Abstract
A chemically-activatable alkynyl steroid analogue probe has been synthesized for visualizing the lipid raft membrane domains by Raman microscopy. The Raman probe, in which ring A of its steroid backbone is replaced with an alkynyl group, was designed to enable activation of the alkyne signal through the Eschenmoser-Tanabe fragmentation reaction of the oxidized cholesterol precursor in lipid bilayer membranes. The alkynyl steroid analogue was observed to form liquid-ordered raft-like domains on a model giant-liposome system in a similar manner as cholesterol, and the large alkyne signal of the accumulated probe at 2120 cm−1 was mapped on the microdomains with a Raman microscope. The alkyne moiety of the probe was confirmed to be converted from the α,β-epoxy ketone group of its precursor by reaction with p-toluensulfonyl hydrazine under a mild condition. Through the reaction, the alkyne signal of the probe was activated on the lipid bilayer membrane of liposomes. Furthermore, the signal activation of the probe was also detected on living cells by stimulated Raman scattering microscopy. The ring-A-opened alkyne steroid analogue, thus, provides a first chemically-activatable Raman probe as a promising tool for potentially unravelling the intracellular formation and trafficking of cholesterol-rich microdomains.
Highlights
Alkynes have recently attracted attention as a label to replace bulky charged fluorescent dyes
When steroids are inserted into a lipid bilayer membrane, ring D, tethering an alkyl chain, locates deep inside the membrane; ring A lies toward the membrane surface
A ring-A-opened alkyne steroid analogue probe has been synthesized as a first chemically-activatable Raman probe
Summary
Alkynes have recently attracted attention as a label to replace bulky charged fluorescent dyes. A wide variety of activatable fluorescent probes that response to various stimuli such as light[17], ions[18,19], chemicals[20] and enzymes[21] have been actively studied By using such probes, the labelled molecules which go through light-irradiated areas or specified environments such as low-pH, highly-reduced and enzyme-rich sites could be selectively visualized. In live-cell Raman imaging, activation of the present probe was observed to be accelerated under a mildly acidic condition due to low pH-enhanced conversion to the alkyne moiety[22] These studies demonstrate the proof of principle that the present activatable probe may visualize the dynamics and trafficking of cholesterol-rich microdomains from the activation point of location in a mildly acidic environment on living systems
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