Ellen M. Sletten

Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality

The study of biomolecules in their native environments is a challenging task because of the vast complexity of cellular systems. Technologies developed in the last few years for the selective modification of biological species in living systems have yielded new insights into cellular processes. Key to these new techniques are bioorthogonal chemical reactions, whose components must react rapidly and selectively with each other under physiological conditions in the presence of the plethora of functionality necessary to sustain life. Herein we describe the bioorthogonal chemical reactions developed to date and how they can be used to study biomolecules.

From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions

Bioorthogonal reactions are chemical reactions that neither interact with nor interfere with a biological system. The participating functional groups must be inert to biological moieties, must selectively reactive with each other under biocompatible conditions, and, for in vivo applications, must be nontoxic to cells and organisms. Additionally, it is helpful if one reactive group is small and therefore minimally perturbing of a biomolecule into which it has been introduced either chemically or biosynthetically. Examples from the past decade suggest that a promising strategy for bioorthogonal reaction development begins with an analysis of functional group and reactivity space outside those defined by Nature. Issues such as stability of reactants and products (particularly in water), kinetics, and unwanted side reactivity with biofunctionalities must be addressed, ideally guided by detailed mechanistic studies. Finally, the reaction must be tested in a variety of environments, escalating from aqueous media to biomolecule solutions to cultured cells and, for the most optimized transformations, to live organisms. Work in our laboratory led to the development of two bioorthogonal transformations that exploit the azide as a small, abiotic, and bioinert reaction partner: the Staudinger ligation and strain-promoted azide–alkyne cycloaddition. The Staudinger ligation is based on the classic Staudinger reduction of azides with triarylphosphines first reported in 1919. In the ligation reaction, the intermediate aza-ylide undergoes intramolecular reaction with an ester, forming an amide bond faster than aza-ylide hydrolysis would otherwise occur in water. The Staudinger ligation is highly selective and reliably forms its product in environs as demanding as live mice. However, the Staudinger ligation has some liabilities, such as the propensity of phosphine reagents to undergo air oxidation and the relatively slow kinetics of the reaction. The Staudinger ligation takes advantage of the electrophilicity of the azide; however, the azide can also participate in cycloaddition reactions. In 1961, Wittig and Krebs noted that the strained, cyclic alkyne cyclooctyne reacts violently when combined neat with phenyl azide, forming a triazole product by 1,3-dipolar cycloaddition. This observation stood in stark contrast to the slow kinetics associated with 1,3-dipolar cycloaddition of azides with unstrained, linear alkynes, the conventional Huisgen process. Notably, the reaction of azides with terminal alkynes can be accelerated dramatically by copper catalysis (this highly popular Cu-catalyzed azide–alkyne cycloaddition (CuAAC) is a quintessential “click” reaction). However, the copper catalysts are too cytotoxic for long-term exposure with live cells or organisms. Thus, for applications of bioorthogonal chemistry in living systems, we built upon Wittig and Krebs’ observation with the design of cyclooctyne reagents that react rapidly and selectively with biomolecule-associated azides. This strain-promoted azide–alkyne cycloaddition is often referred to as “Cu-free click chemistry”. Mechanistic and theoretical studies inspired the design of a series of cyclooctyne compounds bearing fluorine substituents, fused rings, and judiciously situated heteroatoms, with the goals of optimizing azide cycloaddition kinetics, stability, solubility, and pharmacokinetic properties. Cyclooctyne reagents have now been used for labeling azide-modified biomolecules on cultured cells and in live Caenorhabditis elegans, zebrafish, and mice. As this special issue testifies, the field of bioorthogonal chemistry is firmly established as a challenging frontier of reaction methodology and an important new instrument for biological discovery. The above reactions, as well as several newcomers with bioorthogonal attributes, have enabled the high-precision chemical modification of biomolecules in vitro, as well as real-time visualization of molecules and processes in cells and live organisms. The consequence is an impressive body of new knowledge and technology, amassed using a relatively small bioorthogonal reaction compendium. Expansion of this toolkit, an effort that is already well underway, is an important objective for chemists and biologists alike.

Rapid Cu-Free Click Chemistry with Readily Synthesized Biarylazacyclooctynones

Bioorthogonal chemical reactions, those that do not interact or interfere with biology, have allowed for exploration of numerous biological processes that were previously difficult to study. The reaction of azides with strained alkynes, such as cyclooctynes, readily forms a triazole product without the need for a toxic catalyst. Here we describe a biarylazacyclooctynone (BARAC) that has exceptional reaction kinetics and whose synthesis is designed to be both modular and scalable. We employed BARAC for live cell fluorescence imaging of azide-labeled glycans. The high signal-to-background ratio obtained using nanomolar concentrations of BARAC obviated the need for washing steps. Thus, BARAC is a promising reagent for in vivo imaging.

Copper-free click chemistry in living animals

Chemical reactions that enable selective biomolecule labeling in living organisms offer a means to probe biological processes in vivo. Very few reactions possess the requisite bioorthogonality, and, among these, only the Staudinger ligation between azides and triarylphosphines has been employed for direct covalent modification of biomolecules with probes in the mouse, an important model organism for studies of human disease. Here we explore an alternative bioorthogonal reaction, the 1,3-dipolar cycloaddition of azides and cyclooctynes, also known as “Cu-free click chemistry,” for labeling biomolecules in live mice. Mice were administered peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to metabolically label cell-surface sialic acids with azides. After subsequent injection with cyclooctyne reagents, glycoconjugate labeling was observed on isolated splenocytes and in a variety of tissues including the intestines, heart, and liver, with no apparent toxicity. The cyclooctynes tested displayed various labeling efficiencies that likely reflect the combined influence of intrinsic reactivity and bioavailability. These studies establish Cu-free click chemistry as a bioorthogonal reaction that can be executed in the physiologically relevant context of a mouse.

A Hydrophilic Azacyclooctyne for Cu-Free Click Chemistry

Biomolecules labeled with azides can be detected through Cu-free click chemistry with cyclooctyne probes, but their intrinsic hydrophobicity can compromise bioavailability. Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4-yne (DIMAC). Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes. However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection.

Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry

The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called “copper-free click chemistry”, is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARAC’s aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARAC’s aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC’s central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry.

Pictet-spengler ligation for protein chemical modification

Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with α-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C–C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. We used the reaction for site-specific chemical modification of glyoxyl- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a means to generate stable bioconjugates for medical and materials applications.

Thiacycloalkynes for Copper-Free Click Chemistry

Bioorthogonal chemistry enables the interrogation of biomolecules and physiological processes that are inaccessible by using conventional research tools.1 A common experimental protocol starts by labeling a target biomolecule in cells or live organisms with a bioorthogonal functional group. Then, a probe molecule bearing complementary functionality is added to the system and the ensuing bioorthogonal chemical reaction delivers the probe specifically to the targets of interest. For many applications, rapid reaction kinetics are essential. This is particularly true for labeling experiments in live animals, in which reagent concentrations are limited (i.e., nm to low μm), or in which the process that is probed occurs on a fast time scale. Consequently, methodologists interested in the development of bioorthogonal reactions are increasingly focused on kinetic optimization.2

Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition

Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

Difluorobenzocyclooctyne: Synthesis, Reactivity, and Stabilization by β-Cyclodextrin

Highly reactive cyclooctynes have been sought as substrates for Cu-free cycloaddition reactions with azides in biological systems. To elevate the reactivities of cyclooctynes, two strategies, LUMO lowering through propargylic fluorination and strain enhancement through fused aryl rings, have been explored. Here we report the facile synthesis of a difluorobenzocyclooctyne (DIFBO) that combines these modifications. DIFBO was so reactive that it spontaneously trimerized to form two asymmetric products that we characterized by X-ray crystallography. However, we were able to trap DIFBO by forming a stable inclusion complex with β-cyclodextrin in aqueous media. This complex could be stored as a lyophilized powder and then dissociated in organic solvents to produce free DIFBO for in situ kinetic and spectroscopic analysis. Using this procedure, we found that the rate constant for the cycloaddition reaction of DIFBO with an azide exceeds those for difluorinated cyclooctyne (DIFO) and dibenzocyclooctyne (DIBO). Cyclodextrin complexation is therefore a promising approach for stabilizing compounds that possess the high intrinsic reactivities desired for Cu-free click chemistry.