Chem. imaging under numerous modalities. Bioorthogonal reactions for coupling materials in the presence of complex biological milieu are of great interest in biology and medicine. Such reactions have become key components in a variety of applications including protein engineering (1, 2), immunoassay development (3), and cell surface modification (4,5). To date, only a few bioorthogonal reactions have been reported, the most popular being the Staudinger ligation and the [3 + Tamibarotene 2] cycloaddition click reaction between azides and alkynes (6, 7). Here, we report on the use of [4 + 2] DielsCAlder cycloadditions between a tetrazine and olefin as an alternative bioorthogonal reaction. The reaction is extremely selective, high yielding, and proceeds rapidly in aqueous media. The reaction partners show excellent stability in biological Rabbit Polyclonal to IRAK1 (phospho-Ser376) media and are simple to synthesize. The utility of this reaction is demonstrated by the specific labeling of Her2/neu receptors on breast cancer cells. Recently, there has been tremendous interest in the use of the click reaction for biological labeling. The typical click reaction involves copper(I)-catalyzed coupling of an azide and terminal alkyne to generate a stable triazole (7). Until recently, the necessity of the copper catalyst precluded the use of this reaction in biological systems due to concerns regarding toxicity. Bertozzi and others have elegantly solved this problem by developing several new ring-strained cyclooctyne derivatives that do not require a catalyst (4, 8, 9). However, many of these derivatives have poor water solubility or require complex multistep synthesis and are not readily obtainable in large quantities. Despite these shortcomings, the cycloaddition reaction between azides and ring-strained cyclooctynes has been employed for imaging of cells (4) and zebra fish embryos (10). Our search for alternative rapid, selective, and chemically accessible coupling reactions that do not require a catalyst led us to investigate the [4 + 2] DielsCAlder cycloaddition. Not only is the DielsCAlder reaction compatible with aqueous environments, but the second-order rate constants for this reaction are known to be enhanced up to several hundred-fold in aqueous media in comparison to organic solvents (11, 12). Many DielsCAlder reactions are reversible (13) and therefore may not be suitable for biological labeling. The inverse electron demand DielsCAlder cycloaddition of olefins with tetrazines, however, results in irreversible coupling, giving dihydropyridazine products (Scheme 1). During this reaction, dinitrogen is released in a retro DielsCAlder step (14). A variety of tetrazines (15) and dienophiles including cyclic and linear alkenes or alkynes (16) have been studied in this reaction. Selection of the appropriate reaction partners allows for tuning of the coupling rate by several orders of magnitude (15, 16). Open Tamibarotene in a separate window Scheme 1 To probe the feasibility of the tetrazineCdienophile reaction as a tool for biological labeling, a modified norbornene (2) was selected as a model dienophile. Norbornenes offer an excellent balance between facile strain-promoted reactivity with tetrazines and overall chemical stability. Furthermore, a selection of norbornenes with additional conjugation handles are commercially available. In contrast, few tetrazines containing additional reactive groups have been reported. One potential starting point, dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate, has been investigated extensively, but is not stable in aqueous or protic media (17). Other amine-modified tetrazines such Tamibarotene as 1,2,4,5-tetrazine-3,6-diamine (18) or 3,6-bis-(4-aminophenyl)-1,2,4,5-tetrazine (19) have Tamibarotene been described, but have poor reactivity Tamibarotene with dienophiles. In this communication, we detail the synthesis of a stable benzylamine-modified tetrazine with excellent dienophile reactivity and investigate its use in the bioorthogonal pretargeting of live cells. The tetrazine, 3-(4-benzylamino)-1,2,4,5-tetrazine (1), is prepared by reaction of 4-(aminomethyl)benzonitrile with formamidine acetate and anhydrous hydrazine in the presence of elemental sulfur. The initial dihydrotetrazine product is oxidized to the tetrazine by treatment with sodium nitrite in acetic acid in 20% overall yield. The primary amine can be modified via standard amide-coupling procedures to prepare the near-infrared (NIR) fluorophore-modified conjugate, tetrazine-VT680.