Photolabile Protecting Groups Research Articles

Meso-Hydroxylmethyl-1,3,5,7-tetramethylBODIPY: A Visible-Light Photoremovable Protective Group for Carboxyl Groups

AbstractA meso-BODIPY-type photoremovable protecting group for carboxyl group is developed. BODIPYs are well-known for brilliant color, favorable to reaction monitoring and purification. meso-BODIPY esters are achieved via esterification in 44–89% yields, while deprotection is fulfilled under irradiation of blue LED bulb in the presence of photocatalyst and organic hydrogen donor in 43–95% yields. This method is applicable to a variety of acids, such as aromatic, heteroaromatic, alkyl, α,β-unsaturated carboxylic acids, etc. Our BODIPY-deprotective condition is almost neutral that both methyl and tert-butyl esters are compatible.

Seeing and Cleaving: Turn-Off Fluorophore Uncaging and Its Application in Hydrogel Photopatterning and Traceable Neurotransmitter Photocages.

The advancements in targeted drug release and experimental neuroscience have amplified the scientific interest in photolabile protecting groups (PPGs) and photouncaging. The growing need for the detection of uncaging events has led to the development of reporters with fluorescence turn-on upon uncaging. In contrast, fluorescent tags with turn-off properties have been drastically underexplored, although there are applications where they would be sought after. In this work, a rhodamine-based fluorescent tag is developed with signal turn-off following photouncaging. One-photon photolysis experiments reveal a ready loss of red fluorescence signal upon UV (365 nm) irradiation, while no significant change is observed in control experiments in the absence of PPG or with irradiation around the absorption maximum of the fluorophore (595 nm). The two-photon photolysis of the turn-off fluorescent tag is explored in hydrogel photolithography experiments. The hydrogel-bound tag enables the power-, dwell time-, and wavelength-dependent construction of intricate patterns and gradients. Finally, a prominent caged neurotransmitter (MNI-Glu) is modified with the fluorescent tag, resulting in the glutamate precursor named as GlutaTrace with fluorescence traceability and turn-off upon photouncaging. GlutaTrace is successfully applied for the visualization of glutamate precursor distribution following capillary microinjection and for the selective excitation of neurons in a mouse brain model.

Reinvigorating Photo-Activated R-Alkoxysilanes Containing 2-Nitrobenzyl Protecting Groups as Stable Precursors for Photo-Driven Si-O Bond Formation in Polymerization and Surface Modification.

This study aimed to revitalize silicon-based sol-gel chemistry methodologies utilizing photoprotected R-alkoxysilanes to control the synthesis of unique silicon-based materials. We have investigated the synthesis, characterization, light-induced deprotection, and subsequent polymerization/surface functionalization through the use of 2-nitrobenzyloxy-based photoremovable protecting groups (PPGs) as alkoxy reactive groups on ethyl and phenyl (R x -(alkoxy) y silanes, with x = 0-3 and y = 1-3). The photochemical dynamics, relative efficiencies, and kinetics of the novel alkoxysilane-based PPGs were thoroughly investigated using UV light irradiation by NMR and UV/vis methods. We then explored the tin-catalyzed coupling of photodeprotected products (R x -silanols) to form polymers/oligomers. We have found that photoenabled removal of PPGs and conversion to silanols from all silane systems studied is achieved. Furthermore, these deprotected species are polymerizable into siloxanes and effectively used as light-controlled surface modifiers with masking techniques of which proof-of-concept examples are given, enabling promising application as photolithographic reagents.

730 nm Light‐Induced Cleavage of BODIPY Photocages via Entropy‐Driven Triplet Sensitization

AbstractLight‐activated drug delivery systems allow precise spatiotemporal control of a drug release process. However, safe and efficient drug release activation needs a low‐power nonpulsed red/near‐infrared light with high tissue penetration depth. Nevertheless, such systems remain a challenge. Herein, a self‐assembled nanovehicle made of 2,6‐diiodo‐B‐dimethyl‐boron dipyrromethene (BODIPY)‐based photocleavable trigonal molecules bearing Pt(II) meso‐tetraphenyltetranaphthoporhyrin photosensitizer and a fluorescent release marker Nile Red in hydrophobic core is introduced. The system employs endothermic triplet–triplet energy transfer between the photosensitizer and the trigonal molecule, leading to the cleavage of the trigonal molecule followed by cargo release. This allows to engage 730 nm light to cleave BODIPY photoremovable protecting groups (PPGs) instead of 530 nm light that would be needed for direct photocage excitation. Therefore, the approach unleashes the desired activation of drug release via photocleavage with longer wavelengths (within the phototherapeutic window) without any chemical modification of the PPGs. Cell studies demonstrate fast intracellular uptake of the nanovehices by PC3 human prostate cancer cells with accumulation in lysosomes in 2 h. Light irradiation at 730 nm on nanovehicles dispersed in cell media leads to payload release. Remarkably, the system exhibits higher release efficiency at low oxygen concentration than at ambient thus allowing to tackle aggressive hypoxic solid tumors.

Tweezer Dyads for H-Bond Assisted Cooperativity in Tandem Uncaging Systems.

"Tandem" uncaging systems, in which a photolabile protecting group (PPG) is sensitized by an energy-harvesting antenna, may increase the photosensitivity of PPGs by several orders of magnitude for two-photon (2P) photorelease. Yet, they remain poorly accessible because of arduous multi-step synthesis. In this work, we design efficient tandem uncaging systems by (i) using a convenient assembly of the building blocks relying on click chemistry, (ii) introducing H-bonding induced proximity thus facilitating (iii) photoinduced electron transfer (PeT) as a cooperative mechanism. A strong two-photon absorber electron-donating quadrupolar antenna and various electron-accepting PPGs (mDEAC, MNI or MDNI) were clicked stepwise onto a "tweezer-shaped" pyrido-2,6-dicarboxylate platform whose H-bonding and π-stacking abilities were exploited to keep the antenna and the PPGs in close proximity. The different electron-accepting ability of the PPGs led to dyads with wildly different behaviors. Whilst the MDNI and MNI dyads showed poor dark stability or no photo-uncaging ability due to their too high electron-accepting character, the mDEAC dyad benefited from optimum redox potentials to promote PeT and slow down charge recombination, resulting in enhanced uncaging quantum yield (Φu=0.38) compared to mDEAC (Φu=0.014). This unique combination resulted in large 2P photo-sensitivity in the near-infrared window (240 GM at 710 nm).

Study on factors affecting quantum yield for the design of improved ortho-nitrobenzyl photoremovable protecting groups

Abstract The use of photoremovable protecting groups (PPGs) for the protection and deprotection of biologically active substances has become instrumental in elucidating functional mechanisms in cells. However, the development of chromophores responsive to visible and near-infrared (NIR) light often results in reduced quantum yields (Φu) of uncaging reactions. Because a high quantum yield is essential for practical PPGs, it is imperative to understand the factors influencing it and devise strategies to enhance it. In this study, we systematically investigated nitrobiphenyl scaffolds featuring amino substituents as electron donors, potentially serving as visible and NIR excitable chromophores. Among the synthesized structures, PPGs incorporating carbazole and phenothiazine units as amino substituents exhibited the highest Φu values of 0.2 and 0.1, respectively, rendering them promising candidates for physiological studies. Conversely, diphenylamine substituents yielded considerably lower Φu values. Photophysical properties and quantum chemical calculations provided insights into the nature of the lowest excited states and their impact on Φu, highlighting the significance of achieving the balance between local n–π* excitation and charge-transfer excitation for manipulating the uncaging quantum yield in ortho-nitrobenzyl derivatives.

In Search of Visible Light Activatable Photocages: Structure‐Activity Relationship Study on C‐8 Substituted Indene‐Fused‐Coumarinyl Photoremovable Protecting Groups

AbstractA series of C‐8 substituted indeno[1,2‐g]coumarin‐based photoremovable protecting groups (PPGs) were synthesized. para‐Substituted benzoic acids were employed as leaving groups to evaluate their photolytic efficiency. Substitution of phenyl groups was proved to have negative impacts on photochemical properties of the PPGs, including but not limited to: retarded photolysis course, decreased uncaging quantum yield, and unsatisfactory cargo release yield. Electron‐donating diethylamino substituted PPG 3 d, a structural analogue of the widely used 7‐diethylaminocoumarin PPG (DEACM), exhibited red‐shifted absorption maximum and improved optical properties. Photochemical characterization revealed that PPG 3 d not only showed comparable photolytic efficiency to DEACM at 365 nm and 405 nm, but also demonstrated superior sensitivity towards 465 nm wavelength, to which DEACM is unable to absorb and therefore, non‐responsive. The >450 nm photosensitivity makes 3 d a complement to DEACM for long wavelength excitation and a promising PPG for biological applications.

Sulfur atom effect on the photochemical release of benzylamine from caged amines

Abstract Carbamate linkers are commonly used to attach photolabile protecting groups (PPGs) to amine functional groups. However, the release of amines from the carbamate linkage has low photochemical quantum yields because of the low leaving ability of the carbamic anion. In this study, we synthesized a new coumarin-based thiocarbamate-containing caged amine as a model to study the effect of sulfur on the photocleavage of the amine from the PPG. The release of the amine from the thiocarbamate (−SC(O)NHCH2Ph) linkage was ∼20 times faster than from the carbamate (−OC(O)NHCH2Ph) linkage.

Two-Photon-Responsive "TICT + AIE" Active Naphthyridine-BF2 Photoremovable Protecting Group: Application for Specific Staining and Killing of Cancer Cells.

The combined effects of twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE) phenomena have demonstrated a significant influence on excited-state chemistry. These combined TICT and AIE features have been extensively utilized to enhance photodynamic and photothermal therapy. Herein, we demonstrated the synergistic capabilities of TICT and AIE phenomena in the design of the photoremovable protecting group (PRPG), namely, NMe2-Napy-BF2. This innovative PRPG incorporates TICT and AIE characteristics, resulting in four remarkable properties: (i) red-shifted absorption wavelength, (ii) strong near-infrared (NIR) emission, (iii) viscosity-sensitive emission property, and (iv) accelerated photorelease rate. Inspired by these intriguing attributes, we developed a nanodrug delivery system (nano-DDS) using our PRPG for cancer treatment. In vitro studies showed that our nano-DDS manifested effective cellular internalization, specific staining of cancer cells, high-resolution confocal imaging of cancerous cells in the NIR region, and controlled release of the anticancer drug chlorambucil upon exposure to light, leading to cancer cell eradication. Most notably, our nano-DDS exhibited a substantially increased two-photon (TP) absorption cross section (435 GM), exhibiting its potential for in vivo applications. This development holds promise for significant advancements in cancer treatment strategies.

Development of a Two-Photon-Responsive Chromophore, 2-(p-Aminophenyl)-5,6-dimethoxy-1-(hydroxyinden-3-yl)methyl Derivative, as a Photoremovable Protecting Group.

Photoremovable protecting groups (PPGs) are powerful tools that are widely used to investigate biological events in cells. An important requirement for PPGs is the efficient release of bioactive molecules by using visible to near-infrared light in the biological window (650-1350 nm). In this study, we report a new two-photon (2P)-responsive PPG, 2-(p-aminophenyl)-5,6-dimethoxy-1-(hydroxyinden-3-yl)methyl, with a donor-π-donor cyclic stilbene structure. The 2P cross section was approximately 40-50 GM at ∼700 nm. The quantum yield of the uncaging process of caged benzoate was greater than 0.7, demonstrating that the 2P uncaging efficiency was approximately 30 GM at around 700 nm. This newly developed 2P-responsive chromophore can be used in future biological experiments. The mechanism of the photo-uncaging reaction via the carbocation intermediate was elucidated using transient absorption spectroscopy and product analysis.

Effect of Two-Photon Excitation to 8-Azacoumarin Derivatives as Photolabile Protecting Groups.

An improvement of the two-photon excitation was achieved using 8-azacoumarin-type caged compounds, which showed large values of the two-photon uncaging action cross-section (δu >0.1 Goeppert-Mayer (GM)). In particular, the 7-hydroxy-6-iodo-8-azacoumarin (8-aza-Ihc)-caged compound showed an excellent uncaging action cross-section value (δu = 1.28 GM). Therefore, 8-azacoumarin-type photolabile protecting groups (PPGs) can be used as two-photon excitation sources.

Transient Absorption Spectroscopic Investigation of the Photocyclization-Deprotection Reaction of 3',5'-Dimethoxybenzoin Fluoride.

The 3',5'-dimethoxybenzoin (DMB) system has been widely investigated as a photoremovable protecting group (PRPG) for the elimination of various functional groups and has been applied in many fields. The photolysis of DMB fluoride leads to a highly efficient photocyclization-deprotection reaction, resulting in a high yield of 3',5'-dimethoxybenzofuran (DMBF) in a MeCN solution, while there is a competitive reaction that produces DMB in an aqueous solution. The yield of DMB increased as the volume ratio of water increased. To understand the solvent effect of the photolysis of selected DMB-based compounds, a combination of femtosecond to nanosecond transient absorption spectroscopies (fs-TA and ns-TA), nanosecond time-resolved resonance Raman spectroscopy (ns-TR3) and quantum chemical calculation was employed to study the photophysical and photochemical reaction mechanisms of DMB fluoride in different solutions. Facilitated by the bichromophoric nature of DMB fluoride with electron-donating and -withdrawing chromophores, the cyclized intermediates could be found in a pure MeCN solution. The deprotection of a cyclic biradical intermediate results in the simultaneous formation of DMBF and a cyclic cation species. On the other hand, in aqueous solution, fs-TA experiments revealed that α-keto cations could be observed after excitation directly, which could easily produce the DMB through the addition of a hydroxyl within 8.7 ps. This work provides comprehensive photo-deactivation mechanisms of DMB fluoride in MeCN and aqueous conditions and provides critical insights regarding the biomedical application of DMB-based PRPG compounds.

O-Nitrobenzyl-Based Caged exo-16,17-Dihydro-gibberellin A5-13-acetate for Photocontrolled Release of Plant Growth Regulators.

Caged plant growth regulators (caged PGRs) that release bioactive molecules under irradiation are critical in enhancing the efficacy and mitigating the negative environmental effects of PGRs. The synthetically derived plant growth inhibitor exo-16,17-dihydro-gibberellin A5-13-acetate (DHGA5) regulates the development and stress resilience of plants. We report here the conception of novel caged DHGA5 derivatives wherein the photoremovable protecting groups (PRPGs) serve not only to enable light-controlled release but also to protect the carboxyl group during chemical synthesis. Three o-nitrobenzyl-based caged DHGA5 derivatives with different substituents on the nitrobenzyl moiety were obtained and evaluated for their properties in vitro and in vivo. The photolysis half-life values of caged DHGA5 derivatives 7a, 7b, and 7c under a UV lamp were 15.6 h, 1.2 h, and 28.2 h, respectively. Experiments in vivo showed that 0.2 mM of the caged compounds significantly inhibited the growth of the model plant Arabidopsis thaliana and important crop rice in a precise photoactivated form.

Sanger's reagent as a new general phototrigger for organelle imaging

Photoactivatable subcellular organelles imaging offers the desirable spatiotemporal resolution in studying the complex biological processes. Developing proper and simple photoremovable protecting groups (PPGs) is the key for constructing such imaging probes. In this study, we designed and synthesized three Sanger's reagent caged fluorescent probes for specific photoactivated organelle imaging. We showed the photoactivated fluorescence recovery of the probes. The photolysis mechanism was verified by mass spectra (MS) analysis, and the highly efficient photolysis process was monitored by high performance liquid chromatography (HPLC). The probes successfully imaged their respective targeted organelle, including mitochondria, lysosomes, and endoplasmic reticulum (ER), in a photoactivated manner, and exhibited high co-localization coefficients when compared to commercially available probes. Given the high reactivity and efficient photodissociation capability of Sanger's reagent with amino groups, our examples demonstrate the potential of Sanger's reagent as a novel general PPG for constructing photoactivatable fluorescent probes.

5-Chlorocoumaranone-Conjugates as Chemiluminescent Protecting Groups (CLPG) and Precursors to Fluorescent Protecting Groups (FPG)

The introduction and cleavage of protecting groups and the subsequent release of protected molecules is an important tool in synthetic organic chemistry. When polyfunctionalized substrates are involved, the reactivity of similar functional groups must be differentiated and selectively switched on and off. A very useful switching agent is visible or UV-light in photoremovable protecting groups (PRPG), allowing the PG release upon interaction with electromagnetic radiation. The reverse principle is the release of a protected molecule, which is accompanied by emission of light, i.e., chemiluminescent protecting groups (CLPG). This principle is proposed and investigated for phenylalanine (using ureido carboxylic acid 2 and its methyl ester derivative 3) and the carbamate derivatives of paracetamol 4 and L-menthol 7, protected as the corresponding urea-/carbamate-coumaranones 5A, 5E, 6 and 8, respectively. While the carbamate derivative 6 released the protected substrate with a short and strong emission of blue light when treated with a base under atmospheric oxygen, 8 had to be treated additionally with potassium hydroxide in boiling ethanol to completely cleave the PG. Both urea-coumaranone derivatives 5A/5E had a flash-like CL without release of the protected amino acid and, thus, were converted into a fluorescent protecting group (FPG).

Controllable Photocleavage of Blebbistatin Derivatives as Photoremovable Protecting Groups.

Blebbistatin was demonstrated as a promising two-photon near-infrared activated photoremovable protecting group of hydroxyl radicals with various potential applications. However, the photocleavage mechanism of the blebbistatin derivatives remains ambiguous. Herein, blebbistatin derivatives with various electronic characteristic leaving groups were synthesized and studied, and the photocleavage mechanism(s) and the tunable effect of the leaving groups were unveiled by combining photoproduct analysis, reactive oxygen radical species detection, femtosecond transient absorption spectroscopy, and density functional theory calculation. More substantial electron-withdrawing leaving groups facilitate heterolysis of the C-O bond, which results in a cationic intermediate and a corresponding remnant. Weaker electron-withdrawing groups lead to a higher proportion of homolysis of the C-O bond, accompanied by the generation of the reactive oxygen radical species. With this structure-property relationship, the protected groups of the molecules of interest can be rationally chosen to satisfy the different requirements needed for specific applications.

Meso-Methyl BODIPY Photocages: Mechanisms, Photochemical Properties, and Applications.

meso-methyl BODIPY photocages have recently emerged as an exciting new class of photoremovable protecting groups (PPGs) that release leaving groups upon absorption of visible to near-infrared light. In this Perspective, we summarize the development of these PPGs and highlight their critical photochemical properties and applications. We discuss the absorption properties of the BODIPY PPGs, structure-photoreactivity studies, insights into the photoreaction mechanism, the scope of functional groups that can be caged, the chemical synthesis of these structures, and how substituents can alter the water solubility of the PPG and direct the PPG into specific subcellular compartments. Applications that exploit the unique optical and photochemical properties of BODIPY PPGs are also discussed, from wavelength-selective photoactivation to biological studies to photoresponsive organic materials and photomedicine.

Development of Photoremovable Protecting Groups Responsive to Near-Infrared Two-Photon Excitation and Their Application to Drug Delivery Research

Abstract Researchers have been actively pursuing the development of photoremovable protecting groups (PPGs) that respond to near-infrared (NIR) two-photon (2P) excitation and the use of their caged compounds in cellular experiments. Our recent developments are summarized in this Account. This research on 2P excitation reactions will considerably contribute to the elucidation of the mechanism of functional expression of biologically active substances in biological samples and will lead to the development of novel drugs and new disease treatment methods. Other active research areas include imaging of materials that are responsive to 2P excitation and nanomaterial synthesis. We hope that these research fields will be useful for designing molecules sensitive to 2P excitation in the NIR region.

Temporal Release of Cell Cycle Regulator α-Lipoic Acid: An NIR-Light (Two-Photon) Activatable Quinoxaline-Based Nano-Prodrug Delivery System.

The regulation of the cell cycle has recently opened up a new research perspective for cancer treatment. So far, no effort has been made for temporal control of cell cycles using a photocleavable linker. Presented herein is the first report of regulation of disrupted cell cycles through the temporal release of a well-known cell cycle regulator α-lipoic acid (ALA), enabled by a newly designed NIR-active quinoxaline-based photoremovable protecting group (PRPG). The suitable quinoxaline-based photocage of ALA (tetraphenylethelene conjugated) has been formulated as fluorescent organic nanoparticles (FONs) and used effectively as a nano-DDS (drug delivery system) for better solubility and cellular internalization. Fascinatingly, the enhanced TP (two-photon) absorption cross section of the nano-DDS (503 GM) signifies its utility for biological applications. Using green light, we have successfully controlled the time span of cell cycles and cell growth of skin melanoma cell lines (B16F10) by the temporal release of ALA. Further, in silico studies and PDH activity assay supported the observed regulatory behavior of our nano-DDS with respect to photoirradiation. Overall, this approach expands the research path toward a futuristic photocontrolled toolbox for cell cycle regulation.

Design and application of a new julolidine-based photolabile protecting group

Photolabile protecting groups (PPGs) are invaluable tools for protecting a variety of functionalities and serve as alternatives to protecting groups that need chemical cleavage. PPGs are mainly classified into 3 categories namely, 2-nitrobenzyl type PPGs, coumarin-based PPGs, and benzyl type PPGs which have been designed by Wang and co-workers based on the meta effect and computational study observations. This work describes the design and synthesis of a julolidine-based benzyl type PPG 4 and the analysis of its reaction intermediates. The julolidine PPG 4 is applied for the protection of alcohols as carbonates, amines as carbamates, carboxylic acids as esters, and hydroxamic acid. Analysis of the photolyzed samples by HPLC has also been conducted.