Chemodivergent manganese-catalyzed C-H activation: modular synthesis of fluorogenic probes

Abstract

Bioorthogonal late-stage diversification of amino acids and peptides bears enormous potential for drug discovery and molecular imaging. Despite major accomplishments, these strategies largely rely on traditional, lengthy prefunctionalization methods, heavily involving precious transition-metal catalysis. Herein, we report on a resource-economical manganese(I)-catalyzed C-H fluorescent labeling of structurally complex peptides ensured by direct alkynylation and alkenylation manifolds. This modular strategy sets the stage for unraveling structure-activity relationships between structurally discrete fluorophores towards the rational design of BODIPY fluorogenic probes for real-time analysis of immune cell function.

Fig. 1. Divergent and modular assembly of fluorogenic probes via Earth-abundant manganese(I)-catalyzed C–H activation.

a A versatile approach towards diverse fluorogenic probes. b Selected optimization findings for the utilization of bromoalkyne B1 and terminal alkyne B2 to furnish linear alkynylated and bent alkenylated BODIPY amino acid imaging probes. 2-py, 2-pyridyl; SAR, structure–activity relationship; (1-Ad)CO₂H, 1-adamantanecarboxylic acid; DCE, 1,2-dichloroethane; PivOH, pivalic acid.

Fig. 2. Manganese-catalyzed fluorescent labeling of small peptides via late-stage C–H stitching.

Synthesis of peptides 6–18 featuring a linear linker between the BODIPY fluorophore and the tryptophan amino acid via C–H alkynylation.

Fig. 3. Manganese-catalyzed fluorescent labeling of small peptides via late-stage C–H stitching.

Synthesis of peptides 20–45 featuring a bent linker between the BODIPY fluorophore and the tryptophan amino acid via late-stage C–H alkenylation.

Fig. 4. Manganese-catalyzed fluorescent labeling of complex peptides via late-stage C–H stitching.

Synthesis of peptides 47–53 featuring a bent linker between the BODIPY fluorophore and the tryptophan via C–H stitching, late-stage diversification of natural product derivatives featuring the 2,5-diketopiperazine core (54–57) and synthesis of fluorescent cyclic peptides via C–H alkenylation.

Fig. 5. Evaluation of fluidity-sensitive fluorogenic BODIPY probes.

a Spectral properties of the BODIPY amino acids 2, 3, and 60. λ_exc: 450 nm. [^†] Measured in EtOH, [^‡] measured in glycerol:H₂O (6:4) with fluorescein in basic EtOH as a standard, [^§] measured in glycerol:H₂O mixtures with increasing viscosity. b Fluorescence bar plots displaying the fluorescence fold increase in 0–60% glycerol in water (left) and fluorescence emission in 60% glycerol-water (right) of amino acids 2, 3, and 60 (25 µM), λ_exc: 450 nm. Inset) Pictograms of compound 3 under UV excitation in PBS (left) and in liposome suspensions (right). c Fluorescence emission bar plot of lipid-fluorophore conjugates 41–44 (1 µM) after incubation in phosphatidylcholine (PC): cholesterol liposome suspensions (gray) and in PBS (white), λ_exc: 450 nm. d Fluorescence confocal microscopy images of compound 41 (1 µM, green) after incubation with DMPC-based liposomes containing increasing amounts of cholesterol. Scale bar: 10 µm. In all panels, data presented as means ± SEM from experiments performed at least in triplicate. Fluorescence spectra of compound 41 (1 µM) in cholesterol-containing liposomes (green) and in PBS (black). Data presented as means ± SEM (n = 4) for emission in presence of liposomes and (n = 12) for emission in PBS. P values obtained from ONE-ANOVA tests with multiple comparisons. ε, molar extinction coefficient; lipos., liposomes; chol., cholesterol.

Fig. 6. Probe 41 enables fluorescence-based screening of small molecule modulators of CD8⁺ T cells.

a Schematic illustration of our fluorescence-based screening to detect cholesterol fluctuations in T cells using compound 41. b Quantification of the fluorescence emission of Jurkat T cells after incubation with different small molecules and staining with compound 41 (1 µM, λ_exc/_em: 488/525 nm). Data presented as means + SEM (n = 6). c Fluorescence confocal microscopy images of activated CD8⁺ T cells with avasimibe (30 µM) or without avasimibe. Cells were stained with compound 41 (1 µM, green) and Hoechst 33342 (blue, 7 µM) (λ_exc/_em: 405/450 nm (Hoechst), 488/525 nm (4)). White arrows point at the plasma membrane localization of probe 41. Scale bar: 10 µm. d Quantification of the fluorescence intensity (n = 7) and the percentage (n = 6) of 41-stained CD8⁺ T cells under different activation conditions. Flow cytometry analysis of the CD8⁺ T cells markers PD-1 (no avasimibe: n = 6, IL-2 activation: n = 7, IL-2 activation + avasimibe: n = 5) and CD62L (no avasimibe/IL2-activation: n = 6, IL-2 activation+avasimibe: n = 9) before and after treatment with avasimibe (30 µM). In all panels, data presented as means ± SEM from experiments performed at least in triplicate. P values obtained from unpaired two-tailed t tests.