Hiroyoshi Fujioka

Multicolor vibrational imaging in cells and tissues has recently advanced rapidly because of the integration of cutting-edge microscopic techniques with sophisticated chemically engineered probes. Here, we review design strategies for Raman imaging probes, categorized by their chemical structure. Finally, we discuss the potential of multicolored vibrational probes for investigating complex biological phenomena and identify areas requiring further research.

9-Cyanopyronin derivatives have been used as resonance Raman probes for highly sensitive and multiplexed vibrational biological imaging. Here, we synthesized a series of fourteen 9-cyanopyronin derivatives in which the 10th core atom is substituted with various group 13, 14, 15 or 16 elements, and systematically evaluated their properties using electronic pre-resonance stimulated Raman scattering (EPR-SRS) microscopy. Substitution at the 10th position modulates not only the vibrational frequency of the nitrile (C ≡ N) group in the silent region, but also the conjugated double-bond stretching frequency (C = C) in the fingerprint region, and additionally alters the subcellular localization of the probes. By employing different combinations of probes, we were able to utilize the distinct patterns of C ≡ N or C = C bond vibrations together with the different subcellular localizations of the probes to achieve live-cell three-color imaging. These 9-cyanopyronin derivatives greatly expand the vibrational palette available for multiplexed SRS imaging. The alt text for this image may have been generated using AI.

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.

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.

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.

Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.

Although deuterium incorporation into pharmaceutical drugs is an attractive way to expand drug modalities, their physicochemical properties have not been sufficiently examined. This study focuses on examining the changes in physicochemical properties between flurbiprofen (FP) and flurbiprofen-d₈ (FP-d₈), which was successfully prepared by direct and multiple H/D exchange reactions at the eight aromatic C–H bonds of FP. Although the effect of deuterium incorporation was not observed between the crystal structures of FP and FP-d₈, the melting point and heat of fusion of FP-d₈ were lower than those of FP. Additionally, the solubility of FP-d₈ increased by 2-fold compared to that of FP. Calculation of the interaction energy between FP/FP-d₈ and water molecules using the multi-component density functional theory method resulted in increased solubility of FP-d₈. These novel and valuable findings regarding the changes in physicochemical properties triggered by deuterium incorporation can contribute to the further development of deuterated drugs.

Super-resolution vibrational microscopy is promising to increase the degree of multiplexing of nanometer-scale biological imaging because of the narrower spectral linewidth of molecular vibration compared to fluorescence. However, current techniques of super-resolution vibrational microscopy suffer from various limitations including the need for cell fixation, high power loading, or complicated detection schemes. Here, we present reversible saturable optical Raman transitions (RESORT) microscopy, which overcomes these limitations by using photoswitchable stimulated Raman scattering (SRS). We first describe a bright photoswitchable Raman probe (DAE620) and validate its signal activation and depletion characteristics when exposed to low-power (microwatt level) continuous-wave laser light. By harnessing the SRS signal depletion of DAE620 through a donut-shaped beam, we demonstrate super-resolution vibrational imaging of mammalian cells with excellent chemical specificity and spatial resolution beyond the optical diffraction limit. Our results indicate RESORT microscopy to be an effective tool with high potential for multiplexed super-resolution imaging of live cells.