3-Substituted 2-Aminonaphthalene Photocages for Carboxylic Acids and Alcohols; Decaging Mechanism and Potential Applications in Synthesis.

3-Hydroxymethyl-2-aminonaphthalene photocage (photoremovable protecting group) 2 was synthesized and transformed to different ethers and esters to investigate the applicability to decage alcohols and carboxylic acids, respectively. The photoelimination of carboxylic acids takes place relatively efficiently (ΦR = 0.11) upon excitation with near-visible light, contrary to the elimination of alcohols. The scope of the decaging of both alcohols and esters was demonstrated on several examples, including aliphatic and aromatic substrates, carbohydrates, and nonsteroidal anti-inflammatory drugs. The photophysical properties of the photocage and its models, methyl ether 4a and acetyl ester 5a, were investigated. The fluorescence quantum yields (Φf = 0.40-0.002) were found to be reversely proportional to the efficiency of elimination of OH, alcohols, or carboxylic acids. The decaging photochemical reaction mechanism was investigated experimentally by transient absorption techniques with time scales from femtoseconds to seconds and computationally on the TD-DFT level of theory. The photoelimination of carboxylates takes place directly in the singlet excited state by a homolytic cleavage producing a radical pair within 1 ns. The subsequent electron transfer gives rise to aminonaphthalene carbocation and the carboxylate. A wide scope of substrates that can be decaged relatively efficiently with near-visible light and the chromo-orthogonal compatibility of aminonaphthalene and aniline derivatives render these photocages potentially applicable in organic synthesis or biology.

Dipeptides Containing Pyrene and Modified Photochemically Reactive Tyrosine: Noncovalent and Covalent Binding to Polynucleotides.

Dipeptides 1 and 2 were synthesized from unnatural amino acids containing pyrene as a fluorescent label and polynucleotide binding unit, and modified tyrosine as a photochemically reactive unit. Photophysical properties of the peptides were investigated by steady-state and time-resolved fluorescence. Both peptides are fluorescent (Φf = 0.3-0.4) and do not show a tendency to form pyrene excimers in the concentration range < 10-5 M, which is important for their application in the fluorescent labeling of polynucleotides. Furthermore, both peptides are photochemically reactive and undergo deamination delivering quinone methides (QMs) (ΦR = 0.01-0.02), as indicated from the preparative photomethanolysis study of the corresponding N-Boc protected derivatives 7 and 8. Both peptides form stable complexes with polynucleotides (log Ka > 6) by noncovalent interactions and similar affinities, binding to minor grooves, preferably to the AT reach regions. Peptide 2 with a longer spacer between the fluorophore and the photo-activable unit undergoes a more efficient deamination reaction, based on the comparison with the N-Boc protected derivatives. Upon light excitation of the complex 2·oligoAT10, the photo-generation of QM initiates the alkylation, which results in the fluorescent labeling of the oligonucleotide. This study demonstrated, as a proof of principle, that small molecules can combine dual forms of fluorescent labeling of polynucleotides, whereby initial addition of the dye rapidly forms a reversible high-affinity noncovalent complex with ds-DNA/RNA, which can be, upon irradiation by light, converted to the irreversible (covalent) form. Such a dual labeling ability of a dye could have many applications in biomedicinal sciences.

Chromo-Orthogonal Deprotection of Carboxylic Acids by Aminonaphthalene and Aminoaniline Photocages.

Photoremovable protecting groups (photocages) 6b-6i based on 1-amino-2-hydroxymethylnaphthalene were developed, and their applicability to release alcohols and carboxylic acids in photohydrolysis was investigated. Compound 6b cannot release alcohol since N-demethylation takes place instead. However, the photorelease of carboxylic acids from 6c-6i was demonstrated on caged substrates, including some nonsteroidal drugs and a neurotransmitter. A simultaneous use of aniline and aminonaphthalene cages allows for the chromatic orthogonality and selective deprotection by UV-B or near-visible and UV-A light, respectively. The photochemical reaction mechanism of decaging was investigated by fluorescence measurements and laser flash photolysis, indicating that the heterolysis and elimination of carboxylic acids take place in the singlet excited state, delivering carbocation as an intermediate. The photoheterolysis in the singlet excited state, which directly releases caged substrates, is highly applicable for the photocages and has advantages compared to hitherto used nitrobenzyl derivatives.

Excited State Intramolecular Proton Transfer (ESIPT) from -NH2 to the Carbon Atom of a Naphthyl Ring.

Excited state intramolecular proton transfer (ESIPT) has been documented from an amino NH2 group to a carbon atom of an adjacent aromatic ring. This finding changes the paradigm, as hitherto such processes have not been considered as plausible due to slow protonation of carbon and low (photo)acidity of the NH2 group. The ESIPT was studied by irradiation of 2-(2-aminophenyl)naphthalene in CH3CN-D2O, whereupon regiospecific incorporation of deuterium takes place at the naphthalene position 1, with a quantum yield of Φ = 0.11. A synergy of experimental and computational investigations completely unraveled the mechanism of this important photochemical reaction. Upon excitation to the photoreactive S2(La) state, a favorable redistribution of charge sets the stage for ESIPT to the carbon atom in naphthalene position 1. H2O molecules are needed, as they increase the excitation energy and oscillator strength for the population of the S2(La) state. The gain in energy is used to surmount a small energy barrier on the pathway from the Franck-Condon geometry to the conical intersection with the S0, delivering aza-quinone methide.

Photogeneration of quinone methide from adamantylphenol in an ultrafast non-adiabatic dehydration reaction.

The ultrafast photochemical reaction of quinone methide (QM) formation from adamantylphenol was monitored in real time using femtosecond transient absorption spectroscopy and fluorescence upconversion in solution at room temperature. Experiments were complemented by theoretical studies simulating the reaction pathway and elucidating its mechanism. Excitation with sub-20 fs UV pulses and broadband probing revealed ultrafast formation of the long-lived QM intermediate directly in the ground state, occurring with a time constant of around 100 fs. UV-vis transient absorption data covering temporal dynamics from femtoseconds to hundreds of milliseconds revealed persistence of the absorption band assigned to QM and partially overlapped with other contributions tentatively assigned to triplet excited states of the adamantyl derivative and the phenoxyl radical that are clearly distinguished by their evolution on different time scales. Our data, together with the computations, provide evidence of a non-adiabatic photodehydration reaction, which leads to the formation of QM in the ground state via a conical intersection, circumventing the generation of a transient QM excited state.

Wavelength dependent photochemistry of BODIPY-phenols and their applications in the fluorescent labeling of proteins.

A series of BODIPY dyes were synthesized, that were at the 3, or 3 and 5 positions, substituted by photochemically reactive quinone methide (QM) precursor moieties. Fluorescence properties of the molecules were investigated and we demonstrated that the molecules undergo wavelength dependent photochemistry. Photodeamination to deliver QMs takes place only upon excitation to higher excited singlet states, showing unusual anti-Kasha photochemical reactivity. The findings were corroborated by TD-DFT computations. Laser flash photolysis experiments could not reveal QMs due to the low efficiency of their formation, but enabled the detection of phenoxyl radicals. The applicability of the molecules for the fluorescent labeling of bovine serum albumin as a model protein upon photoexcitation at 350 nm was demonstrated.

Non-Covalent Binding of Tripeptides-Containing Tryptophan to Polynucleotides and Photochemical Deamination of Modified Tyrosine to Quinone Methide Leading to Covalent Attachment.

A series of tripeptides TrpTrpPhe (1), TrpTrpTyr (2), and TrpTrpTyr[CH2N(CH3)2] (3) were synthesized, and their photophysical properties and non-covalent binding to polynucleotides were investigated. Fluorescent Trp residues (quantum yield in aqueous solvent ΦF = 0.03-0.06), allowed for the fluorometric study of non-covalent binding to DNA and RNA. Moreover, high and similar affinities of 2×HCl and 3×HCl to all studied double stranded (ds)-polynucleotides were found (logKa = 6.0-6.8). However, the fluorescence spectral responses were strongly dependent on base pair composition: the GC-containing polynucleotides efficiently quenched Trp emission, at variance to AT- or AU-polynucleotides, which induced bisignate response. Namely, addition of AT(U) polynucleotides at excess over studied peptide induced the quenching (attributed to aggregation in the grooves of polynucleotides), whereas at excess of DNA/RNA over peptide the fluorescence increase of Trp was observed. The thermal denaturation and circular dichroism (CD) experiments supported peptides binding within the grooves of polynucleotides. The photogenerated quinone methide (QM) reacts with nucleophiles giving adducts, as demonstrated by the photomethanolysis (quantum yield ΦR = 0.11-0.13). Furthermore, we have demonstrated photoalkylation of AT oligonucleotides by QM, at variance to previous reports describing the highest reactivity of QMs with the GC reach regions of polynucleotides. Our investigations show a proof of principle that QM precursor can be imbedded into a peptide and used as a photochemical switch to enable alkylation of polynucleotides, enabling further applications in chemistry and biology.

Photochemical Reactivity of Naphthol-Naphthalimide Conjugates and Their Biological Activity.

Quinone methide precursors 1a-e, with different alkyl linkers between the naphthol and the naphthalimide chromophore, were synthesized. Their photophysical properties and photochemical reactivity were investigated and connected with biological activity. Upon excitation of the naphthol, Förster resonance energy transfer (FRET) to the naphthalimide takes place and the quantum yields of fluorescence are low (ΦF ≈ 10-2). Due to FRET, photodehydration of naphthols to QMs takes place inefficiently (ΦR ≈ 10-5). However, the formation of QMs can also be initiated upon excitation of naphthalimide, the lower energy chromophore, in a process that involves photoinduced electron transfer (PET) from the naphthol to the naphthalimide. Fluorescence titrations revealed that 1a and 1e form complexes with ct-DNA with moderate association constants Ka ≈ 105-106 M-1, as well as with bovine serum albumin (BSA) Ka ≈ 105 M-1 (1:1 complex). The irradiation of the complex 1e@BSA resulted in the alkylation of the protein, probably via QM. The antiproliferative activity of 1a-e against two human cancer cell lines (H460 and MCF 7) was investigated with the cells kept in the dark or irradiated at 350 nm, whereupon cytotoxicity increased, particularly for 1e (>100 times). Although the enhancement of this activity upon UV irradiation has no imminent therapeutic application, the results presented have importance in the rational design of new generations of anticancer phototherapeutics that absorb visible light.

Photoelimination of Nitrogen from Diazoalkanes: Involvement of Higher Excited Singlet States in the Carbene Formation.

Although diazoalkanes are important carbene precursors in organic synthesis, a comprehensive mechanism of photochemical formation of carbenes from diazoalkanes has not been proposed. Synergies of experiments and computations demonstrate the involvement of higher excited singlet states in the photochemistry of diazoalkanes. In all investigated diazoalkanes, excitation to S1 results in nonreactive internal conversion to S0. On the contrary, excitation to higher-lying singlet states (Sn, n > 1) drives the reaction toward a different segment of the S1/S0 conical intersection seam and results in nitrogen elimination and formation of carbenes.

Substituted adamantylphthalimides: Synthesis, antiviral and antiproliferative activity.

In this study, three groups of adamantylphthalimides, bearing different substituents at the phthalimide moiety, N-(4'-R2 )phthalimidoadamantanes (1-7), 3-[N-(4'-R2 )phthalimido]-1-adamantanols (8-10), and 3-[N-(4'-R2 )phthalimido]adamantane-1-carboxylic acids (11-15), were synthesized and screened against tumor cells and viruses. The most potent compounds are not substituted at the adamantane and bear an OH or NH2 substituent at the phthalimide (compounds 3 and 5). The antiproliferative activities of compounds 3 and 5 are in the micromolar range, much higher than the one of thalidomide. A minor antiviral activity against cytomegalovirus and varicella-zoster virus was found for compounds 3 and 5, but these compounds lacked selectivity. The results presented are important for the rational design of the next-generation compounds with anticancer and antiviral activities.

[3 + 2] Cycloaddition with photogenerated azomethine ylides in ß-cyclodextrin.

Stability constants for the inclusion complexes of cyclohexylphthalimide 2 and adamantylphthalimide 3 with ß-cyclodextrin (ß-CD) were determined by 1H NMR titration, K = 190 ± 50 M-1, and K = 2600 ± 600 M-1, respectively. Photochemical reactivity of the inclusion complexes 2@ß-CD and 3@ß-CD was investigated, and we found out that ß-CD does not affect the decarboxylation efficiency, while it affects the subsequent photochemical H-abstraction, resulting in different product distribution upon irradiation in the presence of ß-CD. The formation of ternary complexes with acrylonitrile (AN) and 2@ß-CD or 3@ß-CD was also essayed by 1H NMR. Although the formation of such complexes was suggested, stability constants could not be determined. Irradiation of 2@ß-CD in the presence of AN in aqueous solution where cycloadduct 7 was formed highly suggests that decarboxylation and [3 + 2] cycloaddition take place in the ternary complex, whereas such a reactivity from bulky adamantane 3 is less likely. This proof of principle that decarboxylation and cycloaddition can be performed in the ß-CD cavity has a significant importance for the design of new supramolecular systems for the control of photoreactivity.

Formation of Quinone Methides by Ultrafast Photodeamination: A Spectroscopic and Computational Study.

Formation of quinone methides (QMs) by photoelimination of an ammonium salt from cresol derivatives was investigated by femtosecond transient absorption spectroscopy (fs-TA) and computationally by time-dependent density functional theory using the PCM(water)/(TD-)B3LYP/6-311++G(d,p) level of theory. The photoelimination takes place in an adiabatic ultrafast reaction on the S1 potential energy surface delivering the corresponding QMs(S1), which were detected by fs-TA. Computations predicted a high-energy cation intermediate in the pathway between the Franck-Condon state of a monoammonium salt and the corresponding QM(S1) that was not detected by fs-TA. On the other hand, elimination from a disalt in H2O takes place in one step, giving directly the QM(S1). The combined experimental and theoretical investigation fully disclosed the formation of QMs by the deamination reaction mechanism, which is important in the application of cresols or similar molecules in biological systems.

Competing photochemical reactions of bis-naphthols and their photoinduced antiproliferative activity.

The photophysical properties and photochemical reactivities of a series of bis-naphthols 4a-4e and bis-anthrols 5a and 5e were investigated by preparative irradiation in CH3OH, fluorescence spectroscopy and laser flash photolysis (LFP). Methanolysis taking place via photodehydration (bis-naphthols: ΦR = 0.04-0.05) is in competition with symmetry breaking charge separation (SB-CS). The SB-CS gave rise to radical ions that were detected for 4a and 4e by LFP. Photodehydration gave quinone methides (QMs) that were also detected by LFP (λmax = 350 nm, τ ≈ 1-2 ms). In the aqueous solvent, excited state proton transfer (ESPT) competes with the abovementioned processes, giving rise to naphtholates, but the process is inefficient and can only be observed in the buffered aqueous solution at pH > 7. Since the dehydration of bis-naphthols delivers QMs, their potential antiproliferative activity was investigated by an MTT test on three human cancer cell lines (NCI-H1299, lung carcinoma; MCF-7, breast adenocarcinoma; and SUM159, pleomorphic breast carcinoma). Cells were treated with 4 or 5 with or without irradiation (350 nm). An enhancement of the activity (up to 10-fold) was observed upon irradiation, which may be associated with QM formation. However, these QMs do not cross-link DNA. The activity is most likely associated with the alkylation of proteins present in the cell cytoplasm, as evidenced by photoinduced alkylation of bovine and human serum albumins by 4a.

Photoelimination of nitrogen from adamantane and pentacycloundecane (PCU) diazirines: a spectroscopic study and supramolecular control.

Photochemical reactivity of pentacycloundecane (PCU) and adamantane diazirines was investigated by preparative irradiation in different solvents, laser flash photolysis (LFP) and quantum chemical computations. In addition, formation of inclusion complexes for diazirines with cucurbit[7]uril, ß- and γ-cyclodextrin (ß- and γ-CD) was investigated by 1H NMR spectroscopy, isothermal microcalorimetry and circular dichroism spectroscopy, followed by the investigation of photochemical reactivity of the formed complexes. Diazirines undergo efficient photochemical elimination of nitrogen (ΦR > 0.5) and deliver the corresponding singlet carbenes. Singlet carbenes react in intra- and intermolecular reactions and we found a rare singlet carbene pathway in CH3OH involving protonation and formation of a carbocation, detected due to the specific rearrangement of the pentacycloundecane skeleton. Singlet diazirines undergo intersystem crossing and deliver triplet carbenes that react with oxygen to form ketones which were isolated after irradiation. Our main finding is that the formation of diazirine inclusion complexes with ß-CD and γ-CD changes the relative ratio of singlet vs. triplet pathways, with singlet carbene products being dominant from the chemistry of the irradiated complexes. Our combined theoretical and experimental studies provide new insights into the supramolecular control of carbene reactivity which has possible applications for the control of product distribution by solvent effects and the choice of constrained media.

Ultrafast Adiabatic Photodehydration of 2-Hydroxymethylphenol and the Formation of Quinone Methide.

The photochemical reactivity of 2-hydroxymethylphenol (1) was investigated experimentally by photochemistry under cryogenic conditions, by detecting reactive intermediates by IR spectroscopy, and by using nanosecond and femtosecond transient absorption spectroscopic methods in solution at room temperature. In addition, theoretical studies were performed to facilitate the interpretation of the experimental results and also to simulate the reaction pathway to obtain a better understanding of the reaction mechanism. The main finding of this work is that photodehydration of 1 takes place in an ultrafast adiabatic photochemical reaction without any clear intermediate, delivering quinone methide (QM) in the excited state. Upon photoexcitation to a higher vibrational level of the singlet excited state, 1 undergoes vibrational relaxation leading to two photochemical pathways, one by which synchronous elimination of H2 O gives QM 2 in its S1 state and the other by which homolytic cleavage of the phenolic O-H bond produces a phenoxyl radical (S0 ). Both are ultrafast processes that occur within a picosecond. The excited state of QM 2 (S1 ) probably deactivates to S0 through a conical intersection to give QM 2 (S0 ), which subsequently delivers benzoxete 4. Elucidation of the reaction mechanisms for the photodehydration of phenols by which QMs are formed is important to tune the reactivity of QMs with DNA and proteins for the potential application of QMs in medicine as therapeutic agents.

Photocyclization of Tetra- and Pentapeptides Containing Adamantylphthalimide and Phenylalanines: Reaction Efficiency and Diastereoselectivity.

A series of tetrapeptides and pentapeptides was synthesized bearing a phthalimide chromophore at the N-terminus. The C-terminus of the peptides was strategically substituted with an amino acid, Phe, Phe(OMe), or Phe(OMe)2 characterized by different oxidation potentials. The photochemical reactivity of the peptides was investigated by preparative irradiation and isolation of photoproducts, as well as with laser flash photolysis. Upon photoexcitation, the peptides undergo photoinduced electron transfer (PET) and decarboxylation, followed by diastereoselective cyclization with the retention of configuration for tetrapeptides or inversion of configuration for pentapeptides. Molecular dynamics (MD) simulations and NOE experiments enabled assignment of the stereochemistry of the cyclic peptides. MD simulations of the linear peptides disclosed conformational reasons for the observed diastereoselectivity, being due to the peptide backbone spatial orientation imposed by the Phe amino acids. The photochemical efficiency for the decarboxylation and cyclization is not dependent on the peptide length, but it depends on the oxidation potential of the amino acid at the C-terminus. The results described herein are particularly important for the rational design of efficient photochemical reactions for the preparation of cyclic peptides with the desired selectivity.

Photodeamination to quinone methides in cucurbit[n]urils: potential application in drug delivery.

We demonstrate a proof of principle for a new approach in the development of a drug delivery system. A positively charged prodrug (phenol) can form a stable inclusion complex with CB[7], which enables more efficient delivery of the prodrug. After photochemical transformation (photoactivation) inside the complex, an active drug quinone methide (QM) is formed and released from the complex, since it is a neutral molecule and forms a less stable complex with CB[7].

Direct Observation of Photoinduced Ultrafast Generation of Singlet and Triplet Quinone Methides in Aqueous Solutions and Insight into the Roles of Acidic and Basic Sites in Quinone Methide Formation.

Femtosecond time-resolved transient absorption spectroscopy experiments and density functional theory computations were done for a mechanistic investigation of 3-(1-phenylvinyl)phenol (1) and 3-hydroxybenzophenone (2) in selected solvents. Both compounds went through an intersystem crossing (ISC) to form the triplet excited states Tππ* and Tnπ* in acetonitrile but behave differently in neutral aqueous solutions, in which a triplet excited state proton transfer (ESPT) induced by the ISC process is also proposed for 2 but a singlet ESPT without ISC is proposed for 1, leading to the production of the triplet quinone methide (QM) and the singlet excited QM species respectively in these two systems. The triplet QM then underwent an ISC process to form an unstable ground state intermediate which soon returned to its starting material 2. However, the singlet excited state QM went through an internal conversion process to the ground state QM followed by the formation of its final product in an irreversible manner. These differences are thought to be derived from the slow vinyl C-C rotation and the moderate basicity of the vinyl C atom in 1 as compared with the fast C-O rotation and the greater basicity of the carbonyl O atom of 2 after photoexcitation. This can account for the experimental results in the literature that the aromatic vinyl compounds undergo efficient singlet excited state photochemical reactions while the aromatic carbonyl compounds prefer triplet photochemical reactions under aqueous conditions. These results have fundamental and significant implications for understanding of the ESPT reactivity in general, as well as for the design of molecules for efficient QM formation in aqueous media with potential applications in cancer phototherapy.

Photochemistry of 1- and 2-Naphthols and Their Water Clusters: The Role of 1 ππ*(La ) Mediated Hydrogen Transfer to Carbon Atoms.

The computational analysis of the isomer- and conformer-dependent photochemistry of 1- and 2-naphthols and their microsolvated water clusters is motivated by their very different excited state reactivities. We present evidence that 1- and 2-naphthol follow distinct excited state deactivation pathways. The deactivation of 2-naphthols, 2-naphthol water clusters, as well as of the anti conformer of 1-naphthol is mediated by the optically dark 1 πσ* state. The dynamics of the 1 πσ* surface leads to the homolytic cleavage of the OH bond. On the contrary, the excited state deactivation of syn 1-naphthol and 1-naphthol water clusters follows an uncommon reaction pathway. Upon excitation to the bright 1 ππ*(La ) state, a highly specific excited state hydrogen transfer (ESHT) to carbon atoms C8 and C5 takes place, yielding 1,8- and 1,5-naphthoquinone methides. The ESHT pathway arises from the intrinsic electronic properties of the 1 ππ*(La ) state of 1-naphthols.

Hydroxymethylaniline Photocages for Carboxylic Acids and Alcohols.

ortho-, meta- and para-Hydroxymethylaniline methyl ethers 3-5-OMe and acetyl derivatives 3-5-OAc were investigated as potential photocages for alcohols and carboxylic acids, respectively. The measurements of photohydrolysis efficiency showed that the decaging from ortho- and meta-derivatives takes place efficiently in aqueous solution, but not for the para-derivatives. Contrary to previous reports, we show that the meta-derivatives are better photocages for alcohols, whereas ortho-derivatives are better protective groups for carboxylic acids. The observed differences were fully disclosed by mechanistic studies involving fluorescence measurements and laser flash photolysis (LFP). Photoheterolysis for the para-derivatives does not take place, whereas both meta- and ortho-derivatives undergo heterolysis and afford the corresponding carbocations 3-C and 4-C. The ortho-carbocation 4-o-C was detected by LFP in aqueous solution (λmax = 410 nm, τ ≈ 90 µs). Moreover, spectroscopic measurements for the meta-acetyl derivative 3-m-OAC indicated the formation of cation in the excited state. The application of an ortho-aniline derivative as a protective group was demonstrated by synthesizing several derivatives of carboxylic acids. In all cases, the photochemical deprotection was accomplished in high yields (>80%). This mechanistic study fully rationalized the photochemistry of aniline photocages which is important for the design of new photocages and has potential for synthetic, biological, and medicinal applications.