A BODIPY-Based Probe Enables Fluorogenicity via Thiol-Dependent Modulation of Fluorophore Aggregation.

Given the popular usage of BODIPY fluorophores in biological research, their propensity to aggregate in aqueous solution and impact their spectroscopic properties arguably warrants more attention. The probe under study herein serves as a case in point. A para-maleimide-substituted meso-phenyl BODIPY (p-MB) had previously been characterized in organic media, where its inherently high fluorescence ruled out its fluorogenic potential. Here, we have found that in aqueous solution, p-MB behaves differently, exhibiting a much-reduced fluorescence as a result of aggregation-caused quenching (ACQ). Additionally, p-MB is capable of responding to complementarily reactive substrates, including thiols and TCEP, to generate a substantial turn-on signal. The fluorescence restoration is largest when it reacts with those containing adjacent ionizable groups. By being part of a polar conjugate, p-MB assumes a disaggregated form, circumventing ACQ and unleashing up to ~1000-fold fluorescence enhancement through apparent disaggregation-induced emission (DIE). While our results support DIE as the turn-on mechanism, we found that the reactivity of the probe is much lower when it is given time to form stable aggregates. Therefore, contrary to the conventional depiction that a DIE probe works by dispersing from preformed aggregates to react with the target, our results suggest that it functions via a target-mediated inhibition of probe aggregation. Altogether, our work highlights the aggregation issue often faced by BODIPY-based probes and demonstrates how that can be exploited for turn-on sensing application. Furthermore, it reconstructs a different pathway for the DIE mechanism.

Expanding the Versatility of Microbial Transglutaminase Using α-Effect Nucleophiles as Noncanonical Substrates.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology, in particular for the attachment of alkyl amines to glutamine-containing peptides and proteins. Here, we expand the substrate repertoire to include hydrazines, hydrazides, and alkoxyamines, resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG. Furthermore, we demonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues. The distinct hydrazide handles can be further coupled with carbonyls, including ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability. The extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme, providing a new approach to facilitate novel applications.

Site-Specific Bioconjugation and Multi-Bioorthogonal Labeling via Rapid Formation of a Boron-Nitrogen Heterocycle.

Precise control of covalent bond formation in the presence of multiple functional groups is pertinent in the development of many next-generation bioconjugates and materials. Strategies derived from bioorthogonal chemistries are contributing greatly in that regard; however, the gain of chemoselectivity is often compromised by the slow rates of many of these existing chemistries. Recent work on a variation of the classical aldehyde/ketone condensation based on ortho-carbonylphenylboronic acids has uncovered markedly accelerated rates compared to those of the simple carbonyl counterparts. The products of these reactions are distinct, often in the form of boron-nitrogen heterocycles. In particular, we have shown that 2-formylphenylboronic acid (2fPBA), when coupled with an α-amino-hydrazide, produces a unique zwitterionic and stable 2,3,1-benzodiazaborine derivative. In this work, we apply this chemistry to generate chemically defined and functional bioconjugates, herein illustrated with immunoconjugates. We show that an antibody and a fluorophore (as payload) equipped with the relevant reactive handles undergo rapid conjugation at near-stoichiometric ratios, displaying a reaction half-life of only ∼5 min with 2 equiv of the linker payload. Importantly, the reaction can be extended to multicomponent labeling by partnering with the popular strain-promoted azide-alkyne cycloaddition and tetrazine- trans-cyclooctene (Tz-TCO) ligation. The mutual orthogonality to both of these chemistries allows simultaneous triple bioorthogonal conjugations, a rare feat thus far that will widen the scope of various multilabeling applications. Further collaboration with the Tz-TCO reaction enables rapid one-pot synthesis of a site-specific dual-payload antibody conjugate. Altogether, we envision that the 2fPBA-α-amino-hydrazide ligation will facilitate efficient assembly of diverse bioconjugates and materials, enabling access to more complex modalities via partnership with other orthogonal chemistries.

Formation of hydrazones and stabilized boron-nitrogen heterocycles in aqueous solution from carbohydrazides and ortho-formylphenylboronic acids.

A recent addition to the suite of fast bioorthogonal reactions combines hydrazines and hydroxylamines with ortho-carbonyl substituted phenylboronic acids. Carbohydrazides are easily incorporated into biomolecules, making them appealing substrates in these reactions. Here we show that simple alkyl carbohydrazides form a single product with ortho-formylphenylboronic acid in an organic solvent and in the solid state. The solution structures of the products formed from the carbohydrazides in buffered aqueous solution, however, are markedly different from those identified in the organic solvent and solid state. The reactants form a mixture of hydrazone and heterocyclic products, the relative composition of which varies with pH. The observed reversibility of bioconjugates using carbohydrazide can thus be explained by the reversibility of the boron-nitrogen bond in the heterocycle. In contrast, the inclusion of an α-amine into the carbohydrazide substrate yields a single product in which both nitrogens are bonded to boron. These tricyclic structures are the same in organic solvent, solid state and aqueous solution from pH 4 to pH 9. Bioconjugates formed with α-amino carbohydrazides are stable to SDS-PAGE, while those formed with simple alkyl carbohydrazides are not. We propose that the inclusion of an intramolecular stabilizing ligand into a carbohydrazide substrate is a generally applicable principle that may be exploited to form boronic acid-based bioconjugates with a defined structure and resistance to hydrolysis.

A Novel Fluorogenic Assay for the Detection of Nephrotoxin-Induced Oxidative Stress in Live Cells and Renal Tissue.

Drug-induced kidney injury frequently leads to aborted clinical trials and drug withdrawals. Sufficiently sensitive sensors capable of detecting mild signs of chemical insult in cell-based screening assays are critical to identifying and eliminating potential toxins in the preclinical stage. Oxidative stress is a common early manifestation of chemical toxicity, and biomolecule carbonylation is an irreversible repercussion of oxidative stress. Here, we present a novel fluorogenic assay using a sensor, TFCH, that responds to biomolecule carbonylation and efficiently detects modest forms of renal injury with much greater sensitivity than standard assays for nephrotoxins. We demonstrate that this sensor can be deployed in live kidney cells and in renal tissue. Our robust assay may help inform preclinical decisions to recall unsafe drug candidates. The application of this sensor in identifying and analyzing diverse pathologies is envisioned.

Visualization of oxidative stress-induced carbonylation in live mammalian cells.

Oxidative stress (OS) is associated with a wide variety of diseases and disorders. Detection of oxidative stress in living systems typically relies on fluorescent probes for reactive oxygen species (ROS), which is challenging because of their short life span and high reactivity. Oxidative damage caused by OS produces a more stable signal, but these biomarkers are usually detected using techniques that are not compatible with live cells. OS-induced biomolecule carbonylation is a stable modification that also possesses a chemically reactive functional group, and its detection typically employs a chemical reaction with a hydrazine-containing probe within the process. These hydrazone-forming reactions require strong acid catalysis or nucleophilic catalysis with an aromatic amine when performed on isolated biomaterial or on fixed cells. In live cells, however, hydrazone-forming reactions are surprisingly facile. Fluorophores possessing hydrazine or hydrazide functional groups can undergo reaction with carbonylated biomolecules in live cells, and these products can be observed using fluorescence microscopy. In this chapter, standard methods for detection of biomolecule carbonylation in cell lysate and in intact cells are enumerated. Protocols for fluorescently labeling biomolecule carbonylation in live cells are provided for commercially available fluorophores. Also described is a one-step protocol that employs one of the hydrazine-modified fluorophores developed in our lab, which are designed to be live-cell compatible and to undergo a spectral change upon hydrazone formation. Finally, a procedure for observing both biomolecule carbonylation and ROS production simultaneously is provided.

Click Chemistry Conjugations.

Click chemistry has found wide application in bioconjugation, enabling control over the site of modification in biomolecules. Demonstrations of this chemistry to construct chemically defined antibody-drug conjugates (ADCs) have increased in recent years, following studies that support benefits of homogeneity and site-specificity of drug placement on the antibody. In this chapter, a brief history of early applications of this chemistry in ADCs is presented. Examples of click chemistries that are utilized for ADC synthesis, including those currently undergoing clinical investigations, are enumerated. Protocols for two common conjugation methods based on carbonyl-aminooxy coupling and strain-promoted azide-alkyne cycloaddition are described.

Benzocoumarin Hydrazine: A Large Stokes Shift Fluorogenic Sensor for Detecting Carbonyls in Isolated Biomolecules and in Live Cells.

Detection and quantification of biomolecule carbonylation, a critical manifestation of oxidative stress, allows better understanding of associated disease states. Existing approaches for such analyses require further processing of cells and tissues, which leads to loss of both spatial and temporal information about carbonylated biomolecules in cells. Live cell detection of these species requires sensors that are nontoxic, sufficiently reactive with the biocarbonyl in the intracellular milieu, and detectable with commonly available instrumentation. Presented here is a new fluorescent sensor for biomolecule carbonyl detection: a hydrazine derivative of a benzocoumarin, 7-hydrazinyl-4-methyl-2H-benzo[h]chromen-2-one (BzCH), which meets these requirements. This probe is especially well suited for live cell studies. It can be excited by a laser line common to many fluorescence microscopes. The emission maximum of BzCH undergoes a substantial red shift upon hydrazone formation (from ∼430 to ∼550 nm), which is the result of fluorophore disaggregation. Additionally, the hydrazone exhibits an exceptionally large Stokes shift (∼195 nm). The latter properties eliminate self-quenching of the probe and the need to remove unreacted fluorophore for reliable carbonyl detection. Thus, biomolecule carbonylation can be detected and quantified in cells and in cell extracts in a one-step procedure using this probe.

Detection of oxidative stress-induced carbonylation in live mammalian cells.

Oxidative stress is often associated with etiology and/or progression of disease conditions, such as cancer, neurodegenerative diseases, and diabetes. At the cellular level, oxidative stress induces carbonylation of biomolecules such as lipids, proteins, and DNA. The presence of carbonyl-containing biomolecules as a hallmark of these diseases provides a suitable target for diagnostic detection. Here, a simple, robust method for detecting cellular aldehydes and ketones in live cells using a fluorophore is presented. A hydrazine-functionalized synthetic fluorophore serves as an efficient nucleophile that rapidly reacts with reactive carbonyls in the cellular milieu. The product thus formed exhibits a wavelength shift in the emission maximum accompanied by an increase in emission intensity. The photochemical characteristics of the fluorophore enable the identification of the fluorophore-conjugated cellular biomolecules in the presence of unreacted dye, eliminating the need for removal of excess fluorophore. Moreover, this fluorophore is found to be nontoxic and is thus appropriate for live cell analysis. Utility of the probe is demonstrated in two cell lines, PC3 and A549. Carbonylation resulting from serum starvation and hydrogen peroxide-induced stress is detected in both cell lines using fluorescence microscopy and a fluorescence plate reader. The fluorescent signal originates from carbonylated proteins and lipids but not from oxidized DNA, and the majority of the fluorescence signal (>60%) is attributed to fluorophore-conjugated lipid oxidation products. This method should be useful for detecting cellular carbonylation in a high-content assay or high-throughput assay format.