Chemistry & Biology Review Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation Craig S. McKay1 and M.G. Finn1,* 1School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA *Correspondence: mgfi[email protected] http://dx.doi.org/10.1016/j.chembiol.2014.09.002 The selective chemical modification of biological molecules drives a good portion of modern drug develop- ment and fundamental biological research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biological molecules so as to achieve selectivity in desired bond-forming events has revolution- ized the field. We provide an update on recent developments in bioorthogonal chemistry that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chemistry that operates in the bio- logical setting. Introduction: Bioorthogonal Click Chemistry oxygen and water (Kolb et al., 2001; Hawker and Wooley, Chemical biology involves the creation of nonbiological mole- 2005; Kolb and Sharpless, 2003; Wu et al., 2004). In vivo bio- cules that exert an effect on, or reveal new information about, orthogonal chemistry and click chemistry therefore overlap quite biological systems. Central to this field is the property of selec- a bit, reflecting the same underlying chemical principles applied tivity: ultimately, one wishes for molecules with perfectly selec- in somewhat different ways toward the discovery or develop- tive biological function, but in practice, one starts with as much ment of molecular function and information. chemical selectivity as possible and tests and refines from there. To meet stringent requirements of rate, selectivity, and Therefore, the ability to make chemical modifications that enable biocompatibility, the development of bioorthogonal reactions the direct detection of, or interaction with, biomolecules in their proceeds through several steps. First, of course, is the identifica- native cellular environments is at the heart of the chemical tion or invention of a highly specific ligation process that works biology enterprise. well in water. Potential problems associated with reactant/prod- Genetically encoded reporters, such as GFP and tetracysteine uct stabilities and reaction biocompatibility must be anticipated motifs, have been used to excellent effect for protein tagging, but and addressed. The reaction is first optimized ‘‘in the flask,’’ other molecules such as glycans, lipids, metabolites, and myriad where the fundamental scope, limitations, and mechanistic posttranslational modifications are not often amenable to this modifications are explored. Then the reaction is tested in a vari- type of labeling. Monoclonal antibodies usually provide sufficient ety of biological environments, escalating in complexity from target specificity, but are laborious to generate and are often un- aqueous media to biomolecule solutions to cultured cells. The able to enter cells and tissues. Covalent chemical modification most optimized transformations are then tested and employed has therefore emerged as an alternative strategy. Bioorthogonal in living organisms and animals (Sletten and Bertozzi, 2011b). reactant pairs, which are most suitable for such applications, are The reactions highlighted in the following section are at different molecular groups with the following properties: (1) they are mutu- stages of development toward the ultimate goal of in vivo appli- ally reactive but do not cross-react or interact in noticeable ways cation. Second-order rate constants for bioorthogonal reactions with biological functionalities or reactions in a cell, (2) they and reported to date span ten orders of magnitude, with the fastest their products are stable and nontoxic in physiological settings, labeling reactions reaching rates up to 105 MÀ1sÀ1. and (3) ideally, their reaction is highly specific and fast (Sletten This perspective provides a critical review of emerging bio- and Bertozzi, 2009). Rate is an often underappreciated factor conjugation strategies with comments on their general utility by the casual user of bioorthogonal chemical technology: very and challenges. We recommend several excellent published re- high rate constants are required for labeling cellular processes views for more comprehensive accounts of the underlying chem- that occur on fast time scales or with low abundance structures istries (Lang and Chin, 2014; Patterson et al., 2014; Prescher and in (or on) the cell. Bertozzi, 2005; Sletten and Bertozzi, 2009). We also provide a Bioorthogonal chemical reactions have emerged as highly few examples of emerging areas of applications such as trig- specific tools that can be used for investigating the dynamics gered release and prodrug activation. and function of biomolecules in living systems (Jewett and Ber- tozzi, 2010; Lang and Chin, 2014; Lim and Lin, 2010b; Patterson Bioorthogonal Conjugation Strategies and Applications et al., 2014; Prescher and Bertozzi, 2005; Sletten and Bertozzi, Typically, bioorthogonal reactions are exothermic by virtue 2009). Click chemistry, inspired by nature’s use of simple and of their highly energetic reactants or strongly stabilized pro- powerful connecting reactions, describes the most specific bio- ducts, and most involve the formation of carbon-heteroatom orthogonal reactions that are wide in scope, easy to perform, and (mostly N, O, and S) bonds. The reactions are typically fusion usually employ readily available reagents that are insensitive to processes that leave no byproducts or release nitrogen, or are Chemistry & Biology 21, September 18, 2014 ª2014 Elsevier Ltd All rights reserved 1075 Chemistry & Biology Review Figure 1. Strategies for Introducing and Conjugating Functionality to Biomolecule Targets (A) The two-step bioorthogonal labeling strategy: in the first step exogenous functionality is introduced genetically, metabolically or chemically, and the second step involves highly specific bioorthogonal reaction. (B) Site-specific bioconjugation based on native functionalities present in proteins. condensation processes that produce water as the only byprod- amino acid residues of peptides or proteins with high site selec- uct. Most applications involve a two-step approach that requires tivity has become important. The addressable functional groups introduction of bioorthogonal reporters by chemical, metabolic, can be introduced as unnatural amino acids (Wang et al., 2006), or genetic means, followed by modification of the biomolecule metabolic precursors, or installed by bioconjugation reactions. with the bioorthogonal labeling reaction (Figure 1). From a In the last category, the traditional processes, which usually pro- practical point of view, a bioorthogonal process finds useful ceed at low coupling rates or with poor chemoselectivity, have application if the reporter group is stable inside the cell and started to yield to new, highly selective organic transformation can be introduced by systemic distribution, metabolically, or and/or mechanistic modifications that improve ligation kinetics through a promiscuous enzymatic pathway (Sletten and Ber- and the stability of the resulting conjugates. tozzi, 2009). Most bioorthogonal reactions reported in the Emerging Approaches for Lysine Modifications. Lysine, argi- literature are chemoselective with respect to many, but not all, nine, histidine, and N-terminal amines contribute to proteins’ biological functionalities, and have been used to label protein positive charge at neutral pH and are often solvent exposed, in vitro or at the cell surface. Only a very few have been used making them amenable to chemical modification via acylation in much more complex systems inside living cells or animals: or alkylation (Table 1, entries 1–4). While primary amines readily the field, therefore, is really only just getting started. undergo reactions with activated esters, anhydrides, carbon- Site-Specific Bioconjugation Based on Native ates, isothiocyanates, and a range of other acylating and alkylat- Functionalities ing agents at slightly alkaline pH, these reactions are neither site Intrinsic bioconjugation reactions selectively target side chains specific nor protein specific. For example, N-hydroxysuccini- and termini of the 20 naturally occurring proteogenic amino acids mide (NHS) esters of a variety of types can cross-react with (Ban et al., 2013). Classical reactions such as thiol-maleimide the side-chain hydroxyl groups of tyrosine, serine, and threonine additions or amine-activated ester acylations are widely used if there is a nearby histidine to take up the NHS ester as an acyl for derivatizing proteins in vitro, but are typically not sufficiently imidazole, which in turn reacts with the –OH group resulting in an specific or rapid to modify a particular biomolecule in a higher ester bond (Hermanson, 2013). complexity cellular environment. Carbodiimide-based methods A variety of promising alternative approaches have recently for coupling of carboxylates are much less popular, probably appeared that take advantage of the special reactivity of aniline for reasons of positional selectivity or the potential for undesired groups, imine/iminium intermediates, and diazonium centers. protein crosslinking, although there are certainly exceptions Thus, a two-stage methodology from Francis and coworkers (Schlick