Bioorthogonal Ligation-Activated Fluorogenic FRET Dyads.

An energy transfer-based signal amplification relay concept enabling transmission of bioorthogonally activatable fluorogenicity of blue-excitable coumarins to yellow/red emitting cyanine frames is presented. Such relay mechanism resulted in improved cyanine fluorogenicities together with increased photostabilities and large apparent Stokes-shifts allowing lower background fluorescence even in no-wash bioorthogonal fluorogenic labeling schemes of intracellular structures in live cells. These energy transfer dyads sharing the same donor moiety together with their parent donor molecule allowed three-color imaging of intracellular targets using one single excitation source with separate emission windows. Sub-diffraction imaging of intracellular structures using the bioorthogonally activatable FRET dyads by STED microscopy is also presented.

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.

Bistetrazine-Cyanines as Double-Clicking Fluorogenic Two-Point Binder or Crosslinker Probes.

Fluorogenic probes can be used to minimize the background fluorescence of unreacted and nonspecifically adsorbed reagents. The preceding years have brought substantial developments in the design and synthesis of bioorthogonally applicable fluorogenic systems mainly based on the quenching effects of azide and tetrazine moieties. The modulation power exerted by these bioorthogonal motifs typically becomes less efficient on more conjugated systems; that is, on probes with redshifted emission wavelength. To reach efficient quenching, that is, fluorogenicity, even in the red range of the spectrum, we present the synthesis, fluorogenic, and conjugation characterization of bistetrazine-cyanine probes with emission maxima between 600 and 620 nm. The probes can bind to genetically altered proteins harboring an 11-amino acid peptide tag with two appending cyclooctyne motifs. Moreover, we also demonstrate the use of these bistetrazines as fluorogenic, covalent cross-linkers between monocyclooctynylated proteins.

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.

Bisazide Cyanine Dyes as Fluorogenic Probes for Bis-Cyclooctynylated Peptide Tags and as Fluorogenic Cross-Linkers of Cyclooctynylated Proteins.

Herein we present the synthesis and fluorogenic characterization of a series of double-quenched bisazide cyanine probes with emission maxima between 565 and 580 nm that can participate in covalent, two-point binding bioorthogonal tagging schemes in combination with bis-cyclooctynylated peptides. Compared to other fluorogenic cyanines, these double-quenched systems showed remarkable fluorescence intensity increase upon formation of cyclic dye-peptide conjugates. Furthermore, we also demonstrated that these bisazides are useful fluorogenic cross-linking platforms that are able to form a covalent linkage between monocyclooctynylated proteins.

A New Approach to Non-Coordinating Anions: Lewis Acid Enhancement of Porphyrin Metal Centers in a Zwitterionic Metal-Organic Framework.

We describe a new strategy to generate non-coordinating anions using zwitterionic metal-organic frameworks (MOFs). By assembly of anionic inorganic secondary building blocks (SBUs) ([In(CO2)4](-)) with cationic metalloporphyrin-based organic linkers, we prepared zwitterionic MOFs in which the complete internal charge separation effectively prevents the potential binding of the counteranion to the cationic metal center. We demonstrate the enhanced Lewis acidity of Mn(III)- and Fe(III)-porphyrins in the zwitterionic MOFs in three representative electrocyclization reactions: [2 + 1] cycloisomerization of enynes, [3 + 2] cycloaddition of aziridines and alkenes, and [4 + 2] hetero-Diels-Alder cycloaddition of aldehydes with dienes. This work paves a new way to design functional MOFs for tunable chemical catalysis.

Preparation and studies of chiral stationary phases containing enantiopure acridino-18-crown-6 ether selectors.

The enantiomeric separation ability of the newly prepared chiral stationary phases containing acridino-18-crown-6 ether selectors was studied by high-performance liquid chromatography (HPLC). The chiral stationary phases separated the enantiomers of selected protonated primary aralkylamines efficiently. The best results were found for the separation of the mixtures of enantiomers of NO2 -PEA.

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.

Green-Light Activatable, Water-Soluble Red-Shifted Coumarin Photocages.

Easily accessible green-light activatable (>500 nm) photocages based on red-shifted, π-extended coumarin scaffolds are developed with uncaging efficiencies similar to those of recently introduced BODIPY derivatives. The photocages possess increased aqueous solubility, high absorption coefficients within the 450-600 nm range, and exceptionally high two-photon cross sections.

Synthesis and fluorescent properties of boroisoquinolines, a new family of fluorophores.

First representatives of a new family of isoquinolines, so called boroisoquinolines, were synthesized and characterized. The synthesis was based on the insertion of the difluoroboranyl group into the 1-methylidene-3,4-dihydroisoquinoline core. The optimization of the 2-difluoroboranyl-3,4-dihydroisoquinoline-1(2H)-ylidene core led to efficient fluorescence in a range of 400-600 nm with outstanding (>100 nm) Stokes shifts. The compounds might be suitable for reversible or irreversible labelling of proteins, particularly the cannabinoid receptor CB2.

Self-Supported BINOL-Derived Phosphoric Acid Based on a Chiral Carbazolic Porous Framework.

The facile synthesis of a porous heterogeneous BINOL-derived chiral phosphoric acid BiCz-POF-1 using the mild, FeCl3-promoted oxidative polymerization is reported. For the first time, carbazole is introduced at the 3,3'-positions of the chiral BINOL-derived phosphoric acid to (1) offer steric hindrance for achieving a high enantioselectivity and (2) serve as a cross-linker for the construction of the porous solid catalyst. BiCz-POF-1 exhibits remarkable catalytic activity and enantioselectivity toward transfer hydrogenation of 1,4-benzoxazine, 1,4-benzoxazinone, and 2-phenylquinolone. Combined with its facile synthesis and excellent recyclability, BiCz-POF-1 represents a new class of heterogeneous chiral phosphoric acid that has wide potential utility in enantioselective organocatalysis.