Abstract
Raman spectroscopy is well-suited to the study of bioorthogonal reaction processes because it is a non-destructive technique, which employs relatively low energy laser irradiation, and water is only very weakly scattered in the Raman spectrum enabling live cell imaging. In addition, Raman spectroscopy allows species-specific label-free visualisation; chemical contrast may be achieved when imaging a cell in its native environment without fixatives or stains. Combined with the rapid advances in the field of Raman imaging over the last decade, particularly in stimulated Raman spectroscopy (SRS), this technique has the potential to revolutionise our mechanistic understanding of the biochemical and medicinal chemistry applications of bioorthogonal reactions. Current approaches to the kinetic analysis of bioorthogonal reactions (including heat flow calorimetry, UV-vis spectroscopy, fluorescence, IR, NMR and MS) have a number of practical shortcomings for intracellular applications. We highlight the advantages offered by Raman microscopy for reaction analysis in the context of both established and emerging bioorthogonal reactions, including the copper(i) catalysed azide-alkyne cycloaddition (CuAAC) click reaction and Glaser-Hay coupling.
Highlights
Bioorthogonal chemistries have been rapidly adopted both in traditional synthetic chemistry and as a means to site-speci cally label biomolecules inside cells.[1,2,3,4,5] More recently their use has been proposed as a means to both construct and de-cage complex drug molecules in vivo as a new therapeutic modality.[6,7] Critical to the success of reactants that couple under bioorthogonal conditions are that they are mutually reactive but do not cross-react, or interact, with other biological functionalities or reactions in a cell; that they are stable and non-toxicPaper in physiological settings; and that their mutual reaction is highly speci c and fast.[1]
Raman spectroscopy is well-suited to the study of bioorthogonal reaction processes because it is a non-destructive technique, which employs relatively low energy laser irradiation, and water is only very weakly scattered in the Raman spectrum enabling live cell imaging
The CuAAC reaction has been studied previously using IR spectroscopy in an organic solvent (DMF) following the decay of the azide peak at 2096 cmÀ1.10 Here, we show that the CuAAC reaction can be followed by IR spectroscopy in an aqueous solvent mixture (Fig. S1†), but not one that is compatible with biological cells
Summary
Bioorthogonal chemistries have been rapidly adopted both in traditional synthetic chemistry and as a means to site-speci cally label biomolecules inside cells.[1,2,3,4,5] More recently their use has been proposed as a means to both construct and de-cage complex drug molecules in vivo as a new therapeutic modality.[6,7] Critical to the success of reactants that couple under bioorthogonal conditions are that they are mutually reactive but do not cross-react, or interact, with other biological functionalities or reactions in a cell; that they are stable and non-toxicPaper in physiological settings; and that their mutual reaction is highly speci c and fast.[1]. Differences arising from intracellular macromolecular crowding,[13] variations in pH across cell populations and within individual cells,[14] and the sequestration of substrates into cellular structures or organelles[15] are poorly accounted for by bulk in vitro solution phase analysis, necessitating the urgent development of techniques which might be applicable in an in cellulo or in vivo microenvironment
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