Neal K. Devaraj

Biomedical Applications of Tetrazine Cycloadditions

Disease mechanisms are increasingly being resolved at the molecular level. Biomedical success at this scale creates synthetic opportunities for combining specifically designed orthogonal reactions in applications such as imaging, diagnostics, and therapy. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k > 10(3) M(-1) s(-1)) and high specificity. Improving kinetics would minimize both the time and amount of labeling agent required to maintain high coupling yields. In this Account, we discuss our recent efforts to design extremely rapid bioorthogonal coupling reactions between tetrazines and strained alkenes. These selective reactions were first used to covalently couple conjugated tetrazine near-infrared-emitting fluorophores to dienophile-modifed extracellular proteins on living cancer cells. Confocal fluorescence microscopy demonstrated efficient and selective labeling, and control experiments showed minimal background fluorescence. Multistep techniques were optimized to work with nanomolar concentrations of labeling agent over a time scale of minutes: the result was successful real-time imaging of covalent modification. We subsequently discovered fluorogenic probes that increase in fluorescence intensity after the chemical reaction, leading to an improved signal-to-background ratio. Fluorogenic probes were used for intracellular imaging of dienophiles. We further developed strategies to react and image chemotherapeutics, such as trans-cyclooctene taxol analogues, inside living cells. Because the coupling partners are small molecules (<300 Da), they offer unique steric advantages in multistep amplification. We also describe recent success in using tetrazine reactions to label biomarkers on cells with magneto-fluorescent nanoparticles. Two-step protocols that use bioorthogonal chemistry can significantly amplify signals over both one-step labeling procedures as well as two-step procedures that use more sterically hindered biotin-avidin interactions. Nanoparticles can be detected with fluorescence or magnetic resonance techniques. These strategies are now being routinely used on clinical samples for biomarker profiling to predict malignancy and patient outcome. Finally, we discuss recent results with tetrazine reactions used for in vivo molecular imaging applications. Rapid tetrazine cycloadditions allow modular labeling of small molecules with the most commonly used positron emission tomography isotope, (18)F. Additionally, recent work has applied this reaction directly in vivo for the pretargeted imaging of solid tumors. Future work with tetrazine cycloadditions will undoubtedly lead to optimized protocols, improved probes, and additional biomedical applications.

Live-Cell Imaging of Cyclopropene Tags with Fluorogenic Tetrazine Cycloadditions

There is growing interest in the use of inverse Diels–Alder tetrazine cycloadditions as rapid catalyst-free bioorthogonal reactions.[1–3] Fluorogenic tetrazines that increase in fluorescence after reaction with dienophiles are particularly useful for live-cell imaging applications.[4] Fluorogenic tetrazines have been recently used for live-cell imaging of small molecules, biomolecules tagged enzymatically with dienophiles, and proteins modified by reactive unnatural amino acids.[4,5] Although fluorogenic tetrazine probes hold great potential for intracellular imaging of small molecules, previous approaches are limited by requiring a large strained dienophile, such as trans-cyclooctene, cyclooctyne, or norbornene.[1,6] This situation is in contrast to Staudinger ligations or strain-promoted azide–cycloalkyne cycloadditions that utilize a small azide functional group.[7,8] This requirement has limited the use of tetrazine reactions in methods that require tags with minimal steric impact or nominal effect on the partition ratio.[2] The development of smaller dienophile partners capable of reacting rapidly with tetrazines would therefore represent a major advance. However, it has been unclear whether small dienophiles could be developed that react rapidly with tetrazines while maintaining their stability. Herein, we demonstrate the applicability of methylcyclopropene tags as dienophiles for reaction with fluorogenic tetrazines. Through systematic synthetic modifications we have optimized the stability, size, and reactivity of the cyclopropene scaffold. We have developed methylcyclopropene derivatives that react rapidly with tetrazines while retaining their aqueous stability and small size. These cyclopropene handles elicit fluorescent responses from quenched tetrazine dyes and are suitable for cellular imaging applications, which we demonstrate by imaging cyclopropene phospholipids distributed in live human breast cancer cells.

Metal Catalyzed One-Pot Synthesis of Tetrazines Directly from Aliphatic Nitriles and Hydrazine

There is rapidly growing interest in the use of 1,2,4,5-tetrazines as bioorthogonal coupling agents.[1-3] Recent applications of tetrazine cycloadditions include intracellular small molecule imaging, genetically targeted protein tagging, post-synthetic DNA labeling, nanoparticle based clinical diagnostics, and in-vivo imaging.[4-7] In addition, tetrazines have seen significant use in materials science[8,9], coordination chemistry, [10,11] and specialty explosives research.[12,13] They are also valuable synthetic intermediates, and have been elegantly deployed on route to several natural product syntheses.[14-16] Despite the promise of tetrazines, the lack of convenient synthetic methods is a significant roadblock to their broader use and study by the scientific community.[17] Here we report that Lewis acid transition metal catalysts, most notably divalent nickel and zinc salts, can catalyze the formation of 1,2,4,5-tetrazines directly from nitriles. To our knowledge, this is the first method utilizing homogenous catalysis to directly synthesize tetrazines from a wide range of unactivated aliphatic nitriles and hydrazine. Symmetric and asymmetric tetrazines were conveniently prepared from multiple precursors including alkyl nitriles, aromatic nitriles, and formamidine salts. This methodology should greatly improve the accessibility of tetrazines and lead to further exploration of their applications, particularly with respect to bioorthogonal conjugations.

Fluorescent Live-Cell Imaging of Metabolically Incorporated Unnatural Cyclopropene-Mannosamine Derivatives

Sugar coated: We recently developed methylcyclopropenes as low-molecular-weight tetrazine coupling partners. Here, we demonstrate that methylcyclopropenes can meet the stringent steric demands required for metabolic imaging of unnatural mannosamines on live cells. Using sequential azide-alkyne chemistry, we also demonstrate multicolor imaging of two different metabolically incorporated unnatural sugars.

In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic Bioorthogonal Live-Cell Imaging Probes†

In spite of the wide application potential of 1,2,4,5-tetrazines, particularly in live-cell and in vivo imaging, a major limitation has been the lack of practical synthetic methods. Here we report the in situ synthesis of (E)-3-substituted 6-alkenyl-1,2,4,5-tetrazine derivatives through an elimination-Heck cascade reaction. By using this strategy, we provide 24 examples of π-conjugated tetrazine derivatives that can be conveniently prepared from tetrazine building blocks and related halides. These include tetrazine analogs of biological small molecules, highly conjugated buta-1,3-diene-substituted tetrazines, and a diverse array of fluorescent probes suitable for live-cell imaging. These highly conjugated probes show very strong fluorescence turn-on (up to 400-fold) when reacted with dienophiles such as cyclopropenes and trans-cyclooctenes, and we demonstrate their application for live-cell imaging. This work provides an efficient and practical synthetic methodology for tetrazine derivatives and will facilitate the application of conjugated tetrazines, particularly as fluorogenic probes for live-cell imaging.

Expanding room for tetrazine ligations in the in vivo chemistry toolbox

There is tremendous interest in developing and refining methods to predictably perform chemical reactions within the framework of living systems. Here we review recent advances in applying tetrazine cycloadditions to live cell and in vivo chemistry. We highlight new syntheses of the tetrazine and dienophile precursors useful for in vivo studies. We briefly overview the use of this reaction in combination with unnatural amino acid technology and discuss applications involving the imaging of glycans on live cells. An emerging area is the use of tetrazine ligations for the development of in vivo imaging probes such as those used for positron emission tomography. We summarize recent applications involving tetrazine cycloadditions performed in live mice for pretargeted imaging of cancer cell biomarkers.

Bioorthogonal tetrazine-mediated transfer reactions facilitate reaction turnover in nucleic acid-templated detection of microRNA.

Tetrazine ligations have proven to be a powerful bioorthogonal technique for the detection of many labeled biomolecules, but the ligating nature of these reactions can limit reaction turnover in templated chemistry. We have developed a transfer reaction between 7-azabenzonorbornadiene derivatives and fluorogenic tetrazines that facilitates turnover amplification of the fluorogenic response in nucleic acid-templated reactions. Fluorogenic tetrazine-mediated transfer (TMT) reaction probes can be used to detect DNA and microRNA (miRNA) templates to 0.5 and 5 pM concentrations, respectively. The endogenous oncogenic miRNA target mir-21 could be detected in crude cell lysates and detected by imaging in live cells. Remarkably, the technique is also able to differentiate between miRNA templates bearing a single mismatch with high signal to background. We imagine that TMT reactions could find wide application for amplified fluorescent detection of clinically relevant nucleic acid templates.

Membrane Assembly Driven by a Biomimetic Coupling Reaction

One of the major goals of synthetic biology is the development of non-natural cellular systems. In this work, we describe a catalytic biomimetic coupling reaction capable of driving the de novo self-assembly of phospholipid membranes. Our system features a coppercatalyzed azide-alkyne cycloaddition that results in the formation of a triazole-containing phospholipid analogue. Concomitant assembly of membranes occurs spontaneously, not requiring preexisting membranes to house catalysts or precursors. The substitution of efficient synthetic reactions for key biochemical processes may offer a general route toward synthetic biological systems.

Rapid oligonucleotide-templated fluorogenic tetrazine ligations

Template driven chemical ligation of fluorogenic probes represents a powerful method for DNA and RNA detection and imaging. Unfortunately, previous techniques have been hampered by requiring chemistry with sluggish kinetics and background side reactions. We have developed fluorescent DNA probes containing quenched fluorophore-tetrazine and methyl-cyclopropene groups that rapidly react by bioorthogonal cycloaddition in the presence of complementary DNA or RNA templates. Ligation increases fluorescence with negligible background signal in the absence of hybridization template. Reaction kinetics depend heavily on template length and linker structure. Using this technique, we demonstrate rapid discrimination between single template mismatches both in buffer and cell media. Fluorogenic bioorthogonal ligations offer a promising route towards the fast and robust fluorescent detection of specific DNA or RNA sequences.

Self-reproducing catalyst drives repeated phospholipid synthesis and membrane growth

Significance We report on the design and synthesis of an artificial cell membrane that sustains continual growth. Lipid membranes are ubiquitous in all domains of life. Numerous studies have exploited the ability of lipids to self-assemble into bilayer vesicles with properties reminiscent of cellular membranes, but previous work has yet to mimic nature’s ability to support persistent phospholipid membrane formation. In this work, we have developed an artificial cell membrane that continually synthesizes all of the components needed to form additional catalytic membranes. These results demonstrate that complex lipid membranes capable of indefinite self-synthesis can emerge when supplied with simpler chemical building blocks. Cell membranes are dynamic structures found in all living organisms. There have been numerous constructs that model phospholipid membranes. However, unlike natural membranes, these biomimetic systems cannot sustain growth owing to an inability to replenish phospholipid-synthesizing catalysts. Here we report on the design and synthesis of artificial membranes embedded with synthetic, self-reproducing catalysts capable of perpetuating phospholipid bilayer formation. Replacing the complex biochemical pathways used in nature with an autocatalyst that also drives lipid synthesis leads to the continual formation of triazole phospholipids and membrane-bound oligotriazole catalysts from simpler starting materials. In addition to continual phospholipid synthesis and vesicle growth, the synthetic membranes are capable of remodeling their physical composition in response to changes in the environment by preferentially incorporating specific precursors. These results demonstrate that complex membranes capable of indefinite self-synthesis can emerge when supplied with simpler chemical building blocks.