Building a Functionalizable, Potent Chemiluminescent Agent: A Rational Design Study on 6,8-Substituted Luminol Derivatives.

Luminol is a prominent chemiluminescent (CL) agent, finding applications across numerous fields, including forensics, immunoassays, and imaging. Different substitution patterns on the aromatic ring can enhance or decrease its CL efficiency. We herein report a systematic study on the synthesis and photophysics of all possible 6,8-disubstituted luminol derivatives bearing H, Ph, and/or Me substituents. Their CL responses are monitored at three pH values (8, 10, and 12), thus revealing the architecture with the optimum CL efficiency. The most efficient pattern is used for the synthesis of a strongly CL luminol derivative, bearing a functional group for further, straightforward derivatization. This adduct exhibits an unprecedented increase in chemiluminescence efficiency at pH = 12, pH = 10, and especially at pH = 8 (closer to the biologically relevant conditions) compared to luminol. Complementary work on the fluorescence of the emissive species as well as quantum chemistry computations are employed for the rationalization of the observed results.

Triplet stabilization for enhanced drug photorelease from sunscreen-based photocages.

Recently, sunscreen-based drug photocages have been introduced to provide UV protection to photoactive drugs, thus increasing their photosafety. Here, combined experimental and theoretical studies performed on a photocage based on the commercial UVA filter avobenzone (AB) and on the photosensitizing non-steroidal anti-inflammatory drug ketoprofen (KP) are presented unveiling the photophysical processes responsible for the light-triggered release. Particular attention is paid to solvent stabilization of the drug and UV filter excited states, respectively, which leads to a switching between the triplet excited state energies of the AB and KP units. Most notably, we show that the stabilization of the AB triplet excited state in ethanol solution is the key requirement for an efficient photouncaging. By contrast, in apolar solvents, in particular hexane, KP has the lowest triplet excited state, hence acting as an energy acceptor quenching the AB triplet manifold, thus inhibiting the desired photoreaction.

Protein Binding of Lapatinib and Its N- and O-Dealkylated Metabolites Interrogated by Fluorescence, Ultrafast Spectroscopy and Molecular Dynamics Simulations.

Lapatinib (LAP) is an anticancer drug generally used to treat breast and lung cancer. It exhibits hypersensitivity reactions in addition to dermatological adverse effects and photosensitivity. Moreover, LAP binds to serum proteins and is readily biotransformed in humans, giving rise to several metabolites, such as N- and O-dealkylated products (N-LAP and O-LAP, respectively). In this context, the aim of the present work is to obtain key information on drug@protein complexation, the first step involved in a number of hypersensitivity reactions, by a combination of fluorescence, femtosecond transient absorption spectroscopy and molecular dynamics (MD) simulations. Following this approach, the behavior of LAP and its metabolites has been investigated in the presence of serum proteins, such as albumins and α1-acid glycoproteins (SAs and AGs, respectively) from human and bovine origin. Fluorescence results pointed to a higher affinity of LAP and its metabolites to human proteins; the highest one was found for LAP@HSA. This is associated to the coplanar orientation adopted by the furan and quinazoline rings of LAP, which favors emission from long-lived (up to the ns time-scale) locally-excited (LE) states, disfavoring population of intramolecular charge transfer (ICT) states. Moreover, the highly constrained environment provided by subdomain IB of HSA resulted in a frozen conformation of the ligand, contributing to fluorescence enhancement. Computational studies were clearly in line with the experimental observations, providing valuable insight into the nature of the binding sites and the conformational arrangement of the ligands inside the protein cavities. Besides, a good correlation was found between the calculated binding energies for each ligand@protein complex and the relative affinities observed in competition experiments.

Photosensitised biphotonic chemistry of pyrimidine derivatives.

Photosensitised biphotonic irradiation of DNA has been rarely addressed, probably due to the difficulties in the experimental design. This is associated with the selection of nucleobases and sensitisers with appropriate absorption spectra and photochemical reactivity, in combination with a laser source emitting intense UVA light of the adequate wavelength. The present paper presents a new strategy involving absorption of a first UVA photon by an adequate sensitiser followed by triplet energy transfer to a pyrimidine (Pyr) derivative and absorption of a second UVA photon by the resulting Pyr triplet excited state. The feasibility of the proposed strategy has been demonstrated using two model reactions: (i) the Norrish-Yang photocyclisation of a tert-butyluracil and (ii) the photohydration of its uracil analogue, lacking the tert-butyl substituent.

Synthesis and Chemiluminescent Properties of Amino-Acylated luminol Derivatives Bearing Phosphonium Cations.

The monitoring of reactive oxygen species in living cells provides valuable information on cell function and performance. Lately, the development of chemiluminescence-based reactive oxygen species monitoring has gained increased attention due to the advantages posed by chemiluminescence, including its rapid measurement and high sensitivity. In this respect, specific organelle-targeting trackers with strong chemiluminescence performance are of high importance. We herein report the synthesis and chemiluminescence properties of eight novel phosphonium-functionalized amino-acylated luminol and isoluminol derivatives, designed as mitochondriotropic chemiluminescence reactive oxygen species trackers. Three different phosphonium cationic moieties were employed (phenyl, p-tolyl, and cyclohexyl), as well as two alkanoyl chains (hexanoyl and undecanoyl) as bridges/linkers. Synthesis is accomplished via the acylation of the corresponding phthalimides, as phthalhydrazide precursors, followed by hydrazinolysis. This method was chosen because the direct acylation of (iso)luminol was discouraging. The new derivatives' chemiluminescence was evaluated and compared with that of the parent molecules. A relatively poor chemiluminescence performance was observed for all derivatives, with the isoluminol-based ones being the poorest. This result is mainly attributed to the low yield of the fluorescence species formation during the chemiluminescence oxidation reaction.

Triplet Energy Transfer versus Excited State Cyclization as the Controlling Step in Photosensitized Bipyrimidine Dimerization.

Polymethylene-linked bipyrimidine models have been designed with different C5 substitutions and bridge lengths. Selective irradiation of 2'-methoxyacetophenone (2M) with the bipyrimidine models affords cyclobutane pyrimidine dimers, even in the presence of bulky substituents. Substitution at C5 affects both the relative triplet energies (ET(rel)) of the pyrimidines (Pyr) and the steric hindrance toward intermolecular energy transfer and intramolecular triplet Pyr* quenching. Photophysical studies showed that alkyl substitution resulted in a significant decrease in the ET(rel) value. Quenching of the triplet excited state of 2M by the Pyr derivatives was proven and established their quenching rate constants (kq). As a general trend, the thymine-containing compounds showed kq values higher than 109 M-1 s-1, while in the uracil and tert-butyluracil analogues, kq was markedly lower. These data are explained considering three different scenarios: (a) triplet energy transfer is the rate controlling step, (b) excited state cyclization is the rate controlling step, and (c) the rate controlling step switches along the reaction. Thus, by introducing variations in the substitution at C5, the length of the linking bridge, or the substrate concentration, it is possible to switch from a process governed by the intrinsic dimerization step to an energy transfer-controlled process.

Transient UV-vis absorption spectroscopic characterisation of 2'-methoxyacetophenone as a DNA photosensitiser.

2'-Methoxyacetophenone (2M) presents improved UVA absorption as compared with other acetophenone derivatives. On the basis of transient infrared spectroscopy it has been previously claimed that 2M is an interesting photosensitiser for cyclobutane pyrimidine dimers (CPDs) formation. In the present paper, a complete UV-Vis transient absorption spectroscopic characterisation of this compound is provided, including triplet-triplet spectra, triplet lifetimes and rate constants for quenching of 2M by a dimeric thymine derivative. Furthermore, generation of singlet oxygen has been proven by time-resolved near IR phosphorescence measurements. Overall, the obtained results confirm the potential of 2M as a DNA photosensitiser, not only for CPDs formation, but also for oxidative damage.

Generation of the Thymine Triplet State by Through-Bond Energy Transfer.

Benzophenone (BP) and drugs containing the BP chromophore, such as the non-steroidal anti-inflammatory drug ketoprofen, have been widely reported as DNA photosensitizers through triplet-triplet energy transfer (TTET). In the present work, a direct spectroscopic fingerprint for the formation of the thymine triplet (3 Thy*) by through-bond (TB) TTET from 3 BP* has been uncovered. This has been achieved in two new systems that have been designed and synthesized with one BP and one thymine (Thy) covalently linked to the two ends of the rigid skeleton of the natural bile acids cholic and lithocholic acid. The results shown here prove that it is possible to achieve triplet energy transfer to a Thy unit even when the photosensitizer is at a long (nonbonding) distance.

Drug-DNA complexation as the key factor in photosensitized thymine dimerization.

The crucial role of photosensitizer@DNA complexation in the formation of cyclobutane pyrimidine dimers (CPDs) has been demonstrated using femtosecond and nanosecond transient absorption and emission measurements in combination with in vitro DNA damage assays. This finding opens the door to re-evaluate the mechanisms involved in CPDs photosensitized by other chemicals.

Blocking cyclobutane pyrimidine dimer formation by steric hindrance.

The efficiency of thymine (Thy) and uracil (Ura) to form cyclobutane pyrimidine dimers (CPDs) in solution, upon UV irradiation differs by one order of magnitude. This could to be partially related to the steric hindrance induced by the methyl at C5 in thymine. The aim of the present work is to establish the influence of a bulky moiety at this position on the photoreactivity of pyrimidines. With this purpose, photosensitization with benzophenone and acetone of a 5-tert-butyl uracil derivative () and the equivalent Thy () has been compared. Introduction of the tert-butyl group completely blocks CPD formation. Moreover, the mechanistic insight obtained by laser flash photolysis is in accordance with the observed photoreactivity.

The (6-4) Dimeric Lesion as a DNA Photosensitizer.

Based on our previous investigations into the photophysical properties of the 5-methyl-2-pyrimidone (Pyo) chromophore, we now extend our studies to the photobehavior of the dimeric (6-4) thymine photoproducts (6-4 PP) to evaluate their capability to act as instrinsic DNA photosensitizers. The lesion presents significant absorption in the UVB/UVA region, weak fluorescence emission, a singlet-excited-state energy of approximately 351 kJ mol(-1) , and a triplet-excited-state energy of 297 kJ mol(-1) . Its triplet transient absorption has a maximum at 420-440 nm, a lifetime of around 7 µs, and a high formation quantum yield, ΦISC =0.86. This species is efficiently quenched by thymidine. Its DNA photosensitizing properties are demonstrated by a series of experiments run on a pBR322 plasmid. The lesion photoinduces both single-strand breaks and the formation of cyclobutane thymine dimers. Altogether, these results show that, the substitution of the pyrimidone ring at C4 by a 5-hydroxy-5,6-dihydrothymine does not cancel out the photosensitization properties of the chromophore.

Understanding of the photoallergic properties of fluoroquinolones: photoreactivity of lomefloxacin with amino acids and albumin.

Although the phototoxic and photoallergic properties of fluoroquinolone antibiotics (FQ) are remarkable, the mechanisms involved in these processes are not completely understood. For this reason, it is considered worthwhile to study in detail the photochemical interactions of lomefloxacin (LFX) and its N-acetyl derivative ALFX, two 6,8-dihalogenated fluoroquinolones, with the most abundant protein in human plasma (human serum albumin, HSA) to analyze their covalent binding. Fluorescence measurements and laser flash photolysis experiments performed in this work have revealed that N-acetylation of the LFX piperazinyl moiety produces an important increase of the drug affinity to albumin. Thus, while the association constant (Ka) for the LFX···HSA complex is below 10(3) M(-1), the Ka for the HSA···ALFX complex resulted in ca. 5 × 10(3) M(-1). Interestingly, LFX is mainly located at site I of HSA, while ALFX shows no preference for site I or II. A high reactivity between the aryl cations generated from (A)LFX dehalogenation and Trp and Tyr together with the generation of covalent adducts between the FQ and these amino acids was observed. However, the interactions between the FQ singlet excited state and albumin in FQ···HSA complexes seem to be the key process of FQ covalent binding to albumin. Moreover, our findings have shown a correlation between the photobinding properties of dihalogenated fluoroquinolones to HSA and their FQ···HSA association constants.

Two-photon chemistry from upper triplet states of thymine.

Photolysis of the benzophenone chromophore by means of high energy laser pulses has been used as a tool to populate upper thymine-like triplet states via intramolecular sensitization. These species undergo characteristic nπ* triplet photoreactivity, as revealed by the Norrish-Yang photocyclization of 5-tert-butyluracil.

Photophysical properties of 5-substituted 2-thiopyrimidines.

The aim of the present work is to determine the influence of C5 substitution on the photophysical properties of 2-thiopyrimidines (2-TPyr). For this purpose, 2-thiouracil, 5-t-butyl-2-thiouracil and 2-thiothymine (TU, BTU and TT, respectively) have been selected as target thionucleobases for the experimental studies and, in parallel, for DFT theoretical calculations. The UV spectra displayed by TU, BTU and TT in EtOH were very similar to each other. They showed a maximum around 275 nm and a shoulder at ca. 290 nm. The three 2-TPyr exhibited a strong phosphorescence emission; from the recorded spectra, triplet excited state energies of ca. 307, 304 and 294 kJ mol(-1) were determined for TU, BTU and TT, respectively. Laser excitation at 308 nm gave rise to a broad transient absorption band from 500 nm to 700 nm, which was in principle assigned to triplet-triplet absorption. This assignment was confirmed by energy transfer experiments using biphenyl (ET = 274 kJ mol(-1)) as an acceptor. The triplet lifetimes were 70 ns, 1.1 µs and 2.3 µs, for TU, BTU and TT, respectively. The obtained photophysical data, both in phosphorescence and transient absorption measurements, point to significantly different properties of the TT triplet excited state in spite of the structural similarities. Theoretical calculations at the B3LYP/aug-cc-pVDZ/PCM level agree well with the experimental range of excited state energies and support the ππ* nature of the lowest triplet states.

Benzophenone photosensitized DNA damage.

Although the carcinogenic potential of ultraviolet radiation is well-known, UV light may interact with DNA by direct absorption or through photosensitization by endogenous or exogenous chromophores. These chromophores can extend the "active" fraction of the solar spectrum to the UVA region and beyond, which means that photosensitizers increase the probability of developing skin cancer upon exposure to sunlight. Therefore researchers would like to understand the mechanisms involved in photosensitized DNA damage both to anticipate possible photobiological risks and to design tailor-made photoprotection strategies. In this context, photosensitized DNA damage can occur through a variety of processes including electron transfer, hydrogen abstraction, triplet-triplet energy transfer, or generation of reactive oxygen species. In this Account, we have chosen benzophenone (BP) as a classical and paradigmatic chromophore to illustrate the different lesions that photosensitization may prompt in nucleosides, in oligonucleotides, or in DNA. Thus, we discuss in detail the accumulated mechanistic evidence of the BP-photosensitized reactions of DNA or its building blocks obtained by our group and others. We also include ketoprofen (KP), a BP-derivative that possesses a chiral center, to highlight the stereodifferentiation in the key photochemical events, revealed through the dynamics of the reactive triplet excited state ((3)KP*). Our results show that irradiation of the BP chromophore in the presence of DNA or its components leads to nucleobase oxidations, cyclobutane pyrimidine dimer formation, single strand breaks, DNA-protein cross-links, or abasic sites. We attribute the manifold photoreactivity of BP to its well established photophysical properties: (i) it absorbs UV light, up to 360 nm; (ii) its intersystem crossing quantum yield (ϕ(ISC)) is almost 1; (iii) the energy of its nπ* lowest triplet excited state (E(T)) is ca. 290 kJ mol(-1); (iv) it produces singlet oxygen ((1)O(2)) with a quantum yield (ϕ(Δ)) of ca. 0.3. For electron transfer and singlet oxygen reactions, we focused on guanine, the nucleobase with the lowest oxidation potential. Among the possible oxidative processes, electron transfer predominates. Conversely, triplet-triplet energy transfer occurs mainly from (3)BP* to thymine, the base with the lowest lying triplet state in DNA. This process results in the formation of cyclobutane pyrimidine dimers, but it also competes with the Paternò-Büchi reaction in nucleobases or nucleosides, giving rise to oxetanes as a result of crossed cycloadditions. Interestingly, we have found significant stereodifferentiation in the quenching of the KP triplet excited state by both 2'-deoxyguanosine and thymidine. Based on these results, this chromophore shows potential as a (chiral) probe for the investigation of electron and triplet energy transport in DNA.

Triplet excimers of fluoroquinolones in aqueous media.

Generation of triplet eximers of 6-fluoro-7-piperazinyl-quinolone-3-carboxylic acids (FQs) have been detected in aqueous media using laser flash photolysis (LFP). These transient species (SS) are generated by self-quenching reactions of FQ triplet excited states such as pefloxacin (PFX), norfloxacin (NFX), the N-acetylated form of NFX (ANFX), and its methyl ester (EANFX) with their ground states. In this context, self-quenching rate constants in the range of (1-7) × 10(8) M(-1) s(-1) were determined. The triplet excimers show transient absorption spectra with λ(max) ca. 710 nm for SS(NFX), 740 nm for SS(PFX), and 620 nm for SS(ANFX) and E(ANFX), which are red-shifted with respect to their predecessors triplet excited states. These excimers can be also observed in the presence of phosphate buffer (PB). Experiments performed with NFX and ANFX at different PB concentrations showed that deprotonation processes are not involved in the generation of SS. The triplet multiplicity of the FQ excimers was confirmed by energy transfer reactions with naproxen. The correlation between fluorescence, intersystem crossing, excimer and photodegradation quantum yields of (A)NFX indicated that FQ self-quenching reactions are mainly a deactivation pathway. On the other hand, generation of FQ radical anions absorbing at λ(max) ca. 620 nm has been observed by an efficient electron transfer reaction from Trp to NFX, PFX, and ANFX (rate constants ca. 1 × 10(9) M(-1) s(-1)).

Enantioselective intramolecular [2 + 2]-photocycloaddition reactions of 4-substituted quinolones catalyzed by a chiral sensitizer with a hydrogen-bonding motif.

Six 2-quinolones, which bear a terminal alkene linked by a three- or four-membered tether to carbon atom C4 of the quinolone, were synthesized and subjected to an intramolecular [2 + 2]-photocycloaddition. The reaction delivered the respective products in high yields (78-99%) and with good regioselectivity in favor of the straight isomer. If conducted in the presence of a chiral hydrogen-bonding template (2.5 equiv) at low temperature in toluene as the solvent, the reaction proceeded enantioselectively (83-94% ee). An organocatalytic reaction was achieved when employing a chiral hydrogen-bonding template with an attached sensitizing unit (benzophenone or xanthone). The xanthone-based organocatalyst proved to be superior as compared to the respective benzophenone. Closer inspection revealed that the reaction of 4-(pent-4-enyloxy)quinolone leading to a six-membered ring, annelated to the cyclobutane, was less enantioselective (up to 41% ee with 30 mol % catalyst) than the reaction of 4-(but-3-enyloxy)quinolone leading to a five-membered ring (90% ee with 5 mol % and 94% ee with 20 mol % catalyst). Photophysical data (emission spectra, laser flash photolysis experiments) proved that the latter photocycloaddition was significantly faster, supporting the idea that the dissociation of the substrate from the catalyst prior to the photocycloaddition is responsible for the decreased enantioselectivity. Under optimized conditions, employing 10 mol % of the xanthone-based organocatalyst at -25 °C in trifluorotoluene as the solvent, three of the other four substrates gave the intramolecular [2 + 2]-photocycloaddition products with high enantioselectivities (72-87% ee). In all catalyzed reactions, the yields based on conversion were moderate to good (40-93%).