Amino acids for Diels-Alder reactions in living cells.

The endeavour to perform tailored chemical reactions in the challenging environment of the intact cell delves deeply into the biological sciences. Requirements include strict bioorthogonality of the reactants and reactions that occur spontaneously and quickly in an aqueous environment or at the interface of membranes. Commonly used reactions that meet these criteria are Staudinger ligations and various forms of click chemistry. The most prominent among the latter is the Huisgen-type [3+2] cycloaddition between azides and alkynes. 2] Through the seminal work of the Bertozzi group, this reaction was stripped of its need for Cu catalysis by straining the alkyne group, thereby making this chemistry (termed strain-promoted alkyne–azide chemistry, SPAAC) viable in intact cells as well as in living animals. These reactions have been widely used to label molecules on cell surfaces and, in a few cases, inside the cell, for instance to label lipids, nucleotides, or carbohydrates. Another exciting click variant is strain-promoted inverse-electrondemand Diels–Alder cycloaddition (SPIEDAC), which can exhibit accelerated reaction rates by using strained reactants and furthermore is irreversible because of the loss of N2 (Scheme 1). This chemistry has been used in cells to label small molecules and is magnitudes faster than the classical Huisgen-type cycloadditions. To date, most biological applications of SPAAC or SPIEDAC do not involve modifications of proteins but instead alter cellular molecules that are not genetically encoded, such as metabolically incorporated sugars. Current tools for site-specific labeling of proteins within the cell use fluorescent protein fusions, self-alkylating protein additions, or high-affinity binding domains. The smallest size of artificial protein modifications currently available to introduce fluorescent labels are tetracysteine motifs consisting of six amino acids. Ideally the modification unit would be only a single artificial amino acid suitable for specific chemistry in cells. The introduction of such unnatural amino acids (UAAs) is possible by codon reassignment or by suppression of the Amber stop codon. For fluorescent labeling, genetically encoded azides can be used, but azides typically suffer from intracellular reduction. Furthermore, encoding the azide jeopardizes the design of a fluorogenic labeling scheme. 16] Fluorogenicity is of particular relevance for high-contrast imaging and super-resolution techniques, since dyes are turned on only after successful labeling, while nonspecifically attached dyes remain quenched. Rather than encoding azides, a more suitable approach is the use of an amino acid that carries the strained reactant, that is, a cyclooctyne group, thereby leaving the nitrogen-bearing reactants to serve as part of a fluorogenic probe. If suppression of the Amber stop codon is used, a single residue in a specified protein can then be replaced with the strained alkyne. This type of protein labeling by using an artificially introduced cyclooctyne amino acid and fluorogenic azides Scheme 1. a) Structures of strained alkene and alkyne UAAs. b) Reaction scheme showing orthogonality and cross-reactivity of SPIEDAC and SPAAC with fluorogenic tetrazine-functionalized dyes (gray sphere) and azide-functionalized dyes (green sphere). Dyes coupled to tetrazine are only fluorescent (green) after successful labeling.