Cell-selective bioorthogonal labeling.

In classic bioorthogonal labeling experiments, the cell's biosynthetic machinery incorporates bioorthogonal tags, creating tagged biomolecules that are subsequently reacted with a corresponding bioorthogonal partner. This two-step approach labels biomolecules throughout the organism indiscriminate of cell type, which can produce background in applications focused on specific cell populations. In this review, we cover advances in bioorthogonal chemistry that enable targeting of bioorthogonal labeling to a desired cell type. Such cell-selective bioorthogonal labeling is achieved in one of three ways. The first approach restricts labeling to specific cells by cell-selective expression of engineered enzymes that enable the bioorthogonal tag's incorporation. The second approach preferentially localizes the bioorthogonal reagents to the desired cell types to restrict their uptake to the desired cells. Finally, the third approach cages the reactivity of the bioorthogonal reagents, allowing activation of the reaction in specific cells by uncaging the reagents selectively in those cell populations.

Pyranthiones/Pyrones: "Click and Release" Donors for Subcellular Hydrogen Sulfide Delivery and Labeling.

Hydrogen sulfide (H2 S), one of the most important gasotransmitters, plays a critical role in endogenous signaling pathways of many diseases. However, developing H2 S donors with both tunable release kinetics and high release efficiency for subcellular delivery has been challenging. Here, we describe a click and release reaction between pyrone/pyranthiones and bicyclononyne (BCN). This reaction features a release of CO2 /COS with second-order rate constants comparable to Strain-Promoted Azide-Alkyne Cycloaddition reactions (SPAACs). Interestingly, pyranthiones showed enhanced reaction rates compared to their pyrone counterparts. We investigated pyrone biorthogonality and demonstrated their utility in protein labeling applications. Moreover, we synthesized substituted pyranthiones with H2 S release kinetics that can address the range of physiologically relevant H2 S dynamics in cells and achieved quantitative H2 S release efficiency in vitro. Finally, we explored the potential of pyranthiones as H2 S/COS donors for mitochondrial-targeted H2 S delivery in living cells.

Self-Patterned Nanoscale Topography of Thin Copolymer Films Prepared by Evaporative Assembly-Resist Early-Stage Bacterial Adhesion.

Biofilm formation on the surfaces of indwelling medical devices has become a growing health threat due to the development of antimicrobial resistance to infection-causing bacteria. For example, ventilator-associated pneumonia caused by Pseudomonas and Staphylococci species has become a significant concern in treatment of patients during COVID-19 pandemic. Nanostructured surfaces with antifouling activity are of interest as a promising strategy to prevent bacterial adhesion without triggering drug resistance. In this study, we report a facile evaporative approach to prepare block copolymer film coatings with nanoscale topography that resist bacterial adhesion. The initial attachment of the target bacterium Pseudomonas aeruginosa PAO1 to copolymer films as well as homopolymer films was evaluated by fluorescence microscopy. Significant reduction in bacterial adhesion (93-99% less) and area coverage (>92% less) on the copolymer films was observed compared with that on the control and homopolymer films [poly(methacrylic acid) (PMAA)─only 40 and 23% less, respectively]. The surfaces of poly(styrene)-PMAA copolymer films with patterned nanoscale topography that contains sharp peaks ranging from 20 to 80 nm spaced at 30-50 nm were confirmed by atomic force microscopy and the corresponding surface morphology analysis. Investigation of the surface wettability and surface potential of polymer films assists in understanding the effect of surface properties on the bacterial attachment. Comparison of bacterial growth studies in polymer solutions with the growth studies on coatings highlights the importance of physical nanostructure in resisting bacterial adhesion, as opposed to chemical characteristics of the copolymers. Such self-patterned antifouling surface coatings, produced with a straightforward and energy-efficient approach, could provide a convenient and effective method to resist bacterial fouling on the surface of medical devices and reduce device-associated infections.

Controlling Rheology of Fluid Interfaces through Microblock Length of Sequence-Controlled Amphiphilic Copolymers.

Previous studies have demonstrated that films of sequence-controlled amphiphilic copolymers display contact angles that depend on microblock size. This suggests that microblock length may provide a means of tuning surface and interfacial properties. In this work, the interfacial rheology of a series of sequence-controlled copolymers, prepared through the addition of bicyclo[4.2.0]oct-1(8)-ene-8-carboxamide (monomer A) and cyclohexene (monomer B) to generate sequences up to 24 monomeric units composed of (A m B n ) i microblocks, where m, n, and i range from 1 to 6. Interfacial rheometry is used to measure the mechanical properties of an air-water interface with these copolymers. As the microblock size increases, the interfacial storage modulus, G', increases, which may be due to an increase in the size of interfacial hydrophobic domains. Small-angle X-ray scattering shows that the copolymers have a similar conformation in solution, suggesting that any variations in the mechanics of the interface are due to assembly at the interface, and not on solution association or bulk rheological properties. This is the first study demonstrating that microblock size can be used to control interfacial rheology of amphiphilic copolymers. Thus, the results provide a new strategy for controlling the dynamics of fluid interfaces through precision sequence-controlled polymers.

Enzyme- or light-triggered cyclopropenes for bioorthogonal ligation.

Since first reported at the beginning of the 21st century, bioorthogonal reactions have become powerful tools for investigating biological systems. Here, we review several classic and current bioorthogonal reactions, including the Staudinger-Bertozzi ligation, strain-promoted azide-alkyne cycloaddition (SPAAC), 1,3-dipolar cycloaddition, and tetrazine-alkene ligation. We discuss the capabilities and limitations of the subset of current bioorthogonal reactions that can be "turned on" by exposure to light or an enzyme. Finally, we focus on our recently developed turn-on cyclopropenes, which can be activated for reaction with tetrazines by exposure to light or enzymes, like nitroreductase, depending on the modular reaction caging group appended to the cyclopropene. We discuss the caged cyclopropene's molecular design and synthesis, and we discuss experiments to evaluate and verify reactivity both in vitro and in vivo.

Visualizing the brain's astrocytes.

Astrocytes are the most abundant cell type in the brain and are a crucial part of solving its mysteries. Originally assumed to be passive supporting cells, astrocyte's functions are now recognized to include active modulation and information processing at the neural synapse. The full extent of the astrocyte contribution to neural processing remains unknown. This is, in part, due to the lack of methods available for astrocyte identification and analysis. Existing strategies employ genetic tools like the astrocyte specific promoters glial fibrillary acidic protein (GFAP) or Aldh1L1 to create transgenic animals with fluorescently labeled astrocytes. Recently, small molecule targeting moieties have enabled the delivery of bright fluorescent dyes to astrocytes. Here, we review methods for targeting astrocytes, with a focus on a recently developed methylpyridinium targeting moiety's development, chemical synthesis, and elaboration to provide new features like light-based spatiotemporal control of cell labeling.

Modular activatable bioorthogonal reagents.

The bioorthogonal reaction toolbox contains approximately two-dozen unique chemistries that permit selective tagging and probing of biomolecules. Over the past two decades, significant effort has been devoted to optimizing and discovering bioorthogonal reagents that are faster, fluorogenic, and orthogonal to the already existing bioorthogonal repertoire. Conversely, efforts to explore bioorthogonal reagents whose reactivity can be controlled in space and/or time are limited. The "activatable" bioorthogonal reagents that do exist are often unimodal, meaning that their reagent's activation method cannot be easily modified to enable activation with red-shifted wavelengths, enzymes, or metabolic-byproducts and ions like H2O2 or Fe3+. Here, we summarize the available activatable bioorthogonal reagents with a focus on our recent addition: modular caged cyclopropenes. We designed caged cyclopropenes to be unreactive to their bioorthogonal partner until they are activated through the removal of the cage by light, an enzyme, or another reaction partner. To accomplish this, their structure includes a nitrogen atom at the cyclopropene C3 position that is decorated with the desired caging group through a carbamate linkage. This 3-N cyclopropene system can allow control of cyclopropene reactivity using a multitude of already available photo- and enzyme-caging groups. Additionally, this cyclopropene scaffold can enable metabolic-byproduct or ion activation of bioorthogonal reactions.

Modular Enzyme- and Light-Based Activation of Cyclopropene-Tetrazine Ligation.

We describe a modular activation strategy for cyclopropene-tetrazine ligation. This activation strategy uses chemically diverse enzyme- or photolabile protecting groups as cyclopropene reactivity cages. The linkages between the caging groups and cyclopropene are through carbamates, thus permitting the application of diverse cages to allow bioorthogonal reactivity by administering enzymes or light.

Caged Cyclopropenes with Improved Tetrazine Ligation Kinetics.

Activatable cyclopropenes are unreactive toward their inverse electron demand Diels-Alder reaction partner (e.g., s-tetrazines) until they are activated. The activation strategy is highly modular due to the cyclopropene's ability to be caged by various light- and enzyme-activatable groups. This work describes the next generation of activatable cyclopropenes with a new core scaffold that maintains the activation modularity of the first generation but improves upon the ligation kinetics with s-tetrazines by ≤270-fold.

Hierarchical assembly in PLA-PEO-PLA hydrogels with crystalline domains and effect of block stereochemistry.

Understanding the development of microstructure (e.g., structures with length scales roughly 0.5-500 µm) in hydrogels is crucial for their use in several biomedical applications. We utilize ultra-small-angle neutron scattering (USANS) and confocal microscopy to explore microstructure of poly(lactide)-poly(ethylene oxide)-poly(lactide) (PLA-PEO-PLA) triblock copolymer hydrogels with varying l/d-lactide ratio. We have previously found that these polymers self-assemble on the nanoscale into micelles. Here, we observe large-scale structures with diverse morphologies, including highly porous self-similar networks with characteristic sizes spanning approximately 120 nm-200 µm. These structural features give rise to power-law scattering indicative of fractal structures in USANS. Mass fractal and surface fractal structures are found for gels with l/d ratios of 80/20 and 50/50, respectively. Confocal microscopy shows microscale water-filled channels and pores that are more clearly evident in gels with a higher fraction of l-lactide in the PLA block as compared to the 50/50 hydrogels. Tuning block stereochemistry may provide a means of controlling the self-assembly and structural evolution at both the nanoscale and microscale, impacting application of these materials in tissue engineering and drug delivery.

Linear Desferrichrome-Linked Silicon-Rhodamine Antibody Conjugate Enables Targeted Multimodal Imaging of HER2 in Vitro and in Vivo.

We report the nuclear and optical in vitro and in vivo imaging of SKOV-3 cells by targeting HER2 with a bimodal trastuzumab conjugate. Previously, we have shown that desferrichrome derivatives provide a robust and versatile radiolabeling platform for the radioisotope zirconium-89. Here, we appended silicon-rhodamine functionalized linear desferrichrome to trastuzumab. This construct was radiolabeled and used to image cellular binding and antibody uptake in vitro and in vivo. The robust extinction coefficient of the SiR deep-red emissive fluorophore enables direct quantification of the number of appended chelators and fluorophore molecules per antibody. Subsequent radiolabeling of the multifunctional immunoconjugate with 89Zr was achieved with a 64 ± 9% radiochemical yield, while the reference immunoconjugate desferrioxamine (DFO)-trastuzumab exhibited a yield of 84 ± 9%. In vivo PET imaging (24, 48, 72, and 96 h post injection) and biodistribution experiments (96 h post injection) in HER2+ tumor bearing mice revealed no statistically significant difference of the two 89Zr-labeled conjugates at each time point evaluated. The bimodal conjugate permitted successful in vivo fluorescence imaging (96 h post injection) and subsequent fluorescence-guided, surgical resection of the tumor mass. This report details the first successful application of a fluorophore-functionalized desferrichrome derivative for targeted imaging, motivating further development and application of this scaffold as a multimodal imaging platform.

Design Principles for Cationic, Astrocyte-Targeted Probes.

The brain's astrocytes play key roles in normal and pathological brain processes. Targeting small molecules to astrocytes in the presence of the many other cell types in the brain will provide useful tools for their visualization and manipulation. Herein, we explore the functional consequences of synthetic modifications to a recently described astrocyte marker composed of a bright rhodamine-based fluorophore and an astrocyte-targeting moiety. We altered the nature of the targeting moiety to probe the dependence of astrocyte targeting on hydrophobicity, charge, and pKa when exposed to astrocytes and neurons isolated from the mouse cortex. We found that an overall molecular charge of +2 and a targeting moiety with a heterocyclic aromatic amine are important requirements for specific and robust astrocyte labeling. These results provide a basis for engineering astrocyte-targeted molecular tools with unique properties, including metabolite sensing or optogenetic control.

Lipidated cyclopropenes via a stable 3-N spirocyclopropene scaffold.

Lipidated cyclopropenes serve as useful bioorthogonal reagents for imaging cell membranes due to the cyclopropene's small size and ability to ligate with pro-fluorescent tetrazines. Previously, the lipidation of cyclopropenes required modification at the C3 position because methods to append lipids at C1/C2 were not available. Herein, we describe C1/C2 lipidation with the biologically active lipid ceramide and a common phospholipid using a cyclopropene scaffold whose reactivity with 1,2,4,5-tetrazines has been caged.

Caged cyclopropenes for controlling bioorthogonal reactivity.

Bioorthogonal ligations have been designed and optimized to provide new experimental avenues for understanding biological systems. Generally, these optimizations have focused on improving reaction rates and orthogonality to both biology and other members of the bioorthogonal reaction repertoire. Less well explored are reactions that permit control of bioorthogonal reactivity in space and time. Here we describe a strategy that enables modular control of the cyclopropene-tetrazine ligation. We developed 3-N-substituted spirocyclopropenes that are designed to be unreactive towards 1,2,4,5-tetrazines when bulky N-protecting groups sterically prohibit the tetrazine's approach, and reactive once the groups are removed. We describe the synthesis of 3-N spirocyclopropenes with an appended electron withdrawing group to promote stability. Modification of the cyclopropene 3-N with a bulky, light-cleavable caging group was effective at stifling its reaction with tetrazine, and the caged cyclopropene was resistant to reaction with biological nucleophiles. As expected, upon removal of the light-labile group, the 3-N cyclopropene reacted with tetrazine to form the expected ligation product both in solution and on a tetrazine-modified protein. This reactivity caging strategy leverages the popular carbamate protecting group linkage, enabling the use of diverse caging groups to tailor the reaction's activation modality for specific applications.

Visualizing the Brain's Astrocytes with Diverse Chemical Scaffolds.

Astrocytes are the most abundant cells in the brain. They support neurons, adjust synaptic strength, and modulate neuronal signaling, yet the full extent of their functions is obscured by the dearth of methods for their visualization and analysis. Here, we report a chemical reporter that targets small molecules specifically to astrocytes both in vitro and in vivo. Fluorescent versions of this tag are imported through an organic cation transporter to label glia across species. The structural modularity of this approach will enable wide-ranging applications for understanding astrocyte biology.

Neuronal Calcium Recording with an Engineered TEV Protease.

Technologies for measuring the transient Ca2+ spikes that accompany neural signaling have revolutionized our understanding of the brain. Nevertheless, microscopic visualization of Ca2+ spikes on the time scale of neural activity across large brain regions or in thick specimens remains a significant challenge. The recent development of stable integrators of Ca2+, instead of transient reporters, provides an avenue to investigate neural signaling in otherwise challenging systems. Here, we describe an engineered Ca2+-sensing enzyme consisting of a split Tobacco Etch Virus (TEV) protease with each half tethered to a calmodulin or M13 Ca2+ binding domain. This Split TEV, Ca2+ Activated Neuron Recorder (SCANR) remains separate and catalytically incompetent until a spike in cellular Ca2+ triggers its reconstitution and the subsequent turnover of a caged, genetically encoded reporter substrate. We report the identification of a successful Ca2+-sensing split TEV from a library of chimeras and deployment of the enzyme in primary rat hippocampal neurons.

Inexpensive multigram-scale synthesis of cyclic enamines and 3-N spirocyclopropyl systems.

Cyclic enamines are important synthons for many synthetic and pharmacological targets. Here, we report an inexpensive, catalyst-free, multigram-scale synthesis for cyclic enamines with exocyclic double bonds and four- to seven-membered rings. This strategy is more conducive to scale up, permissive of functionalization around the cyclic system, and less sensitive to the nature of the N-protecting group than previously-described methods for cyclic enamine synthesis. Further, we explore application of these enamines to the synthesis of highly-strained spirocyclic 3N-cyclopropyl scaffolds.

Light-Activated Staudinger-Bertozzi Ligation within Living Animals.

The ability to regulate small molecule chemistry in vivo will enable new avenues of exploration in imaging and pharmacology. However, realization of these goals will require reactions with high specificity and precise control. Here we demonstrate photocontrol over the highly specific Staudinger-Bertozzi ligation in vitro and in vivo. Our simple approach, photocaging the key phosphine atom, allows for the facile production of reagents with photochemistry that can be engineered for specific applications. The resulting compounds, which are both stable and efficiently activated, enable the spatial labeling of metabolically introduced azides in vitro and on live zebrafish.

A photocaged, cyclopropene-containing analog of the amino acid neurotransmitter glutamate.

Substituted cyclopropenes serve as compact biorthogonal appendages that enable analysis of biomolecules in complex systems. Neurotransmitters, a chemically diverse group of biomolecules that control neuron excitation and inhibition, are not among the systems that have been studied using biorthogonal chemistry. Here we describe the synthesis of cyclopropene-containing analogs of the excitatory amino acid neurotransmitter glutamate starting from a Garner's aldehyde-derived alkyne. The deprotected cyclopropene glutamate was stable in solution but decomposed upon concentration. Appending a light-cleavable group improved the stability of the cyclopropene while simultaneously caging the neurotransmitter. This strategy has the potential to permit deployment of cyclopropene-modified glutamate as a bioorthogonal probe of the neurotransmitter glutamate in vivo with spatiotemporal precision.

Imaging the sialome during zebrafish development with copper-free click chemistry.

The sialome comprises sialylated glycoproteins and glycolipids that play essential roles in cell-cell communication. Using azide-modified molecular precursors of sialic acids and copper-free click chemistry, we visualized the spatiotemporal dynamics of the sialome in live zebrafish embryos.