Language：English   Publishing type：Research paper (scientific journal)  

Visualizing specific enzyme activities in living systems is important in biomedical research, and Raman imaging probes are particularly suitable for simultaneous multiplexed detection of plural enzyme activities due to their narrow signal peak widths. Here, we present a molecular design strategy for enzyme-activity-detecting Raman probes based on control of aggregation. The probe itself is soluble and diffusive in aqueous solution due to its hydrophilic substrate moiety, but hydrolysis by the target enzyme affords a hydrophobic product that forms aggregates, thereby increasing the local dye concentration, which in turn results in a stronger Raman signal that enables visualization of the enzyme activity. We validated the molecular design by developing Raman probes targeting aminopeptidase, glycosidase and carboxypeptidase. The vibrational frequency can be shifted by isotope-editing, and the developed probes were successfully applied to visualize the activities of aminopeptidase and glycosidase in live cultured cells and spheroids.

DOI： 10.1021/acs.analchem.5c02231

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Language：English   Publishing type：Research paper (scientific journal)  

Coumarin-hemicyanine (CHC) hybrid fluorophores bearing an intramolecular nucleophile have been reported as ratiometric fluorescent probes based on the equilibrium between the π-conjugated open and cyclic closed forms. Here, we report red-shifted CHC derivatives that can serve as scaffold dyes of activated fluorescent probes and activated Raman probes. We prepared a series of cyano-substituted CHC derivatives and found that substitution at C-4 of the coumarin moiety is effective in extending the absorption wavelength. The near-infrared (NIR) emission of these 4CN-CHC derivatives exhibits high tissue penetration. Dual-color imaging with 4CN-CHC enabled ratiometric monitoring of the lysosomal pH changes. Furthermore, the coherent anti-Stokes Raman scattering (CARS) signals of 4CN-CHC derivatives could be regulated by modifying the open/closed equilibrium, and the different patterns of CARS spectra enabled us to perform multicomponent imaging. Thus, these 4CN-CHC derivatives expand the repertoire of functional probes for NIR and multiplexed vibrational imaging.

DOI： 10.1021/acs.analchem.5c02714

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Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

DOI： 10.1021/jacs.5c04193

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Language：English   Publishing type：Research paper (scientific journal)  

Deuterated compounds used in drug discovery and live-cell imaging have recently gained the attention of various scientific fields. Although hydrogen-deuterium (H/D) exchange reactions are straightforward deuteration methods, achieving perfect chemoselectivity is challenging. We report the highly chemoselective deuteration of α-thio C(sp3)-H bonds using a thioxanthone or anthraquinone organic photocatalyst bearing an aromatic ketone skeleton and D2O as an inexpensive deuterium source under 390 nm irradiation. Notably, incorporation of deuterium at the α-positions of the O/N atoms, benzylic positions, and aromatic rings was not observed. The present chemoselectivity was accomplished via a single electron transfer mechanism between the photocatalyst and S-containing substrates, as proven by laser-induced time-resolved transient absorption spectroscopic measurements. Furthermore, the proposed deuteration method could be applied to various S-containing substrates, including pharmaceuticals and biologically active compounds with high regioselectivities. The available deuterated compounds as novel deuterated alkylation reagents for future drug discovery and materials for Raman imaging were also demonstrated.

DOI： 10.1021/jacs.5c01894

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Publisher：Cold Spring Harbor Laboratory  

Abstract

Combinatorial use of an antibody-reporter enzyme conjugate and a fluorescence probe activated by the enzyme is a powerful strategy for fluorescence-guided cancer surgery. However, conventional probes for typical reporter enzymes are insufficiently bioorthogonal, resulting in high background signals in non-target tissues. We screened a library of HMRef (rhodol derivative)-based fluorescence probes bearing various sugar moieties, and discovered that HMRef-β-D-fucose is bioorthogonal in mammalian systems, but is activated by a metagenomic glycosidase, Td2F2. Directed evolution generated a mutant with ak_cat/K_mvalue for HMRef-β-D-fucose of 3.3 x 10⁵/M/sec, 7.3 times higher than that of wild-type Td2F2 and comparable to that of β-galactosidase (LacZ) with a corresponding probe. Theoretical calculation suggested that E296G mutation in Td2F2 causes structural changes that facilitate the probe’s access to the enzyme’s active site. In a proof-of-concept study, cancer cells were visualized with a minimal background in the mesentery of a mouse model of peritoneally disseminated human-ovarian-cancer-derived SKOV-3 cells, which endogenously express HER2, by using HMRef-β-D-fucose together with engineered Td2F2 conjugated/fused to a HER2-binding antibody/nanobody.

DOI： 10.1101/2025.03.04.641560

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