trans-Cyclooctene groups incorporated into proteins via non-canonical amino acids (ncAAs) are emerging as specific handles for bioorthogonal chemistry. Here, we present a highly improved synthetic access to the axially and the equatorially linked trans-cyclooct-2-ene isomers (1 a,b). We further show that the axially connected isomer has a half-life about 10 times higher than the equatorial isomer and reacts with tetrazines much faster, as determined by stopped-flow experiments. The improved properties resulted in different labeling performance of the insulin receptor on the surface of intact cells.
Several
diseases are associated with disturbed redox signaling
and altered metabolism of sulfur-containing metabolites and proteins.
Importantly, oxidative degradation of fresh-frozen tissues begins
within the normal time scale of MALDI MSI sample preparation. As a
result, analytical methods that preserve the redox state of the tissue
are urgently needed for refined studies of the underlying mechanisms.
Nevertheless, no derivatization strategy for free sulfhydryl groups
in tissue is known for MALDI MSI. Here, we report the first derivatization
reagent, (E)-2-cyano-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-(4-hydroxyphenyl)acrylamide (CHC-Mal),
for selective detection of free thiols using MALDI MSI. We performed
in situ derivatization of free thiol groups from thiol-containing
metabolites such as glutathione and cysteine and reduced proteins
such as insulin and imaged their spatial distribution in porcine and
mouse xenograft tissue. Derivatization of thiol-containing metabolites
with CHC-Mal for MALDI MSI was also possible when using aged tissue
in the presence of excess reducing agents. Importantly, CHC-Mal-derivatized
low mass-metabolites could be detected without the use of a conventional
MALDI matrix.
Freisetzung in Zellen: Das Signalmolekül PI(3,4,5)P3 wurde chemisch in einer Form zugänglich gemacht, die seine zelluläre Aufnahme und anschließende Aktivierung durch Licht ermöglicht (siehe Bild). Die zügige achtstufige Synthesesequenz liefert ein stabiles Derivat des Signalmoleküls mit hoher biologischer Aktivität nach Blitzlichtphotolyse in lebenden Zellen.
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