapsulated inside the liposomes and the lipid membrane is permeable to this compound. The formation of conjugated dienes and trienes, upon UVA irradiation, was followed by absorption at 234 and 270 nm, respectively. The photoproducts were characterized by mass spectrometry and oxygenation of SoyPC was demonstrated. In addition, analysis of MS/MS spectra suggested the formation hydroperoxides. Finally, the biological implications of the findings are discussed.

LL37 peptide@silver nanoparticles: combining the best of the two worlds for skin infection control.

Capping silver nanoparticles with LL37 peptide eradicates the antiproliferative effect of silver on primary skin cells, but retains the bactericidal properties of silver nanoparticles with activities comparable to silver nitrate or silver sulfadiazine. In addition, LL37 capped silver nanoparticles have anti-biofilm formation activity.

Intra- and extra-cellular DNA damage by harmine and 9-methyl-harmine.

It is known that ß-carbolines are able to produce photosensitized damage in cell-free DNA, but there is little information on their effects on cellular DNA. Therefore, we have analyzed the DNA damage produced by harmine and 9-methyl-harmine under UVA irradiation in V79 cells, together with the associated generation of micronuclei and photocytotoxicity. The results indicate that the most frequent photoproducts generated in the cellular DNA are modified purines such as 8-oxo-7,8-dihydroguanine. Only relatively few single-strand breaks were observed. CPDs were absent, although they were generated in cell-free DNA irradiated under the same conditions. The overall extent of DNA damage in the cells was considerably smaller than the one observed in cell free DNA. The generation of cellular DNA damage was associated with a significant generation of micronuclei and decreased cell proliferation. The data indicate that ß-carbolines act as photosensitizers in mammalian cells. The spectrum of DNA modification, and therefore the mechanism of DNA damage generation, differs considerably from that observed with cell-free DNA.

Mechanisms of DNA damage by photoexcited 9-methyl-ß-carbolines.

It has been well documented that ß-carboline alkaloids, particularly the 9-methyl derivatives, are efficient photosensitizers. However, structure-activity relationships are missing and the photochemical mechanisms involved in the DNA photodamage still remain unknown. In the present work, we examined the capability of three 9-methyl-ß-carbolines (9-methyl-norharmane, 9-methyl-harmane and 9-methyl-harmine) to induce DNA damage upon UVA excitation at physiological pH. The type and extent of the damage was analyzed together with the photophysical and binding properties of the ß-carboline derivatives investigated. The results indicate that even at neutral pH most of the DNA damage is generated from the protonated form of the excited ß-carbolines in a type-I reaction. Oxidized purine residues are produced in high excess over oxidized pyrimidines, single-strand breaks and sites of base loss. In addition, the excited neutral form of the ß-carbolines is responsible for significant generation of cyclobutane pyrimidine dimers (CPDs) by triplet-triplet-energy transfer. In the case of 9-methyl-norharmane, the yield of CPDs is increased in D2O, probably due to less rapid protonation in the deuterated solvent.

Characterization and reactivity of photodimers of dihydroneopterin and dihydrobiopterin.

7,8-Dihydrobiopterin (H(2)Bip) and 7,8-dihydroneopterin (H(2)Nep) belong to a class of heterocyclic compounds present in a wide range of living systems. H(2)Bip accumulates in the skin of patients suffering from vitiligo, whereas H(2)Nep is secreted by human macrophages when the cellular immune system is activated. We have investigated the photochemical reactivity of both compounds upon UV-A irradiation (320-400 nm), the chemical structures of the products and their thermal stability. The study was performed in neutral aqueous solutions. The reactions were followed by UV/Visible spectrophotometry and HPLC and the products were analyzed by means of electrospray ionization mass spectrometry and (1)H-NMR. Excitation of H(2)Bip and H(2)Nep leads to the formation, in each case, of two main isomeric dimers. The latter compounds undergo a thermal process that may consist in a retro [2 + 2]-cycloaddition and hydrolysis to yield the reactant (H(2)Bip or H(2)Nep) and a product that has incorporated a molecule of H(2)O.

Photosensitization of DNA by ß-carbolines: kinetic analysis and photoproduct characterization.

ß-Carbolines (ßCs) are a group of alkaloids present in many plants and animals. It has been suggested that these alkaloids participate in a variety of significant photosensitized processes. Despite their well-established natural occurrence, the main biological role of these alkaloids and the mechanisms involved are, to date, poorly understood. In the present work, we examined the capability of three important ßCs (norharmane, harmane and harmine) and two of its derivatives (N-methyl-norharmane and N-methyl-harmane) to induce DNA damage upon UV-A excitation, correlating the type and extent of the damage with the photophysical characteristics and DNA binding properties of the compounds. The results indicate that DNA damage is mostly mediated by a direct type-I photoreaction of the protonated ßCs after non-intercalative electrostatic binding. Reactive oxygen species such as singlet oxygen and superoxide are not involved to a major extent, as indicated by the only small influence of D(2)O and of superoxide dismutase on damage generation. An analysis with repair enzymes revealed that oxidative purine modifications such as 8-oxo-7,8-dihydroguanine, sites of base loss and single-strand breaks (SSB) are generated by all ßCs, while only photoexcited harmine gives rise to the formation of cyclobutane pyrimidine dimers as well.