Enhancement of the Photosensitizing Properties of 6-Carboxypterin through Covalent Binding to the pH-Responsive and Biocompatible Poly(allylamine Hydrochloride).

A polymeric photosensitizer was synthesized through covalent attachment of the natural photosensitizer 6-carboxypterin (Cap) to a poly(allylamine hydrochloride) (PAH) polymer. The optimization of the functionalization steps and purification procedure is described. The overall yield of the functionalization reaction was 67% to generate the modified polymer (PAH-Cap), featuring a Cap substitution degree of approximately 1% and advantageous spectroscopic properties. Photosensitizing properties of PAH-Cap were observed to occur via both photooxidation mechanisms, i.e., type I and type II. This feature was demonstrated using a biologically relevant target molecule, 2'-deoxyguanosine (dG). The spectroscopic, photophysical, and photochemical behaviors in aqueous environments were studied and compared to Cap. To explore possible further relevant biological applications, experiments with PAH-Cap and dG were carried out at physiological pH. PAH-Cap can generate singlet molecular oxygen and initiate an electron transfer process at pH 7 in air-saturated solutions upon UVA irradiation. Moreover, based on its spectroscopic features, visible light can be used to initiate the photooxidation of biological compounds in water, with many interesting advantages compared to free Cap and other related pteridines. These advantages include an enhancement of the photosensitizing effect at physiological pH and the potential of PAH-Cap for its use as a building block in supramolecular assemblies. The functionalization strategy hereby described can be employed for the preparation of robust photoactive polymers with great potential for its application in photodynamic therapy (PDT) and disinfection technologies.

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.

Practical Aspects in the Study of Biological Photosensitization Including Reaction Mechanisms and Product Analyses: A Do's and Don'ts Guide†.

The interaction of light with natural matter leads to a plethora of photosensitized reactions. These reactions cause the degradation of biomolecules, such as DNA, lipids, proteins, being therefore detrimental to the living organisms, or they can also be beneficial by allowing the treatment of several diseases by photomedicine. Based on the molecular mechanistic understanding of the photosensitization reactions, we propose to classify them in four processes: oxygen-dependent (type I and type II processes) and oxygen-independent [triplet-triplet energy transfer (TTET) and photoadduct formation]. In here, these processes are discussed by considering a wide variety of approaches including time-resolved and steady-state techniques, together with solvent, quencher, and scavenger effects. The main aim of this survey is to provide a description of general techniques and approaches that can be used to investigate photosensitization reactions of biomolecules together with basic recommendations on good practices. Illustration of the suitability of these approaches is provided by the measurement of key biomarkers of singlet oxygen and one-electron oxidation reactions in both isolated and cellular DNA. Our work is an educational review that is mostly addressed to students and beginners.

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.

Pterin-lysine photoadduct: a potential candidate for photoallergy.

Photoallergy is a photosensitivity disorder associated with a modified ability of the skin to react to the combined effect of drugs and sunlight. It has been attributed to the covalent conjugation of proteins with a photosensitizer, yielding modified macromolecules that can act as antigen provoking the immune system response. The potential role of some endogenous compounds as photoallergens has not been fully established. It has been previously proposed that pterins, which are endogenous photosensitizers present in human skin under pathological conditions, are able to covalently bind to proteins. Here, we evaluated the capability of pterin (Ptr) to form photoadducts with free Lysine (Lys) and poly-L-lysine (poly-Lys). The findings obtained using chromatographic and spectroscopic tools, confirm the formation of photoadducts of Ptr with Lys residues. With poly-Lys the resulting adduct retains the spectroscopic properties of the photosensitizer, suggesting that the aromatic Ptr structure is conserved. On the other hand, the photoproduct formed with free Lys does not behave like Ptr, which suggests that if this product is a photoadduct, a chemical modification may have occurred during the photochemical reaction that alters the pterin moiety.

Photosensitized Dimerization of Tyrosine: The Oxygen Paradox.

In electron-transfer initiated photosensitization processes, molecular oxygen (O2 ) is not involved in the first bimolecular event, but almost always participates in subsequent steps giving rise to oxygenated products. An exception to this general behavior is the photosensitized dimerization of tyrosine (Tyr), where O2 does not participate as a reactant in any step of the pathway yielding Tyr dimers (Tyr2 ). In the pterin (Ptr) photosensitized oxidation of Tyr, O2 does not directly participate in the formation of Tyr2 and quenches the triplet excited state of Ptr, the reactive species that initiates the process. However, O2 is necessary for the dimerization, phenomenon that we have named as the oxygen paradox. Here, we review the literature on the photosensitized formation of Tyr2 and present results of steady-state and time resolved experiments, in search of a mechanistic model to explain the contradictory role of O2 in this photochemical reaction system.

Pterin-photosensitization of thymine under anaerobic conditions in the presence of guanine.

Pterin (Ptr) is a model photosensitizer that acts mainly through type I mechanism and is able to photoinduce the one-electron oxidation of purine and pyrimidine nucleobases. However, under anaerobic conditions Ptr reacts with thymine (T) to form photoadducts (Ptr-T) but does not lead to the photodegradation of guanine (G), which is the nucleobase with the lowest ionization potential. Accordingly, G is thermodynamically able to reduce the radicals of the other nucleobases and has been described in this sense as the "hole sink" of the DNA double helix. Here we analyze by steady-state and time-resolved studies the effect of G in the anaerobic photosensitization of T by Ptr, using nucleotides and oligonucleotides of different sequences. We demonstrated that G is able to reduce T radicals but does not prevent the formation of Ptr-T adducts. Our results suggest that after the encounter between the excited Ptr and T, and completion of the electron transfer step, part of the radicals escape from the solvent cage, to further react with other species. However, a proportion of radicals do not escape and evolve to photoadducts before separation. We provide new evidence that contributes to understand the photosensitizing properties of Ptr in the absence of O2, the mechanism of formation of photoadducts in the DNA and the protective role of G towards the photodamage in other nucleobases.

Photosensitization Reactions of Biomolecules: Definition, Targets and Mechanisms.

Photosensitization reactions have been demonstrated to be largely responsible for the deleterious biological effects of UV and visible radiation, as well as for the curative actions of photomedicine. A large number of endogenous and exogenous photosensitizers, biological targets and mechanisms have been reported in the past few decades. Evolving from the original definitions of the type I and type II photosensitized oxidations, we now provide physicochemical frameworks, classifications and key examples of these mechanisms in order to organize, interpret and understand the vast information available in the literature and the new reports, which are in vigorous growth. This review surveys in an extended manner all identified photosensitization mechanisms of the major biomolecule groups such as nucleic acids, proteins, lipids bridging the gap with the subsequent biological processes. Also described are the effects of photosensitization in cells in which UVA and UVB irradiation triggers enzyme activation with the subsequent delayed generation of superoxide anion radical and nitric oxide. Definitions of photosensitized reactions are identified in biomolecules with key insights into cells and tissues.

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.

Type I Photosensitized Oxidation of Methionine†.

Methionine (Met) is an essential sulfur-containing amino acid, sensitive to oxidation. The oxidation of Met can occur by numerous pathways, including enzymatic modifications and oxidative stress, being able to cause relevant alterations in protein functionality. Under UV radiation, Met may be oxidized by direct absorption (below 250 nm) or by photosensitized reactions. Herein, kinetics of the reaction and identification of products during photosensitized oxidation were analyzed to elucidate the mechanism for the degradation of Met under UV-A irradiation using pterins, pterin (Ptr) and 6-methylpterin (Mep), as sensitizers. The process begins with an electron transfer from Met to the triplet-excited state of the photosensitizer (Ptr or Mep), to yield the corresponding pair of radicals, Met radical cation (Met•+ ) and the radical anion of the sensitizer (Sens•- ). In air-equilibrated solutions, Met•+ incorporates one or two atoms of oxygen to yield methionine sulfoxide (MetO) and methionine sulfone (MetO2 ), whereas Sens•- reacts with O2 to recover the photosensitizer and generate superoxide anion (O2 •- ). In anaerobic conditions, further free-radical reactions lead to the formation of the corresponding dihydropterin derivatives (H2 Ptr or H2 Mep).

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.

Role of Tryptophan Residues in the Toxicity and Photosensitized Inactivation of Escherichia coli α-Hemolysin.

α-Hemolysin (HlyA) is an extracellular protein toxin secreted by uropathogenic strains of Escherichia coli that inserts into membranes of eukaryotic cells. The main goal of this work was to investigate the involvement of tryptophan (W) residues in the hemolytic activity of HlyA. We investigated the hemolytic activity of six single-point mutant proteins, in which one of the four Ws was replaced by cysteine (C) or leucine (L). We also analyzed the photoinactivation of HlyA with pterin (Ptr), an endogenous photosensitizer, as a method of unspecific oxidation of W and tyrosine (Y) residues. HlyA photoinactivation was analyzed by ultraviolet-visible spectrophotometry, hemolytic activity measurement, fluorescence spectroscopy, and electrophoretic analysis. The results indicate that Ws are important in the hemolytic process. Specifically, the chemical structure of the amino acid at position 578 is important for the acylation of HlyA at residue K563. Furthermore, the exposure of HlyA to ultraviolet radiation, with energy similar to that experienced under sun exposure, in the presence of Ptr induces the inactivation of the toxin, causing chemical changes in, at least, W and Y, the rate of damage to W residues being faster than that observed for Y residues. This work not only deepens our understanding of the structure-function relationship of the toxin but also introduces the possibility of using photoinactivation of HlyA for potential applications such as obtaining innocuous molecules for vaccine production and the elimination of the toxin from contaminated surfaces and drinking water.

Synergistic effect of carboxypterin and methylene blue applied to antimicrobial photodynamic therapy against mature biofilm of Klebsiella pneumoniae.

The control of multidrug-resistant (MDR) bacteria is a growing public health problem, and new strategies are urgently needed for the control of the infections caused by these microorganisms. Notoriously, some MDR microorganisms generate complex structures or biofilms, which adhere to surfaces and confer extraordinary resistance properties that are fundamental challenges to control infections. One of the promising strategies for the control of MDR bacteria is antimicrobial photodynamic therapy (aPDT), which takes advantage of suitable photosensitizers (PS), oxygen and radiation to eradicate microorganisms by the generation of highly reactive species, including reactive oxygen species (ROS) that cause cytotoxic damage and cell death. Habitual aPDT treatments use only methylene blue (MB), but MDR microorganism eradication is not completely achieved. The key result of this study revealed that a combination of two known PSs, 6-carboxypterin (Cap, 100 µM) and MB (2.5-10 µM) exposed to ultraviolet and visible radiation, presents a synergistic effect on the eradication of a MDR Klebsiella pneumoniae strain. Similar effect was observed when the treatment was performed either with planktonic or biofilm growing cells. Moreover, it was found that after treatment the killing action continues in the absence of irradiation leading to the eradication of the microorganisms growing in biofilm. Therefore, the combined aPDT represents a promising strategy for the management of clinical contact surfaces, disinfection of surgical instruments, biofouling and even antimicrobial wastewater treatment.

Chemical Modifications of Globular Proteins Phototriggered by an Endogenous Photosensitizer.

The main goal of the present work was to investigate the damages photoinduced by pterin (Ptr), an endogenous photosensitizer present in human skin under pathological conditions, on a globular protein such as ubiquitin (Ub). Particular attention has been paid on the formation of covalent adducts between Ptr and the protein that can behave as photoantigen and provoke an immune system response. Here, a multifaceted approach including UV-visible spectrophotometry, fluorescence spectroscopy, electrophoresis, size exclusion chromatography, and mass spectrometry is used to establish the Ub changes triggered by UV-A irradiation in the presence of Ptr. Under anaerobic conditions, the only reaction corresponds to the formation of a covalently bound Ptr-Ub adduct that retains the spectroscopic properties of the free photosensitizer. A more complex scheme is observed in air-equilibrated solutions with the occurrence of three different processes, that is, formation of a Ptr-Ub adduct, dimerization, and fragmentation of the protein.

Evidence of the effectiveness of Resveratrol in the prevention of guanine one-electron oxidation: possible benefits in cancer prevention.

Over the past few years, the interest in Resveratrol (3,4',5,-trihydroxystilbene, RSV) has increased due to the evidence found of its antioxidant action that protects biomolecules and cells from oxidative damage. The interest has been further exacerbated by the natural presence of RSV in some fruits and derivatives, especially in red wine. In this paper we present evidence of RSV capacity in protecting a deoxynucleotide, an essential constituent of DNA, from one-electron oxidation. This article evaluates the mechanism responsible for the antioxidant action of RSV, after one-electron oxidation of 2'-deoxyguanosine 5'-monophosphate (dGMP), by kinetic analysis during steady-state irradiation and laser flash photolysis experiments. Results showed that RSV protects dGMP by recovering the nucleotide from its radical, which is formed after the reaction of dGMP with the triplet excited state of the photosensitizer. In the absence of RSV, dGMP is irremediably oxidized, and if the damage occurs in dGMP located in DNA molecules, the consequences can be as serious as mutations and subsequent carcinogenic lesions.

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.