Red‐Shifted Fluorogenic Substrate for Detection of lacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

Abstract

The Escherichia coli lacZ gene encoding b-galactosidase is awidely used reporter,but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples.O ur recently reported fluorogenic substrate SPiDER-bGal is suitable for this purpose,b ut its hydrolysis product shows green fluorescence emission, and ar ed-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers.Herein, we describe the development of ar ed-shifted fluorogenic substrate for bgalactosidase,S PiDER-Red-bGal, based on as ilicon rhodol scaffold and ac arboxylic group as the intramolecular nucleophile.L acZ-positive cells were successfully labeled with SPiDER-Red-bGal at single-cell resolution in living samples, which enabled us to visualized ifferent cell types in combination with GFP markers.The Escherichia coli lacZg ene,w hich encodes b-galactosidase,i sapowerful reporter in combination with synthetic substrates, [1] and many lacZ-expressing animals have been established for experimental use.H owever,a lthough several substrates are available for detecting b-galactosidase activity, [2] selective labeling of lacZ-positive cells with single-cell resolution in living tissues or organisms is not feasible with most existing probes.W er ecently developed an ew class of functionalized fluorogenic substrate for b-galactosidase, SPiDER-bGal, [3] which is suitable for this purpose;w e controlled the fluorescence through intramolecular spirocyclization and achieved cellular retention by application of quinone methide chemistry.However,the utility of SPiDER-bGal is limited because it emits green fluorescence at around 550 nm, and therefore it is difficult to use in combination with green fluorescent protein (GFP), [4] one of the most widely used fluorescent tags for biological studies.A lthough red fluorescent proteins have been widely used as reporters in the red wavelength region, their tendency to form aggregates when expressed in neurons in mouse brain may limit their applicability. [5] Therefore,d evelopment of ar ed fluorescent probe for b-galactosidase with single-cell resolution should extend the utility of the lacZreporter.Herein, we describe the development of an ew fluorogenic substrate for b-galactosidase,w hich emits red fluorescence at above 600 nm without loss of the advantageous properties of SPiDER-bGal. We show that the new probe,SPiDER-Red-bGal, can label lacZpositive cells in combination with GFP markers and can successfully visualize multiple cell types at single-cell resolution in living samples.In order to develop red-shifted b-galactosidase probes,we first selected 2'Me-Sirhodol (Supporting Information, Scheme S1) as the core fluorophore,i nw hich the oxygen atom at the 10th position of rhodol is replaced with as ilicon atom;t his replacement has been reported to extend the absorption and fluorescence wavelengths by about 100 nm. [6] Indeed, 2'Me-Sirhodol seemed to have favorable properties for our purpose;i ts hows absorption/emission maxi...

An Ionic Liquid Medium Enables Development of a Phosphine-Mediated Amine–Azide Bioconjugation Method

“…Bioconjugation technologies have become a cornerstone of the multifaceted fields of chemistry and biology for various applications spanning therapeutics, , enzyme activity profiling, elucidation of cellular processes, , fluorescence imaging, − and material preparation . Despite the wide array of available chemical tools enabling the rapid growth of such research fields, the development of novel chemical labeling methods remains inevitable, as individual experimental design necessitates particular technologies for diverse aims .…”

Recent Advances in Quinone Methide Chemistry for Protein-Proximity Capturing

“…To improve its viability, they have further disclosed the red-shifted fluorogenic substrate for β-galactosidase SPiDER-Red-βGal, which was built based on a silicon rhodol scaffold and a carboxylic group as the intramolecular nucleophile. 16 LacZ -positive cells were successfully labeled with SPiDER-Red-βGal at single-cell resolution in living cells, displaying great potential in visualization of different cells along with different GFP dyes.…”

A Versatile Linker for Probes Targeting Hydrolases via In Situ labeling

“…25,26 It can be generated by external stimuli under physiological conditions from a stable masked phenol precursor bearing a leaving group (such as a fluorine atom) on the methyl group at the para-or ortho-position. 27 This strategy has been used in inhibitors and prodrugs, [28][29][30] self-immolative linkers in dendrimers, 31 chemical selection of catalytic antibodies, 32,33 activity-based probes for various enzymes, including phosphatase, 34 glycosidases, 22,23,[35][36][37][38][39][40][41] , tyrosine phosphatase, 42 steroid sulfatase, 43 and beta-lactamase, 44 cell imaging 45,46 and probing protein-protein/DNA interactions. 47 Among these applications, the fluorine on either the monofluoromethyl or difluoromethyl group was used as a leaving group to form the active o-quinone methide intermediate via spontaneous elimination of a molecule of HF.…”