Turning Red without Feeling Embarrassed─Xanthenium-Based Photocages for Red-Light-Activated Phototherapeutics.

Herein, we present high-yielding, concise access to a set of xanthenium-derived, water-soluble, low-molecular-weight photocages allowing light-controlled cargo release in the green to red region. Very importantly, these new photocages allow installation of various payloads through ester, carbamate, or carbonate linkages even at the last stage of the synthesis. Payloads were uncaged with high efficiency upon green, orange, or red light irradiation, leading to the release of carboxylic acids, phenols, and amines. The near-ideal properties of a carboxanthenium derivative were further evaluated in the context of light-controlled drug release using a camptothecin-derived chemotherapeutic drug, SN38. Notably, the caged drug showed orders of magnitude lower efficiency in cellulo, which was reinstated after red light irradiation. The presented photocages offer properties that facilitate the translation of photoactivated chemotherapy toward clinical applications.

Microscope laser assisted photooxidative activation of bioorthogonal ClickOx probes.

A photoactivatable fluorogenic tetrazine-rhodaphenothiazine probe was synthesized and studied in light-assisted, bioorthogonal labeling schemes. Experimental results revealed that the bioorthogonally conjugated probe efficiently sensitizes 1O2 generation upon illumination with green or orange light and undergoes self-oxidation leading to an intensely fluorescent sulfoxide product. An added value of the present probe is that it is also suitable for STED super-resolution microscopy using a 660 nm depletion laser.

Bioothogonally applicable, π-extended rhodamines for super-resolution microscopy imaging for intracellular proteins.

A set of new, bioorthogonally applicable tetrazine and polarity modulated double fluorogenic π-extended rhodamine probes were synthesized. Fluorogenicity and cell labeling experiments suggest that combination of the two quenching mechanisms allows low background labeling schemes even for probes with poor reactivity based fluorogenicity. Two of the new probes were tested in biological labeling schemes of intracellular proteins both in fixed and live cells. The labeled cells were subsequently subjected to confocal and STED imaging. These studies revealed that the rhodaindanes tested are membrane permeable, can stand the challenging environment of live cells and suitable for bioorthogonal, site-specific labeling of intracellular proteins. Furthermore, we found that both probes are suitable for subdiffraction imaging of the labeled structures using STED microscopy.

A rapid and concise setup for the fast screening of FRET pairs using bioorthogonalized fluorescent dyes.

One of the most popular means to follow interactions between bio(macro)molecules is Förster resonance energy transfer (FRET). There is large interest in widening the selection of fluorescent FRET pairs especially in the region of the red/far red range, where minimal autofluorescence is encountered. A set of bioorthogonally applicable fluorescent dyes, synthesized recently in our lab, were paired (Cy3T/Cy5T; Cy1A/Cy3T and Cy1A/CBRD1A) based on their spectral characteristics in order to test their potential in FRET applications. For fast elaboration of the selected pairs we have created a bioorthogonalized platform based on complementary 17-mer DNA oligomers. The cyclooctynylated strands were modified nearly quantitatively with the fluorophores via bioorthogonal chemistry steps, using azide- (Cy1; CBRD1) or tetrazine-modified (Cy3; Cy5) dyes. Reactions were followed by capillary electrophoresis using a method specifically developed for this project. FRET efficiencies of the fluorescent dye pairs were compared both in close proximity (5' and 3' matched) and at larger distance (5' and 5' matched). The specificity of FRET signals was further elaborated by denaturation and competition studies. Cy1A/Cy3T and Cy1A/CBRD1A introduced here as novel FRET pairs are highly recommended for FRET applications based on the significant changes in fluorescence intensities of the donor and acceptor peaks. Application of one of the FRET pairs was demonstrated in live cells, transfected with labeled oligos. Furthermore, the concise installation of the dyes allows for efficient fluorescence modification of any selected DNA strands as was demonstrated in the construction of Cy3T labeled oligomers, which were used in the FISH-based detection of Helicobacter pylori.