A Palette of Minimally Tagged Sucrose Analogues for Real‐Time Raman Imaging of Intracellular Plant Metabolism

Abstract

Sucrose is the main saccharide used for long‐distance transport in plants and plays an essential role in energy metabolism; however, there are no analogues for real‐time imaging in live cells. We have optimised a synthetic approach to prepare sucrose analogues including very small (≈50 Da or less) Raman tags in the fructose moiety. Spectroscopic analysis identified the alkyne‐tagged compound 6 as a sucrose analogue recognised by endogenous transporters in live cells and with higher Raman intensity than other sucrose derivatives. Herein, we demonstrate the application of compound 6 as the first optical probe to visualise real‐time uptake and intracellular localisation of sucrose in live plant cells using Raman microscopy.

Mitokyne: A Ratiometric Raman Probe for Mitochondrial pH

“…Stimulated Raman scattering (SRS) microscopy is a powerful, cutting-edge technology for visualizing endogenous cellular biomolecules in a label-free manner with excellent spatial resolution and video rate image acquisition . The recent advent of commercial vendors of instruments for SRS has accelerated the integration of this spectroscopic method into pharmaceutical and drug discovery research. , The technique can be used to track and monitor the cellular milieu and its response to external stimuli in a spatiotemporal manner, and in combination with chemical probes, it can be used to image the distribution of drugs, − natural products, − sugars, − and lipids. − Raman-active tags have also been metabolically incorporated into nucleic acids and proteins significantly expanding the potential uses of this instrumentation . The majority of probes reported so far for SRS imaging possess constant signal intensity and frequency, which limits their applications to staining. − An exciting yet overlooked area of Raman imaging is the development of reversible sensors which permit the quantification and monitoring of cellular dynamics.…”

Local Crowd, Local Probe: Strengths and Drawbacks of Azidohomoalanine as a Site-Specific Crowding Probe

“…14,23−26 These probes have been demonstrated as effective tools in various biologically relevant spectroscopic studies including the measurement of protein folding, ligand binding, side-chain solvation, local electrostatics, and metabolism. 14,16,17,24,27 Among nitriles, azidohomoalanine (AHA) is a particularly appealing spectroscopic probe due to its relatively large oscillator strength. 15,16 In addition to being minimally perturbative, vibrational probes provide localized information, and can be installed at various locations in a protein, providing a view of the nearby chemical environment.…”

“…Furthermore, these modes are often delocalized, restricting their efficacy in interrogating the local environment. One effective solution to these problems has been the implementation of local vibrational probes–minimally perturbative chemical motifs appearing in otherwise unoccupied spectral regions–including nitriles, azides, and thiocyanates. ,− These probes have been demonstrated as effective tools in various biologically relevant spectroscopic studies including the measurement of protein folding, ligand binding, side-chain solvation, local electrostatics, and metabolism. ,,,, Among nitriles, azidohomoalanine (AHA) is a particularly appealing spectroscopic probe due to its relatively large oscillator strength. , In addition to being minimally perturbative, vibrational probes provide localized information, and can be installed at various locations in a protein, providing a view of the nearby chemical environment.…”