Bioorthogonal Bioconjugation

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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 development 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 biological 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 M1s1. 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

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 et al., 2005). As applications of bioconjugates become connects the primary amine of lysine to an aniline center by more complex, the development of reliable chemoselective virtue of a reasonably selective reaction with isatoic anhydride, methods for introducing functional molecules into the natural followed by a highly selective oxidative coupling mediated by

NaIO4 (Table 1, entry 5) (Hooker et al., 2006). Aniline modifica- N-terminal acylation (entry 7) (Dawson et al., 1994; Dawson tions to proteins can also be introduced at N termini by acylation and Kent, 2000). However, a few examples of recent variations (Behrens et al., 2011) or can be genetically introduced using on these themes have appeared. amber codon suppression (Mehl et al., 2003) and can undergo Thus, Rao and coworkers reported the development of a thiol- oxidative couplings with aminophenols mediated by NaIO4 (Beh- based ligation between 2-cyanobenzothiazole (CBT) and rens et al., 2011) or more recently by ferricyanides (Obermeyer D-cysteine (Table 2, entry 8) (Ren et al., 2009; Van de Bittner et al., 2014b). et al., 2013). The reaction works well under physiological condi- The venerable reductive amination reaction has never been tions with a second order rate constant of 9.1 M1s1 (Ren et al., much use under biological conditions, because it requires acidic 2009; Van de Bittner et al., 2013). It was subsequently applied to conditions to preserve the cyanoborohydride (or equivalent) protein labeling (Ren et al., 2009) and imaging protease activity in reductant. However, the use of an iridium complex has been living cells (Liang et al., 2010) and, most recently, for the self as- found to make this unnecessary, opening this pathway for the sembly of a fluorescent molecule in apoptotic cells for imaging decoration of lysine amines (Table 1, entry 6) (Gildersleeve chemotherapeutic efficacy in vivo (Ye et al., 2014). An intriguing et al., 2008; Jentoft and Dearborn, 1979). The acidic conditions NCL variation uses salicylaldehyde as an adaptor to marry N-ter- required for NaBH3CN reduction of the aldiminium intermediate minal serine with a PEG derivative (entry 9) (Levine et al., 2014). can be avoided with iridium-catalyzed transfer hydrogenation Chemoselective Connections to Arginine, Histidine, Trypto- using [Cp*Ir-(bipy)(H2O)]SO4. Iminium intermediates can also phan, and Tyrosine Side Chains. Site-specific conjugation to be intercepted intramolecularly with an adjacent diene group, re- these amino acids is less common and seems even today to sulting in intramolecular tandem 6p electrocyclization followed be dominated by new applications of old chemistry—a charac- by auto-oxidation and hydrolysis to yield zwitterionic pyridinium teristic of many click and bioconjugation protocols. For example, products (Table 1, entry 7) (Tanaka et al., 2008). This strategy has strategies for the addressing of arginine residues are rare, as the been employed for peptide labeling in conjugation to a cyclic strongly basic guanidino group (pKa > 12.0) is almost always pro- RGDyK motif for visualizing avb3-integrins displayed on cell sur- tonated under physiological conditions. Among the oldest faces (Tanaka et al., 2014), but it seems also to have promise in known reactions at arginine is condensation with a,b-dicarbonyl more complex situations if undesired conjugate addition can be compounds, but this is usually much too slow for efficient avoided. Furthermore, an adjacent boronic acid center can sta- bioconjugation: bovine serum albumin requires 14 days for bilize iminoboronate intermediates derived from lysine or N-ter- room-temperature Maillard reactions with 100 mM methyl- minal amines; such bonds can be reversed upon addition of fruc- glyoxal at pH 7.4 (Oya et al., 1999). In spite of its low rate, tose, dopamine, or glutathione (Table 1, entry 8) (Cal et al., 2012). this process has been applied to the labeling of arginine residues Lastly in this category, diazonium salts are one of the most of lysozymes (Table 3, entry 1) (Gauthier and Klok, 2011). reactive types of compounds easily accessible in aqueous me- While the long-known reactivity of histidine with vinylsulfones dia. While diazonium salts have been increasingly exploited for continues to receive productive attention (del Castillo et al., their reaction with tyrosine (see below), it has been something 2014), histidine residues are most commonly exploited in a of a surprise that they have not been more widely explored for chemoselective sense for the connection of proteins to com- productive ligation reactions with other biological nucleophiles. plexed metal ions (Co3+,Ni2+,Cu2+), metal surfaces, and metal This lack of action has recently ended with a report from Carreira nanoparticles; such binding interactions are beyond the scope and coworkers describing the use of simple aminoterephthalate of this review. derivatives (Diethelm et al., 2014). Diatozation and reaction with More exciting are the renewed appreciation of the mild nucle- protein amines is followed by intramolecular trapping of triazenyl ophilicity of methionine (thioether), tryptophan (indole), and tyro- intermediates with an adjacent ester group to give hydrolytically sine (phenol) side chains as potential sites for selective bond stable triazin-4(3H)-ones. Although rate constants were not re- formation. Kramer and Deming have returned to simple alkyl- ported, the reaction appears to be roughly between the speed ation as a method to address methionine, finding that the result- of amine-NHS ester and thiol-maleimide couplings, but with ing sulfonium ions are often quite stable to aqueous base, heat, excellent chemoselectivity. One can expect modifications of and other extreme conditions (Table 3, entry 2) (Kramer and this already productive methodology in the near future. Deming, 2012, 2013). So far, this chemistry has not found wide N-Terminal Modifications The N-terminal amine of peptides application, being restricted in the reported cases to polypep- and proteins can be selectively modified by a variety of reactions tides containing many methionine residues, but it is promising. (Table 2, entries 1-6). The a-amino group reacts with moderate to The Francis lab has developed a tryptophan modification using high selectively with ketenes (4:1 to > 99:1 preference for rhodium carbenoids in aqueous solution (Antos and Francis, a-amino group over ε-amino groups) under mild conditions 2004) over a broad pH range (entry 3) (Antos et al., 2009). This (Chan et al., 2012). N-terminal serine or threonine residues can rhodium catalysis has been applied to site-specific modification be oxidized with periodate (Geoghegan and Stroh, 1992), and of aromatic side chains (Trp, Tyr and Phe) using metallopeptides N-terminal amines can undergo a variety of reactions with alde- capable of molecular recognition (Antos et al., 2009; Chen et al., hydes, including pyridoxal phosphate mediated N-terminal 2011). transamination (Gilmore et al., 2006; Scheck et al., 2008; Witus Tyrosine contains an ionisable phenolic side chain (pKa 9.7– et al., 2010), thiazolidine ring formation (Bernardes et al., 2013; 10.1) that is often solvent exposed (Koide and Sidhu, 2009) Wade et al., 2002), and Pictet-Spengler cyclization (Sasaki and can be site-specifically modified by a number of reactions et al., 2008). And, of course, native chemical ligation (NCL) and (Jones et al., 2014). For obvious reasons of selectivity, the direct its variants exploit S-to-N acyl migration for rapid and selective alkylation of the phenolic oxygen is quite rare, but it has been

Table 1. Recent Developments in the Bioconjugation of Amino Groups in Proteins Entry Residue Reagent(s) Product Reference 1 Lys Standard reagents; see (Hermanson, 2013)

2 Lys

3 Lys

4 Lys

5 Lys (Hooker et al., 2006)

6 Lys (McFarland and Francis, 2005)

7 Lys (Tanaka et al., 2008; Tanaka et al., 2014)

(Continued on next page)

Table 1. Continued Entry Residue Reagent(s) Product Reference 8 Lys, N-term. (Cal et al., 2012)

9 Lys, N-term. (Diethelm et al., 2014)

recently managed by Pd-catalyzed alkylation (Jones et al., 2014; amine and anisidene moieties has been accomplished with cer- Tilley and Francis, 2006) or prenylation58,59 via Claisen ium(IV) ammonium molybdate (entry 11) (Seim et al., 2011). A rearrangement of the O-prenylated precursor, mediated by pre- milder oxidant, potassium ferricyanide, has also been shown to nyltransferase enzymes (Table 3, entry 4) (McIntosh et al., 2013; be capable of coupling anilines with o-aminophenols on pro- Rudolf and Poulter, 2013). The aromatic ring is a more distinctive teins, but without contaminating thiol oxidation (Obermeyer nucleophile, however, and diazonium salts have long been et al., 2014b). Electrophilic iodination of tyrosines with 125I and known to react with phenols to make diazo dyes. Variations on 131I is also useful (Schumacher and Tsomides, 2001), because this reaction have also been used to label tyrosine aromatic rings the products can undergo aqueous Suzuki-Miyaura cross with moderate rate and high selectivity (Table 3, entries 5 and 6) coupling (Vilaro´ et al., 2008). Tyrosine can also be iodinated at (Bruckman et al., 2008; Hooker et al., 2004; Jones et al., 2012; the ortho position using bis(pyridine)iodinium tetraﬂuoroborate

Schlick et al., 2005). The resulting diazo intermediate can be pro- (IPy2BF4, entry 12) (Chalker et al., 2011; Espun˜ a et al., 2006) cessed in a number of ways, such as by oxidation to an o-imino- for subsequent palladium catalyzed cross coupling (Chalker quinone for hetero-Diels-Alder reaction (Hooker et al., 2004), or et al., 2009b, 2011; Espun˜ a et al., 2006). Particularly attractive simply kept as a connector to another functional group that for potential in vivo applications is oxidation by tyrosinase to can be addressed by a second bioorthogonal reaction (such as the corresponding o-quinones, followed by selective nucleo- oxime formation) (Gavrilyuk et al., 2013; Schlick et al., 2005). philic trapping (Long and Hedstrom, 2012). Similarly, a three component Mannich reaction between tyrosine Cysteine Modifications. The thiol group of cysteine side chains and imines formed from aldehydes and electron rich anilines has is one of the most widely used functional groups for the synthetic also been described (entry 7) (Joshi et al., 2004), although it is modification of peptides and proteins (Chalker et al., 2009a), relatively slow and requires a large excess of regents (McFarland since thiols are less abundant and more reactive than amines et al., 2008; Romanini and Francis, 2008). The aqueous ene-type (Fodje and Al-Karadaghi, 2002). Cysteine residues are rarely reaction of tyrosine with cyclic diazodicarboxamides seems found on highly solvent-accessible surfaces of a protein; most more generally promising (Ban et al., 2010, 2013; Jessica conjugation reactions therefore require pretreatment with a et al., 2013), because it proceeds selectively at the o-position reducing agent such as dithiothreitol (DTT) to cleave an acces- of the phenol side chain and was shown to provide thermally sible disulfide or reduce an oxidatively passivated cysteine thiol and hydrolytically stable products (entry 8). (as in a sulfenic acid). Many of the electrophiles traditionally used Quite a number of tyrosine functionalization processes rely on with thiols (iodoacetamides, maleimides, vinyl sulfones, vinylpyr- one-electron oxidation as an initial activating step, selectivity idines, epoxides; Table 4, entries 1–4) (Chalker et al., 2009a; arising from the fact that phenol is the most easily oxidized com- Stenzel, 2012) are subject to competing reactions with other mon side chain. Thus, nickel(II)-catalyzed oxidative coupling of nucleophilic amino acids (usually lysine and histidine) to an two phenols can be used for protein crosslinking (Kodadek extent that can be highly variable and occasionally substantial et al., 2005) and has been followed by bioconjugation on tyrosine (Baldwin and Kiick, 2011; Heukeshoven, 1980; Lewis and residues present on the capsid proteins of virus-like particles Shively, 1998; Alley et al., 2008; Shen et al., 2012). Disulfide (entry 9) (Meunier et al., 2004). Similarly, ligand-directed, sin- exchange is another common option for modifying cysteine 2+ gle-electron transfer from a photocatalyst ([Ru(bpy)3] ) bound (entry 5) (Pollack and Schultz, 1989), provided that there is ther- to a protein ligand results in the efficient formation of tyrosyl modynamic preference for the disulfide on the protein. However, radicals, which can be trapped by agents such as N0-acetyl- this method suffers from sluggish rates and limited control of N,N-dimethyl-1,4-phenylenediamine (entry 10) (Sato and product distributions. Nakamura, 2013). Oxidative conjugation of tyrosines, including Maleimide chemistry remains the subject of continual investi- O-alkylation, with various peptides containing phenylenedi- gation and modification. Frequently regarded as a reliable

Table 2. Recent Developments in the Bioconjugation of N-Terminal Amines in Proteins Entry Residue Reagent(s) Product Reference 1 Any (Chan et al., 2012)

2 Ser, Thr (Geoghegan and Stroh, 1992) NaIO4

3 Pro (Obermeyer et al., 2014a)

4 Any (Gilmore et al., 2006; Scheck et al., 2008; Witus et al., 2010)

5 Cys (Bernardes et al., 2013; Wade et al., 2002)

6 Trp (Sasaki et al., 2008)

7 Cys (Dawson et al., 1994; Dawson and Kent, 2000; Esser-Kahn and Francis, 2008)

(Continued on next page)

Table 2. Continued Entry Residue Reagent(s) Product Reference 8 Cys (Ren et al., 2009)

9 Ser (Levine et al., 2014)

connection (Bednar, 1990; Kim et al., 2008), the thiol-maleimide borohydride. These reagents are highly selective for cysteine conjugate addition product has recently been shown by Baldwin over lysine. and Kiick to be labile or exchangeable under physiologically rele- The range of thiol-reactive functional groups explored for bio- vant conditions (Baldwin and Kiick, 2011). While uncontrolled or conjugation continues to expand. Thiols undergo highly spe- contaminating cleavage can often be a problem, engineered cific conjugation with allenes (in the presence of gold catalyst, cleavability can be an advantage for applications that require entry 13) (Chan et al., 2013) or allyl selenosulfate salts (entry 14) the release of a cargo from a carrier protein, for example. The (Crich et al., 2006). Positional selectivity has been elegantly incorporation of a leaving group such as alkoxide into the malei- achieved by genetically encoding reactive azobenzene-electro- mide changes the reaction mechanism with thiol from conjugate phile groups onto proteins (entry 15). Site-specific linkage can addition to addition-elimination reaction (Table 4, entry 6), occur with a nearby cysteine residue in proximity-enabled creating a class of maleimides that offers reversible modification fashion (Hoppmann et al., 2012, 2014). Such bridge formation and up to three conjugation points. Introducing two leaving is highly specific even in the presence of other thiols groups groups (entry 7) allows a disulfide linkage to be conveniently re- on the scaffold, and the light-driven isomerization of the azo- placed with a bridging cyclic structure (Bryden et al., 2014). benzene bridge from the trans to the cis conformation can be Cysteine thiol groups also undergo addition reactions with elec- used to change protein conformation and activity. Among the tron-deficient alkynes (Shiu et al., 2009) such as alkynones and most interesting electrophiles are perfluoroaromatic com- alkynoate amides or esters, the adducts of which also have the pounds, identified by Pentelute and coworkers to react cleanly capability to undergo exchange with additional thiol. Modifica- with cysteine residues under mild conditions (entry 16). This tions to these electrophiles, such as 3-aryl propionitriles (entry strategy has been applied to site-selective modification of 8) (Koniev et al., 2014) and allenamides (entry 9) (Abbas et al., cysteine residues in unprotected peptides, serving as a molec- 2014) are designed to retain high chemoselective cysteine ular staple (Spokoyny et al., 2013) for the synthesis of hybrid tagging in complex aqueous media, while stabilizing the resulting macrocyclic peptides (Zou et al., 2014). Remarkably, SNAr re- adducts toward thiol exchange and hydrolysis. actions can also be catalyzed by naturally occurring glutathione Another alternative to maleimides are electron-deficient oxa- S-transferase for efficient ligation between a polypeptide norbornadienes (entry 10), which display thiol addition rates and an N-terminal glutathione sequence (Zhang et al., 2013). similar to those of maleimides but have greater aqueous stability In this way, unique chemical orthogonality can be achieved, (Hong et al., 2009a; Kislukhin et al., 2011; Kislukhin et al., 2012). modifying multiple cysteine sites with different chemical probes An additional fragmentation feature of this linkage is discussed while avoiding protecting groups and additional synthetic at the end of this article. Julia-Kocienski-like sulfone derivatives procedures. (Toda et al., 2013) based on methylsulfonyl-functionalized heter- The cysteine residue can be rendered electrophilic as well. oaromatic derivatives (entry 11) have been used for rapid pro- Oxidative elimination by treatment with O-mesitylenesulfonyl tein/peptide conjugation, and their conjugates were found to hydroxylamine (MSH) under basic conditions affords dehydroa- be more stable than the maleimide-cysteine conjugates in lanine (Bernardes et al., 2008), which can be modified by conju- human plasma. Similarly, latently reactive PEG-di-sulfone gate addition of thiols (entry 17) (Chalker et al., 2009a) or can (Balan et al., 2007) and PEG-mono-sulfone (Badescu et al., undergo olefin cross-metathesis with ruthenium catalysts (Lin 2014) have been used for chemoselective cysteine conjugation. et al., 2008). Additionally, thiol-ene click reactions that are so The PEG mono-sulfone reagent undergoes facile elimination useful in materials contexts have also been used for bio- of toluene sulfinic acid under mild conditions to generate conjugation (entry 18), as in the glycosylation of a single cysteine the reactive a,b-unsaturated PEG reagent for conjugation moiety on glutathione (Dondoni et al., 2009) or the labeling of (entry 12). Retro-Michael de-conjugation can be prevented by alkyne-bearing proteins expressed in E. coli (Li et al., 2012, treating the ketone with a mild reducing agent such as sodium 2013b).

Table 3. Recent Developments in Bioconjugation at Arg, Met, Trp, and Tyr Residues Entry Residue Reagent(s) Product Reference 1 Arg (Gauthier and Klok, 2011)

2 Met (Kramer and Deming, 2012)

3 Trp (Antos and Francis, 2004; Antos et al., 2009)

4 Tyr (Chen et al., 2009; Tilley and Francis, 2006)

5 Tyr (Hooker et al., 2004)

6 Tyr (Gavrilyuk et al., 2013; Schlick et al., 2005)

7 Tyr (Joshi et al., 2004; McFarland et al., 2008; Romanini and Francis, 2008)

8 Tyr (Ban et al., 2010; Ban et al., 2013; Jessica et al., 2013)

(Continued on next page)

Table 3. Continued Entry Residue Reagent(s) Product Reference 9 Tyr (Kodadek et al., 2005; Meunier et al., 2004)

10 Tyr (Sato and Nakamura, 2013)

11 Tyr (Sato and Nakamura, 2013; Seim et al., 2011)

12 Tyr (Vilaro´ et al., 2008)

13 Tyr (Long and Hedstrom, 2012)

Bioorthogonal Reactions Based on Extrinsic et al., 2012). Aldehydes can also be introduced into proteins by Functionalities periodate oxidation of N-terminal Ser/Thr residues (Geoghegan Transimination Reactions of Ketones/Aldehydes with Hydra- and Stroh, 1992), site-specific protein transaminations (Geoghe- zides/Alkoxyamines. Among the first functionalities to be gan et al., 1993; Gilmore et al., 2006; Witus et al., 2010, 2013), explored as extrinsic bioorthogonal reactants were ketones periodate oxidation of sialic acids displayed on proteins (Hage and aldehydes (Rideout, 1986). Although they are not completely et al., 1997), and by addition of ketone-containing small mole- abiotic and are present in some intracellular metabolites, their cules to protein C-terminal thioesters generated by expressed synthetic accessibility and small size has made them amenable protein ligation (Esser-Kahn and Francis, 2008). to incorporation into biomolecules. Popular routes include up- Once incorporated, the carbonyl group can be addressed by a take and processing of unnatural intermediates of glycan biosyn- number of highly specific methods, as illustrated in Figure 2. The thesis (Jacobs et al., 2000; Luchansky et al., 2004; Mahal et al., discovery by Dawson and coworkers of aniline catalysis of the 1997; Sadamoto et al., 2004), genetically controlled incorpora- click reaction with a-effect nucleophiles such as hydrazine and tion into proteins in a residue-specific (Datta et al., 2002; Ngo aminooxy groups at pH 4–6 has made this popular process and Tirrell, 2011; Tang et al., 2009) or site-specific manner (Car- even more effective (Figure 2A) (Gaertner et al., 1992; Jencks, rico et al., 2007; Chin et al., 2003; Hudak et al., 2011, 2012; Liu 1959; Sander and Jencks, 1968), with the organocatalytic pro- and Schultz, 2010; Wang et al., 2003a; Wu et al., 2009), and cess proceeding through a highly reactive Schiff base (Cornish use in peptide tags that direct enzymatic ligation of aldehyde et al., 1996; Dirksen and Dawson, 2008; Dirksen et al., 2006a, or ketone bearing small molecules (Chen et al., 2005; Rashidian 2006b; Jencks, 1959; Rashidian et al., 2013; Ulrich et al.,

Table 4. Recent Developments in Bioconjugation at Cys Residues Entry Residue Reagent(s) Product Reference 1 Cys Standard reagents; see (Chalker et al., 2009a; Hermanson, 2013; Kim et al., 2008)

2 Cys

3 Cys

4 Cys

5 Cys

6 Cys (Marculescu et al., 2014)

7 Cys (Bryden et al., 2014)

8 Cys (Koniev et al., 2014)

9 Cys (Abbas et al., 2014)

(Continued on next page)

Table 4. Continued Entry Residue Reagent(s) Product Reference 10 Cys, Lys (Hong et al., 2009a; Kislukhin et al., 2011; Kislukhin et al., 2012)

11 Cys (Toda et al., 2013)

12 Cys (Badescu et al., 2014)

13 Cys (Chan et al., 2013)

14 Cys (Crich et al., 2006)

15 Cys (Spokoyny et al., 2013)

16 Cys (Hoppmann et al., 2012; Hoppmann et al., 2014)

17 Cys (Bernardes et al., 2008)

18 Cys (Dondoni et al., 2009; Li et al., 2012)

Figure 3. Nontraceless and Traceless Staudinger Ligations

Azide-Based Bioorthogonal Reactions. Azides are essentially absent from biological systems (Griffin, 1994) and are unreactive with biological functionality and so have become one of the pre- miere bioorthogonal participants (Debets et al., 2010b). Azides are small, not very polar, and incapable of significant hydrogen bonding, making them unlikely to significantly change the prop- Figure 2. Polar Condensation Reactions of Carbonyl Compounds erties of structures to which they are attached. They can be easily introduced into biomolecules such as glycans (Baskin et al., 2007; Chang et al., 2010; Laughlin et al., 2008), proteins 2014). To improve biocompatibility and increase reaction rate at (Beatty et al., 2010), lipids (Hang et al., 2007; Kho et al., 2004), pH 7, substituted anilines (Dirksen and Dawson, 2008; Dirksen and nucleic acids by biosynthetic pathways. Azide modified pro- et al., 2006b; Rashidian et al., 2013; Wendeler et al., 2014) teins have been used to label biomolecules in living systems and such as water-soluble 5-methoxyanthranilic, 3,5-diaminoben- animals. Likewise, alkynes are abiotic and can be easily intro- zoic (Crisalli and Kool, 2013b), and 2-aminobenzenephosphonic duced into biological molecules (Grammel and Hang, 2013; acid (Crisalli and Kool, 2013a) have been found to accelerate the Johnson et al., 2010; Ngo and Tirrell, 2011). condensation reaction by up to 40-fold at pH 7 as compared to While only about 15 years old (Saxon et al., 2000), the Stau- the aniline-catalyzed reaction. Although the electronic and acid/ dinger-inspired reaction between azides and phosphines to yield base properties of the nucleophilic reactants strongly influence amide linkages developed by Bertozzi and coworkers is now a the rate at biological pH (Kool et al., 2014), it must be remem- classic among bioorthogonal processes and remains highly use- bered that all of these ligations are reversible and that hydra- ful (Kiick et al., 2002; van Berkel et al., 2011). The byproduct zones are especially prone to dissociation at low concentrations phosphine oxide can remain attached to, or can be released (Dirksen et al., 2006a; Dirksen et al., 2006b; Kalia and Raines, from, the final product (Figure 3)(Nilsson et al., 2000; Saxon

2008), whereas oximes are more stable (Kalia and Raines, et al., 2000). Although the kinetics are sluggish (k2 approximately 2008). Hydrazone and oxime condensations are therefore best 3 3 103 M1 s1)(Lin et al., 2005) and the phosphine reagents suited for in vitro studies or reactions at cell surfaces (Tuley prone to oxidation by air or metabolic enzymes, the Staudinger et al., 2014), because the necessary reactant concentrations ligation is a popular choice for in vivo work due to its remarkable can be too high to achieve intracellularly and a-effect nucleo- selectivity and compatibility with everything from cells (Agard philes can undergo reactions with carbonyl-bearing metabolites et al., 2006; Chang et al., 2007; Saxon and Bertozzi, 2000)to such as pyruvate, oxaloacetate, sugars, and various cofactors. animals (Prescher et al., 2004). The Pictet-Spengler reaction between aldehydes and tryptamine Copper-catalyzed azide-alkyne cycloaddition (CuAAC, nucleophiles generates hydrolysis-resistant oxacarbolines via Figure 4)(Rostovtsev et al., 2002; Tornøe et al., 2002; Worrell an intermediate oxyiminium ion in acidic environments, followed et al., 2013) has emerged as a highly versatile ligation process by spontaneous cyclization (Figure 2B) (Agarwal et al., 2013a, for bioconjugation applications of all types, except in living cells 2013b; Sto¨ ckigt et al., 2011). A recent variant that joins and tissues where free copper ions are toxic. Such operations as glyoxyl-modified peptides with an aminobenzamidoxime pro- the immobilization, labeling, or capture of proteins, nucleic ceeds at rates that are comparable to the hydrazino-Pictet acids, and polysaccharides (Moses and Moorhouse, 2007) Spengler reaction (3 M1s1 at pH 6.0, Figure 2C) (Kitov et al., benefit from the small size and unobtrusive nature of the terminal 2014). Additionally, aldehydes have been shown to undergo alkyne and azide moieties. Among many other applications, the oxidative condensation with aryldiamines in the presence of CuAAC process has been used to label the outer (Banerjee et al., Cu(II) or Zn(II); this process was employed for the chemical modi- 2010, 2011; Hong et al., 2009b; Wang et al., 2003b) and inner fication of a arylenediamine-containing T4 lysozome (Figure 2D) (Abedin et al., 2009; Hovlid et al., 2014) surfaces of azide- and (Ji et al., 2014). alkyne-functionalized virus particles, modify proteins in vitro

Figure 4. Key Features of the CuI-Catalyzed Azide-Alkyne Cycloaddition Reaction (A and B) Overall process and mechanistic overview of the catalyzed azide-alkyne cycloaddition (CuAAC) catalytic cycle. (C) Accelerating ligands commonly used in the CuAAC reaction. (D) Strategies to accelerate rates by chelation of the azide component. (E) Cu-promoted generation of reactive oxygen species. (F) Dehydroascorbic acid mediated cross-linking of protein side chains. and in vivo (Deiters et al., 2003; Liu and Schultz, 2010; Ngo and Oxidative stress and biological damage associated with Tirrell, 2011), tag azide-modified lipids (Hang et al., 2011), and CuAAC has been attributed to CuI-promoted generation of reac- label azide- or alkyne-modified nucleic acids. The CuAAC reactive oxygen species (ROS) from O2 (Brewer, 2010; Hong et al., tion proceeds at considerably faster rates than the Staudinger 2010; Wang et al., 2003b). Catalytically active CuI is not stable ligation (more than 25-fold) and is usually 10–100 times faster under physiological conditions, and the oxidation of CuI to CuII than copper-free strain-promoted azide-alkyne cycloaddition by either O2 or H2O2 (via Fenton processes) facilitates the (discussed below) (Jewett et al., 2010; Presolski et al., 2010), production of superoxide and hydroxyl radicals, respectively with second-order rate constants of 10-200 M1s1 in the pres- (Figure 4E) (Biaglow et al., 1997; Tabbı` et al., 2001). The presence ence of 20–500 mMCuI. of ROS can affect the structural and functional integrity of bio- With the proper selection of copper-binding ligand (Hong molecules, causing degradation of amino acids and cleavage et al., 2009b; Presolski et al., 2010), CuAAC reactions proceed of peptide chains (Stadtman, 2006), and this has been observed rapidly within richly functionalized biological environments under CuAAC conditions (Hong et al., 2009b; Kennedy et al., such as cell lysates. Indeed, the labeling of groups incorporated 2011; Kumar et al., 2011). Furthermore, the ascorbate-reducing onto the outer surface of live cells can be managed very effi- agent used in these reactions can also do damage by virtue of ciently with the best catalysts (Besanceney-Webler et al., the electrophilic properties of its oxidized form (dehydroascor- 2011; Hong et al., 2010). Note, however, that CuAAC bio- bate), which can react with lysine, arginine (Reihl et al., 2004), conjugations usually require greater amounts of CuI and acceler- and cysteine (Kay et al., 2013) side chains, leading to protein ating ligand than the biomolecular azide and alkyne, which are crosslinking (Figure 4F) (Corti et al., 2010). Additives such as usually present in nanomolar-micromolar concentrations. excess Cu-binding ligand and aminoguanidine are used as

Figure 5. Strain-Promoted 1,3-Dipolar Cycloaddition Reactions Useful for Bioconjugation (A) Strain-promoted azide-alkyne cycloaddition (SPAAC). (B) Common cyclooctyne reagents and associated rate constants for reaction with benzyl azide. (C and D) Side reactions of strain-promoted click chemistry reagents or products. (E) Alternative strain-promoted 1,3-dipolar cycloaddition reactions.

ROS and dehydroascorbate scavengers, respectively (Hong providing 25-fold enhancement in specific labeling rate relative et al., 2009b). to conventional nonchelating azides at low copper concentra- CuAAC is a ligand-accelerated process (Chan et al., 2004; tions (10–100 mM) (Jiang et al., 2014; Uttamapinant et al., 2012, Hong et al., 2009b), and a variety of ligands have been found 2013). Azides bearing tighter binding ligands such as tetraden- to be effective under different conditions. In general, such tate bis(triazolyl)amino azide have also exhibited outstanding ligands enhance cell compatibility by stabilizing the CuI oxidation reactivity with alkynes under dilute conditions in the presence state and increasing its catalytic efficiency. The most widely of complex cellular media and were used for the tracking of used ligands (Figure 4C) include sulfonated bathrophenanthro- paclitaxel inside living cells (Bevilacqua et al., 2014). line (BPDS) (Lewis et al., 2004), tris(benzimidazole) (BimC4A)3 The most effective way to improve the biocompatibility of (Rodionov et al., 2007b), tris-triazole TBTA (Chan et al., 2004), azide-alkyne cycloaddition has been to eliminate the require- water soluble THPTA (Hong et al., 2009b), BTTES (Soriano Del ment of CuI catalysis and accelerate the reaction with alkynes Amo et al., 2010), BTTAA (Besanceney-Webler et al., 2011), that are activated by ring strain. The strain-promoted azide- and bis(L-histidine) (Kennedy et al., 2011). Chelation-assisted alkyne cycloaddition (SPAAC, Figure 5A) (Agard et al., 2004, copper catalysis is a recent and complementary approach to 2006) has been used effectively in a variety of in vivo contexts, ligand-based acceleration (Brotherton et al., 2009; Kuang including in mammalian cells (Agard et al., 2004; Baskin et al., et al., 2010, 2011), designed to enhance the ‘‘weakest link’’ in 2007) and animals (Baskin et al., 2007; Laughlin et al., 2008). Sig- the CuAAC mechanism: the association of azide with the metal nificant effort has been focused on increasing the rate, biocom- center (Rodionov et al., 2007a). For example, a bidentate picolyl patibility, and pharmacokinetic properties of the cycloooctyne azide (shown in Figure 4D) was used in combination with BTTAA reagents used in SPAAC. An optimal balance between reactivity for site-specific conjugations of proteins on the surface of live and stability must be achieved for effective performance in vivo. cells with metabolically labeled RNA and protein molecules, The reactivity of the cyclooctyne can be modulated (Figure 5B)

uses of SPAAC methodology are the derivatization of purified proteins in vitro (Agard et al., 2004), glycans on cell surfaces for probing sialic acid biosynthesis (Beatty et al., 2010), and in vivo experiments with zebrafish (Dehnert et al., 2012), the C. elegans nematode (Laughlin and Bertozzi, 2009), and mice (Chang et al., 2010) following biological uptake of ManNAz, now also commercially available. Alternative 1,3-dipoles such as nitrones (McKay et al., 2010, 2011; Ning et al., 2010), nitrile oxides (Sanders et al., 2011; Singh and Heaney, 2011), diazoalkanes (McGrath and Raines, 2012), and syndones (Wallace and Chin, 2014) have also been explored as means for improving the kinetics and biocompatibility of reactions with multiple-bond partners (primarily strained alkynes, Figure 5E). Strain-promoted alkyne-nitrone cycloaddition (SPANC) reactions demonstrate rate constants up to 60 M1s1 (McKay et al., 2010, 2012; Ning et al., 2010) and have been used for N-terminal peptide modification (Ning et al., 2010), direct protein labeling, and pretargeted labeling of ligand-receptor interactions on cell surfaces (McKay et al., 2011). Isoxazolines bearing N-phenyl groups are prone to thermal rearrangements (Baldwin Figure 6. Strain-Promoted Cycloadditions et al., 1968), but this can be minimized by using electron-donating Strain-promoted cycloadditions of (A) azides and (B) nitriles oxides with groups (e.g., Me, Et) at this position. Cyclic nitrones display strained alkenes; (C) quadricyclanes with a nickel complex. greater stability toward hydrolysis and faster kinetics than their acyclic counterparts, and nitrone reactivity is tunable to by appending electron withdrawing groups at the propargylic allow for simultaneous SPANC reactions for multiplex labeling position (MOFO, DIFO) (Agard et al., 2006; Baskin et al., 2007) (MacKenzie and Pezacki, 2014; MacKenzie et al., 2014). Nitrile ox- or by augmentation of strain energy through aryl ring (DIBO, ides have also been explored as alternative 1,3-dipoles in reac- DIBAC and BARAC) (Debets et al., 2010a; Jewett et al., 2010; tions with cyclooctynes to yield isoxazoles, and the reaction Ning et al., 2008) or cyclopropyl (BCN) (Dommerholt et al., has been applied for generating oxime-containing nucleotides 2010) ring fusion (Sletten et al., 2014). The superior reaction and peptides (Jawalekar et al., 2011) as well as carbohydrates rate of BARAC is counterbalanced by its instability toward hydro- (Sanders et al., 2011). Nitrile oxides must be generated in situ lysis in phosphate buffered saline (t1/2 = 24 h) and its tendency for due to their more reactive nature. Additionally, syndones have intramolecular rearrangement under acidic conditions (Chigri- been used as 1,3-dipoles in CuI catalyzed cycloadditions with ter- nova et al., 2013). Additionally, cyclooctynes have been reported minal alkynes (Kolodych et al., 2013) and more recently in strain- to undergo nucleophilic addition with cellular nucleophiles such promoted cycloadditions with bicyclononyne (rate constant = as glutathione (Beatty et al., 2010; Chang et al., 2010), homotri- 0.054 M1s1, comparable with that of azides). This method merization (Sletten et al., 2010), and reaction with cysteine sul- was applied to the efficient fluorescent tagging of cyclooctyne- fenic acids (with trapping rates that are an order of magnitude modified proteins (Wallace and Chin, 2014). Recently, the rate faster than SPAAC and exceed the rates of previously sulfenic of this cycloaddition process has been improved 30-fold by using 1 1 acid capture reactions by more than 100-fold) (Figures 5C and 4-chlorosyndones (k2 = 1.6 M s )(Plougastel et al., 2014). 5D) (Poole et al., 2014). Azide modified proteins have also been found to undergo [3+2] The major constraint on the use of SPAAC in bioconjugation cycloaddition with strained olefin probes such as oxanorborna- derives from the relatively large size and hydrophobic nature of dienes, which form 1,2,3-triazoles upon retro-Diels-Alder frag- the cyclooctyne component, which can affect their distribution mentation (Figure 6A) (van Berkel et al., 2007). While relatively and change the biological properties of the species to which slow (rate constants in the 104 M1s1 range), the reaction has they are attached. Despite the synthetic tractability of the diben- been used for functionalizing azide-modified peptides (van Berkel zocyclooctynes and kinetic benefits of aza analogs thereof, the et al., 2007) and remains attractive due to the relatively easy syn- original difluorinated cyclooctynes remain useful reagents for thetic accessibility of the oxanorbornadiene fragment. Nitrile copper-free click chemistry for this reason. DIFO is less sterically oxides have also been used in 1,3-diplar cycloaddition with nor- encumbered than the benzannulated variants and has been bornenes leading to a regioisomeric mixture of 2-isoxazolines shown to be more effective for site-specific incorporation into (Figure 6B). This approach has been implemented for the fluores- proteins. In one case, the larger dibenzocyclooctyne could not cent modification of oligonucleotides (Gutsmiedl et al., 2009; be site-specifically incorporated into proteins through amber Singh and Heaney, 2011). While in situ generated nitrile imines stop codon techniques (Plass et al., 2011). A different early lim- are prone to hydrolysis, their 1,3-dipolar cycloaddition is faster itation of SPAAC, the requirement for independent synthesis of and has been applied to the fluorescent labeling of acrylamide cyclooctynes, has been largely eliminated for most users by containing proteins (Wang et al., 2014). This reaction, however, the commercial availability of a variety of derivatives, including is strongly influenced by pH and chloride ion concentration. those with pendant and orthogonal reactive functionality such An alternative strain-promoted reaction involving the compact as NHS ester or maleimide. Among the many recent productive quadricyclane structure was recently reported by Sletten and

Figure 7. Photo-Induced 1,3-Dipolar Cycloadditions of 2,5-Diaryltetrazoles and Alkenes

Tetrazine Ligations: Inverse-Electron Demand Diels-Alder Reactions. In the quest for faster rates, and therefore lower usable concentrations, for bioorthogonal connecting reactions, a new standard has been set by the inverse-electron- demand Diels-Alder reaction between Bertozzi (Sletten and Bertozzi, 2011a). In its initial form, the 1,2,4,5-tetrazines and strained alkenes (Table 5)(Devaraj and desired [2+2+2] cycloaddition was found to occur most produc- Weissleder, 2011). Although only recently developed, this tively with the p-system of a Ni-bis(dithiolene) complex process has received wide attention, including use of trans- (Figure 6C, rate constant = 0.25 M1s1). Quadricyclanes are cyclooctene (TCO) (Blackman et al., 2008; Taylor et al., 2011), quite easy to make (photochemical reaction with norborna- cyclooctyne (Chen et al., 2012), cyclopropene (Patterson et al., dienes), are stable in aqueous media at room temperature, and 2012; Yang et al., 2012b, 2014a), norbornene (Devaraj et al., are not known to react with biomolecules. Using this reaction, 2008; Han et al., 2010; Vrabel et al., 2013), cyclobutene (Pipkorn quadricyclane modified bovine serum albumin (BSA) was effi- et al., 2009), azetidine (Engelsma et al., 2014), and isonitrile ciently labeled in the presence of cell lysates, but the reaction (Stairs et al., 2013) dienophiles. Reaction rates with TCO are truly has yet to achieve wider use. spectacular (in the 106 M1s1 range); other dienophiles react Photo-Inducible Click Chemistry. The supreme chemoselec- with slower, but still useful, speed (Table 5)(Lang et al., 2012b). tivity of bioorthogonal reactions makes them attractive for the As these examples illustrate, the tetrazine ligation reaction selective probing of molecular events in biological environments. has undergone significant optimization to improve the reaction A key component of such investigations is time, since learning kinetics, product stability, and cell permeability of the tetrazine when a species appears or reacts in a cell is often as useful as (Karver et al., 2011) and trans-cyclooctene (Taylor et al., 2011) knowing where it is. The temporal component can be controlled derivatives. Increasing the strain in the trans-cyclooctene moiety to some degree by triggering the expression of a protein or nu- through cyclopropyl fusion resulted in a 50-fold rate enhance- cleic acid with genetic promotors, but such modulation of the ment (Lang et al., 2012b; Seitchik et al., 2012). Additionally, chemistry is less common. For this reason, the recent develop- p-conjugated tetrazines exhibit strong fluorescence upon ment of photochemically triggered bioorthogonal reactions is cycloadditions with dienophiles such as cyclopropenes and exciting, and is certainly in its infancy (Lim and Lin, 2011). A trans-cycloctene (Wu et al., 2014). Applications of tetrazine- light-induced reaction between diaryl tetrazoles and alkenes TCO ligations have included labeling of newly synthesized pro- was first developed for bioconjugation by Lin et al., involving teins (Lang et al., 2012a) and cancer cells (Devaraj et al., 2009; the photochemical ejection of dinitrogen from the tetrazole fol- 2010), in vivo cancer imaging with 111In (Rossin et al., 2010) lowed by rapid trapping of the resulting nitrile imine by alkene and 18F radiolabeling (Keliher et al., 2011; Li et al., 2010; Reiner (Figure 7A) (Song et al., 2008b; Wang et al., 2008). Similarly, et al., 2011), cancer cell detection (Haun et al., 2010), fluorescent azirines can be photoactivated as nitrile ylides (Figure 7B), a imaging of cytoskeletal proteins within living mammalian cells technique that has been used for PEG modification of lysozomes (Liu et al., 2012), and recently the methodology has been used (Lim and Lin, 2010a). with amino acids modified by tetrazine (Seitchik et al., 2012) Further improvements have been made by tailoring the tetra- and trans-cyclooctene (Lang et al., 2012b) for genetic incorpora- zole precursor. Electron-donating groups serve to increase the tion into proteins. The favorable kinetics of tetrazine-trans-cyclo- energy of the highest occupied molecular orbital of the nitrile- octene ligation will make it particularly useful for tracking fast imine, leading to increased reactivity with terminal alkenes biological processes and labeling low-abundance proteins. (Wang et al., 2009) and extending the tetrazole conjugation The availability of the tetrazine component has been dramati- with unsaturated substituents shifts the photochemical event cally improved by new synthetic methods (Wu et al., 2014; Yang to longer wavelength light, minimizing biomolecule damage. et al., 2012a, 2014a) and by the commercial availability of a Introducing strain in the alkene component results in further few derivatives. The trans-cyclooctene component, like cyclooc- rate enhancement (rates up to 34000 M1s1)(Lim and Lin, tynes used for SPAAC, suffers from some disadvantages of size 2010a; Yu and Lin, 2014). The power of the technique is just and hydrophobicity. However, tetrazines react reasonably well starting to be explored, with initial reports of the time and posi- with the much smaller moieties listed above (Table 5), making tionally controlled labeling of alkene-containing (homoallyl the components of the tetrazine ligation reaction not much glycine) proteins overexpressed in E. coli cells (Lim and Lin, more perturbing than azides and alkynes used in CuAAC, 2011; Song et al., 2008a). In addition, tetrazole-amino acids although probably a bit more cross-reactive with other functional site-specifically incorporated into proteins expressed in E. coli groups in biology. (Wang et al., 2010) and mammalian cells have also been Bio-orthogonal Organometallic Reactions. Although the appli- addressed (Song et al., 2010; Yu et al., 2012). cation of transition metals in medicine has been investigated for

Table 5. Tetrazine-Based Inverse-Electron Demand Diels-Alder Reactions with Strained Alkenes and Alkynes Tetrazine Dienophile Product Rate Constant Reference 210 – 2,800,000 M-1s-1 (Blackman et al., 2008; Taylor et al., 2011)

0.12 – 9.5 M-1s-1 (Devaraj et al., 2008; Han et al., 2010; Vrabel et al., 2013)

not reported (Pipkorn et al., 2009)

3.3 – 41 M-1s-1 (Chen et al., 2012)

0.03 – 13 M-1s-1 (Patterson et al., 2012; Yang et al., 2014a; Yang et al., 2012b)

0.39 M-1s-1 (Engelsma et al., 2014)

not reported (Stairs et al., 2013)

centuries, their use to mediate chemoselective transformations CuAAC chemistry). Much more challenging for the practical is much more recent (Yang et al., 2014b). The nature of late tran- application of organometallic reactions in biology, however, is sition metals makes them well suited to the manipulation of un- the presence of relatively high (mid-micromolar) concentrations saturated and polarizable functional groups (oleﬁns, alkynes, of thiols in cellular media, principally in the form of glutathione. aryl iodides, arylboronic acids, etc.) and to the retention of reac- General solutions to this problem remain to be developed. tivity in aqueous environments. The presence of oxygen and In 2006, Meggers and Streu used a ruthenium catalyst to other biological oxidants might have been anticipated as a gen- mediate allyl carbamate deprotection of a caged ﬂuorophore in- eral impediment, but a variety of procedures are known (and can side living cells (Streu and Meggers, 2006). The lack of apparent be anticipated) that are not sensitive to oxidation. Notable toxicity associated with this procedure prompted the investiga- successes at present are catalytic cycles mediated by PdII/ tion of heterogeneous palladium chemistry in cells. Pd(0)-func- PdIV species and the preservation of CuI species in reducing en- tionalized microspheres were shown to be able to enter cells vironments (although not without cost, as described above for and mediate allyl carbamate deprotections and Suzuki-Miyaura

Figure 8. Examples of Bioorthogonal Reactions Mediated by Organometallic Complexes cross-couping in the cytoplasm (Sasmal et al., 2013; Yusop followed by much better conditions for related Sonagashira et al., 2011). Applications of palladium-based applications in reactions of homoparpargylglycine-containing substrates (Fig- cell culture include copper-free Sonagashira coupling (Li et al., ure 8C) (Li et al., 2011). The same type of water-soluble Pd 2011), extracellular Suzuki coupling on the surface of E. coli cells complex was also used for an efficient Suzuki-Miyaura coupl- (Spicer et al., 2012), and conjugation of thiol groups with allyl se- ing reactions (Spicer et al., 2012) (including the example lenosulfate salts (Crich et al., 2006). in Figure 8D) (Chalker et al., 2009b), following the pioneering While Pd-catalyzed cross-coupling reactions have promise in use of a standard organometallic catalyst (Pd0-dibenzylidene the bioconjugation arena because of their excellent functional acetone, Pd-DBA) under moderately forcing conditions (pH 8.5, group tolerance and compatibility with water (Shaughnessy, 70C) (Brustad et al., 2008; Lim and Lin, 2010b). 2006)(Nicolaou et al., 2005), some optimization is always Because it uses the small bioorthogonal alkene group, olefin required; examples are shown in Figure 8. Thus, an early report metathesis is of particular interest for potential use in biological of a Mizoroki-Heck modification of a genetically engineered systems. Ruthenium complexes currently dominate this appli- Ras protein bearing an p-iodophenylalanine with a biotin-alkene cation, due to an advantageous combination of reaction rate, reagent proceeded in very low yield (Figure 8A) (Kodama et al., accessibility, tailorability, lack of toxicity, and functional group 2006). The reaction was facilitated by the water soluble tolerance (Lin et al., 2009, 2010, 2013). S-allylcysteine can be Pd-TPPTS (triphenylphosphine-3,30,300-trisulfonate) catalyst, easily introduced into proteins by a variety of methods, although the outcome and requirement for inert atmosphere including conjugate addition of allyl thiol to dehydroalanine, highlights limitations displayed by many early examples of direct allylation of cysteine, desulfurization of allyl disulfide, or transition metal-catalyzed bioconjugation reactions. The same metabolic incorporation as a methionine surrogate in methio- modified protein substrate was later derivatized more success- nine auxotrophic E. coli (van Hest et al., 2000) or by reassign- fully with a propargyl-tethered biotin under aqueous Sonaga- ment of the amber stop codon (Ai et al., 2010). The resulting shira coupling conditions (Figure 8B) (Kodama et al., 2007), allyl sulphides (Lin et al., 2008) are effective substrates for

Figure 9. Examples of Responsive Linkages Enabled by Bioorthogonal Ligations

tutes a rich area of application of bioorthogonal chemistry. An example is the use of abiotic bioorthgonal reactions to provoke drug activation (Figure 10A) in contrast to the traditional reliance on endogeneous processes such as hydrolysis or enzymatic cleavage (Figure 10B). In a relatively simple example, the reduction of an azide by phosphine (van Brakel et al., 2008) has been used as the prodrug activator in vivo, producing the corresponding aromatic amine that releases the cytotoxic cargo by 1,6- benzylic fragmentation (Figure 10C). The Staudinger ligation was also used to trigger drug liberation by virtue of a cross metathesis using standard (Figure 8E) and new catalysts similar process triggered by O-to-N acyl migration to the developed for aqueous-phase applications (Burtscher and iminophosphorane intermediate in the Staudinger process Grela, 2009; Skowerski et al., 2012; Tomasek and Schatz, (Figure 10D) (Azoulay et al., 2006). Although conceptually 2013). attractive, these approaches suffer from slow reaction kinetics Responsive Strategies: Ligation And Release. Whereas com- and the oxidative sensitivity of the phosphine reagent. The plete, or even representative, coverage of the concept of installation by Robillard and coworkers of a carbamate drug cleavable linkers is beyond the scope of this article, it is useful linkage next to the double bond of a trans-cyclooctene dieno- to mention that bioorthogonal click reactions have an impor- phile created a clever solution to these problems for anti- tant role to play in the creation of molecules and materials body-drug conjugates (Versteegen et al., 2013). Fast tetrazine that create linkages, release active ingredients, or perform ligation was followed by facile rearrangement to the corre- other functions in response to changes in biological location sponding pyridazine with ejection of the allylic carbamate or cellular condition. Figure 9A shows an example of such a leaving group, thus releasing the drug only upon receipt of triggered bioorthogonal ligation process: the rapid hetero- the bioorthogonal reaction signal (Figure 10E). Diels-Alder ligation of a quinolinone-based quinone methide, generated in situ, with vinyl thioethers (Li et al., 2013a). The Conclusions process has been used quite successfully for site-specific This examination of the recent literature of bioorthogonal click protein labeling as well as imaging bioactive small molecules reactions has revealed continuing exciting activity in ‘‘tradi- inside live cells. A particularly interesting recent example of tional’’ ligations to protein termini and side chains, as well as controlled bond formation and release (Figure 9B) takes advan- a wealth of new chemistry that has pushed the boundaries of tage of an oxidation event to create an electrophile suitable for rate and selectivity. However, new reactions have yet to find capture as an oxime ether (Park et al., 2014). Reduction of the much application to other biological substrates; ligations to nu- adduct then sets the stage for facile N-O bond cleavage, cleic acids, lipids, and glycans are almost exclusively done releasing the captured molecule in a manner that is not the with well-established methods, reflecting both the ubiquity of reverse of the original bond-forming step. Such sequential applications for peptide and protein derivatization and the diverse chemistry provides more opportunities for control of fact that proteins have more functional groups to address. each step of the process. Another variant on this theme is pro- But these chemically more challenging biomolecular substrates vided by the aforementioned oxanorbornadiene linkages (Table are just as important for applications in medicine, materials sci- 4, entry 10). These feature a built-in cleavage trigger, because ence, and fundamental chemical biology, and so define several their Michael adducts undergo retro-Diels-Alder fragmentation aspects of the future growth of the field of bioorthogonal at rates predetermined over several orders of magnitude by chemistry. their substitution pattern (Figure 9C) (Kislukhin et al., 2012). The overall dream shared by many investigators is the precise If the resulting furan and thiomaleate moieties do not com- insertion of nonbiological chemical reactions into ever-more- promise the function of the released functional molecules, complex biological systems, up to and including higher organ- this provides a well-controlled triggered cleavage pathway for isms. In this quest, we choose to dance with the dazzling drug release. molecular and functional complexity of evolution, without step- The development of prodrugs, molecules activated in the ping on Nature’s toes. The music is sublime, the destination body to reveal therapeutic function in order to limit off-target rewarding, and we have a few good moves. Finding and using toxicity or enhance properties such as biodistribution, consti- new ones is an endeavor most worthwhile.

Figure 10. Prodrug Activation Strategies Using Bioorthogonal Chemistry (A) Overall strategy. (B) Examples of traditional endogenous prodrug activation mechanisms. (C and D) Phosphine-based activation examples. (E) Activation assisted by tetrazine ligation.

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