Modern Synthetic Avenues for the Preparation of Functional Fluorophores

Abstract

Biomedical research relies on the fast and accurate profiling of specific biomolecules and cells in a non-invasive manner. Functional fluorophores are powerful tools for such studies. As these sophisticated structures are often difficult to access through conventional synthetic strategies, new chemical processes have been developed in the past few years. In this Minireview, we describe the most recent advances in the design, preparation, and fine-tuning of fluorophores by means of multicomponent reactions, C-H activation processes, cycloadditions, and biomolecule-based chemical transformations.

Keywords: C−H activation; fluorescent probes; imaging; microscopy; multicomponent reactions.

Figure 1

Classical and modern synthetic strategies for the preparation of functional fluorophores.

Figure 2

Representative examples of functional fluorophores obtained by MCRs. A) A polarity‐dependent boron‐containing fluorophore. B) Bis(imidazole) heavy‐metal probes. C) N‐Heterocyclic carbene derived optical probes. D) Fluorophores that bind to benzodiazepine receptors in mitochondria. E) Coumarin‐containing fluorescent peptoids that target mitochondria. F) Rhodamine‐based tags for bioorthogonal chemistry and protein profiling. G) Dansyl‐based protein reactive polymers. H) PhagoGreen as a pH‐sensitive BODIPY fluorophore for in vivo imaging of phagocytic macrophages. I) Blue‐emitting furoisoquinolines. The fragments originating from the various precursor building blocks are colored to highlight the multicomponent nature of the syntheses.

Figure 3

Synthesis and application of acyl fluoride mesoionic fluorophores. Isoquinoline‐based mesoionic fluorides for the A) fluorescence detection of histamine (Histamine Blue) and B) fluorescence labeling of oligonucleotides. C) Green‐fluorescent mesoionic BODIPY conjugated to natamycin for imaging fungal cells. Fluorescence images of different fungal species after incubation with the natamycin analogue (5 μm) for 20 min and time‐course analysis. Scale bars: 20 μm. F. oxysporum red, A. flavus green, F. solani blue. TFAA=trifluoroacetic anhydride. Reproduced with permission from the Royal Society of Chemistry17 (A) and the American Chemical Society (C).19.

Figure 4

Top: Synthesis of a fluorogenic Trp–BODIPY amino acid by C−H activation. The Trp–BODIPY amino acid was incorporated into antimicrobial peptides to image the fungal pathogen A. fumigatus in ex vivo human tissue by multi‐photon microscopy. Bottom: Fluorescence images of a) the fluorogenic peptide, b) RFP‐expressing A. fumigatus, and c) merged (a) and (b) together with the second harmonic generation from collagen fibers from lung tissue. Scale bar: 10 μm. DMF=N,N‐dimethylformamide, Fmoc=9‐fluorenylmethoxycarbonyl, MW=microwave, SPPS=solid‐phase peptide synthesis, TFA=trifluoroacetic acid. Reproduced with permission from Springer Nature.22

Figure 5

Preparation of fluorescent compounds by C−H activation. A) Donor–acceptor–donor thienopyrazines as NIR dyes. B) Tunable tetraaryl pyrazole fluorophores. C) Seoul‐Fluors (pyrroloindolizinone fluorophores). D) Pyridotriazole approach to metal probes. E) Palladium‐catalyzed cascade reactions towards solid‐state‐emitting xanthenes. F) A C−H activation approach to fluorescent polyheteroaromatic systems. G) C−H activation to derivatize the BODIPY core. H) CDCs towards indazole‐based fluorescent dyads. I) Fluorescent probes obtained by functionalization of N‐phenylpyridinium ions. J) An Fe³⁺ probe obtained by two CDCs. The chemical bonds formed upon C−H activation are highlighted in red. OTf=trifluoromethanesulfonate, TFE=2,2,2‐trifluoroethanol.

Figure 6

Synthesis of functional fluorophores by means of cycloadditions. A) Preparation of redox‐sensitive coumarins by azide–alkyne 1,3‐dipolar cycloaddition. B) Solvatochromic naphthalenes obtained by intramolecular didehydro‐Diels–Alder reactions. DCE=1,2‐dichloroethane. C) Photoclick reaction between tetrazoles and alkenes to generate environmentally sensitive fluorophores. Inset: Absorbance and fluorescence (λ _ex=405 nm) spectra of a representative pyrazoline adduct in different solvents. ACN=acetonitrile, DCM=dichloromethane, EA=ethyl acetate, PBS=phosphate‐buffered saline. Reproduced with permission from the American Chemical Society.48

Figure 7

Fluorogenic probes with emission enhancement upon cycloaddition. A) Fluorescein‐based Calfluors with intramolecular photoinduced electron transfer (PeT) quenching and fluorescence emission after CuAAC reaction. Fluorescence spectra of Calfluors covering the whole spectral range. B) A representative example of a BODIPY–tetrazine fluorogen and its Diels–Alder condensation with trans‐cyclooctenes (TCO) to produce superbright fluorophores. Fluorescence microscopy images of A‐431 live cells after incubation with anti‐EGFR TCO‐conjugated monoclonal antibodies and BODIPY–tetrazines. C) Modifications of protein surfaces by cycloadditions with sydnones. Left: Dibenzoazacyclooctyne (DIBAC) and 5‐norbornene‐2‐acetic acid (Nor) are attached to the proteins BSA and OVA by amide formation. The labeled proteins BSA–DIBAC and OVA–Nor simultaneously react with sydnone–BODIPY (Syd‐630) and tetrazine–BODIPY (Tz‐504). Right: Gel analysis of BSA–DIBAC and OVA–Nor after incubation with either Syd‐630, Tz‐504, both reagents simultaneously, or no reagent (−). Reproduced with permission from the American Chemical Society54 (A), Wiley‐VCH56 (B), and the Royal Society of Chemistry57 (C).

Figure 8

Functional fluorophores using biomolecule‐based approaches. A) Protein labeling by inverse‐electron‐demand Diels–Alder cycloadditions. Structures of genetically encoded unnatural amino acids and tetrazine‐containing fluorophores. B) Photoactivatable phalloidin conjugate of 5‐carboxy‐NVOC2‐SiRhQ. a) Super‐resolution microscopy image of a COS‐7 cell stained with the phalloidin conjugate. b) Expanded image of the boxed region in (a), showing a protruding filopodial structure, and the line‐scan intensity across the filopodial structure in (b) (shown in black) and a Gaussian fit (red). C) Two‐step procedure for subcellular labeling of the Golgi apparatus in live cells; cells are treated first with Cer‐TCO, a trans‐cyclooctene‐containing ceramide lipid, and then reacted with the tetrazine fluorophore SiR‐Tz for 3D confocal and stimulated emission depletion (STED) super‐resolution microscopy. Reproduced with permission from Springer Nature68 (A) and Wiley‐VCH74, 77 (B, C).