Huisgen 1,3-dipolar cycloaddition between a terminal alkyne group and an azide, provide new possibilities for the bioorthogonal labeling of molecules like amino acids

This article is available online at http://www.jlr.org to radioactive labeling. They are convenient and sensitive reporters, but in general contain spacious tags of high impact on the structure, likely to infl uence specifi city and kinetic parameters of enzymatic reactions. Although there are reports of assays with unchanged affi nity of the enzyme to fl uorescently labeled substrates, notably nitrobenzoxadiazole (NBD)-sphingolipids [e.g., NBD-sphinganine ( 2 ), NBDsphingosine-1-phosphate ( 3 )] the applicability of a substrate has to be validated thoroughly. For instance, when the boron dipyrromethene (BDP) analog, BDP-sphingosine-1phosphate, was developed as a more photostable and hydrophobic alternative to NBD-sphingosine-1-phosphate, the BDP-, but not the NBD-derivate displayed a higher K m value than the natural substrate ( 4 ). A smaller nonradioactive universally applicable tag with less impact on the substrate structure would hence be benefi cial. Click chemistry ( 5 ), and especially the copper-catalyzed Huisgen 1,3-dipolar cycloaddition between a terminal alkyne group and an azide, provide new possibilities for the bioorthogonal labeling of molecules like amino acids ( 6 ), sugars ( 7 ), and nucleotides ( 8 ), which have already triggered many applications in life sciences ( 9, 10 ). In lipid biology, alkyne and azide analogs and precursors are already applied broadly, e.g., in protein lipidation analyses [reviewed in ( 11 )], receptor binding studies ( 12 ), tracing of fatty acid metabolism ( 13 ), and the labeling of lipids in Abstract Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specifi c detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fl uorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thinlayer chromatography and fl uorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays. —Gaebler, A., R. Milan, L. Straub, D. Hoelper, L. Kuerschner, and C. Thiele. Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays. J. Lipid Res. 2013. 54: 2282–2290.