Towards Selective Delivery of a Ruthenium(II) Polypyridyl Complex-Containing Bombesin Conjugate into Cancer Cells.

An increasing number of novel Ru(II) polypyridyl complexes have been successfully applied as photosensitizers (PSs) for photodynamic therapy (PDT). Despite recent advances in optimized PSs with refined photophysical properties, the lack of tumoral selectivity is often a major hurdle for their clinical development. Here, classical maleimide and versatile NHS-activated acrylamide strategies were employed to site-selectively conjugate a promising Ru(II) polypyridyl complex to the N-terminally Cys-modified Bombesin (BBN) targeting unit. Surprisingly, the decreased cell uptake of these novel Ru-BBN conjugates in cancer cells did not hamper the high phototoxic activity of the Ru-containing bioconjugates and even decreased the toxicity of the constructs in the absence of light irradiation. Overall, although deceiving in terms of selectivity, our new bioconjugates could still be useful for advanced cancer treatment due to their nontoxicity in the dark.

BASHY Dyes Are Highly Efficient Lipid Droplet-Targeting Photosensitizers that Induce Ferroptosis through Lipid Peroxidation.

Ferroptosis is an iron-dependent lipid-peroxidation-driven mechanism of cell death and a promising therapeutic target to eradicate cancer cells. In this study, we discovered that boronic acid-derived salicylidenehydrazone (BASHY) dyes are highly efficient singlet-oxygen photosensitizers (PSs; ΦΔ up to 0.8) that induce ferroptosis triggered by photodynamic therapy. The best-performing BASHY dye displayed a high phototoxicity against the human glioblastoma multiform U87 cell line, with an IC50 value in the low nanomolar range (4.40 nM) and a remarkable phototoxicity index (PI > 22,700). Importantly, BASHY dyes were shown to accumulate in lipid droplets (LDs) and this intracellular partition was found to be essential for the enhanced phototoxicity and the induction of ferroptosis through lipid peroxidation. The safety and phototoxicity of this platform were validated using an in vivo zebrafish model (Danio rerio).

Engineering the BASHY Dye Platform toward Architectures with Responsive Fluorescence.

A set of nine boronic-acid-derived salicylidenehydrazone (BASHY) complexes has been synthesized in good to very good chemical yields in a versatile three-component reaction. In an extension to previous reports on this dye platform, the focus was put on the electronic modification of the "vertical" positions of the salicylidenehydrazone backbone. This enabled the observation of fluorescence quenching by photoinduced electron transfer (PeT), which can be reverted by the addition of acid in organic solvent (OFF-ON fluorescence switching). The resulting emission is observed in the green-to-orange spectral region (maxima at 520-590 nm). In contrast, under physiological pH conditions in water, the PeT process is inherently decativated, thereby enabling the observation of fluorescence in the red-to-NIR region (maxima at 650-680 nm) with appreciable quantum yields and lifetimes. The latter characteristic supported the application of the dyes in fluorescence lifetime imaging (FLIM) of live A549 cells.

Interactive Bioconjugation at N-Terminal Cysteines by Using O-Salicylaldehyde Esters towards Dual Site-Selective Functionalization.

N-terminal Cys modification has been intensively studied to produce homogeneous bioconjugates essentially through two modes of reaction: reversible modification with the equilibrium shifted towards the formation of the desired conjugate or stable and irreversible conjugates. Herein, we report a new method of N-terminal cysteine modification using O-salicylaldehyde esters (OSAEs) through fast conjugation and irreversible deconjugation. These reagents can rapidly react with N-terminal Cys at low-micromolar concentration to form thiazolidines with subsequent hydrolysis of the ester moiety to the phenolic derivative. These phenolic thiazolidines can be hydrolyzed at acidic pH (≈4.5) to recover the intact N-terminal Cys. Bioconjugation reactions using OSAEs offer controlled reversibility to as act as a protecting group for N-terminal cysteines, allowing the modification of in-chain residues without perturbing the N-terminal Cys, which can then be deprotected and used as a conjugation site.

Cysteine-Assisted Click-Chemistry for Proximity-Driven, Site-Specific Acetylation of Histones.

Post-translational modifications of histones are essential in the regulation of chromatin structure and function. Among these modifications, lysine acetylation is one of the most established. Earlier studies relied on the use of chromatin containing heterogeneous mixtures of histones acetylated at multiple sites. Differentiating the individual contribution of single acetylation events towards chromatin regulation is thus of great relevance. However, it is difficult to access homogeneous samples of histones, with a single acetylation, in sufficient quantities for such studies. By engineering histone H3 with a cysteine in proximity of the lysine of interest, we demonstrate that conjugation with maleimide-DBCO followed by a strain-promoted alkyne-azide cycloaddition reaction results in the acetylation of a single lysine in a controlled, site-specific manner. The chemical precision offered by our click-to-acetylate approach will facilitate access to and the study of acetylated histones.

Dual Stimuli-Responsive Dynamic Covalent Peptide Tags: Toward Sequence-Controlled Release in Tumor-like Microenvironments.

Dynamic covalent chemistry (DCvC) has emerged as a versatile synthetic tool for devising stable, stimuli-responsive linkers or conjugates. The interplay of binding affinity, association and dissociation constants exhibits a strong influence on the selectivity of the reaction, the conversion rate, as well as the stability in aqueous solutions. Nevertheless, dynamic covalent interactions often exhibit fast binding and fast dissociation events or vice versa, affecting their conversion rates or stabilities. To overcome the limitation in linker design, we reported herein dual responsive dynamic covalent peptide tags combining a pH responsive boronate ester with fast association and dissociation rates, and a redox-active disulfide with slow formation and dissociation rate. Precoordination by boronic acid-catechol interaction improves self-sorting and selectivity in disulfide formation into heterodimers. The resulting bis-peptide conjugate exhibited improved complex stability in aqueous solution and acidic tumor-like extracellular microenvironment. Furthermore, the conjugate responds to pH changes within the physiological range as well as to redox conditions found inside cancer cells. Such tags hold great promise, through cooperative effects, for controlling the stability of bioconjugates under dilution in aqueous media, as well as designing intelligent pharmaceutics that react to distinct biological stimuli in cells.

Unveiling the Potential of Transition Metal Complexes for Medicine: Translational in Situ Activation of Metal-Based Drugs from Bench to in Vivo Applications.

The development of metal-based anticancer drugs has been hampered, among other reasons, by their lack of selectivity for cancer cells. In a recent article, Zou and co-workers presented the successful intracellular activation of organogold(I) complexes for potential cancer treatment through Pd(II)-mediated transmetallation, overcoming some off-target activity of novel gold-based drugs. This unique strategy builds the perfect bridge between metallodrug usage and bioorthogonal intracellular catalysis for more advanced and selective therapies. Such an approach will hopefully pave the way for forthcoming studies in medicinal inorganic chemistry.

Correction: A 2-formylphenylboronic acid (2FPBA)-maleimide crosslinker: a versatile platform for Cys-peptide-hydrazine conjugation and interplay.

Correction for 'A 2-formylphenylboronic acid (2FPBA)-maleimide crosslinker: a versatile platform for Cys-peptide-hydrazine conjugation and interplay' by João P. M. António et al., Org. Biomol. Chem., 2021, 19, 6221-6226, DOI: 10.1039/D1OB00917F.

A 2-formylphenylboronic acid (2FPBA)-maleimide crosslinker: a versatile platform for Cys-peptide-hydrazine conjugation and interplay.

In this work, we describe the preparation of a heterobifunctional 2-formylphenylboronic acid (2-FPBA)-maleimide crosslinker and explore its versatility in the preparation of various bioconjugates. We demonstrate the straightforward attachment of hydrazine payloads to cysteine residues in peptides, as well as the crosslinking of different thiol-bearing peptides or payloads with N-terminal cysteine peptides. Importantly, the dynamic nature of the 2-FPBA handle enables an interplay between the thiazolidine and diazaborine forms, which allows obtaining various products controlled by (and in some cases independent of) the order of addition of the components.

Correction: Boronic acids as building blocks for the construction of therapeutically useful bioconjugates.

Correction for 'Boronic acids as building blocks for the construction of therapeutically useful bioconjugates' by João P. M. António et al., Chem. Soc. Rev., 2019, 48, 3513-3536, DOI: .

Efficient Amino-Sulfhydryl Stapling on Peptides and Proteins Using Bifunctional NHS-Activated Acrylamides.

Widely used reagents in the peptide functionalization toolbox, Michael acceptors and N-hydroxysuccinimide (NHS) activated esters, are combined in NHS-activated acrylamides for efficient chemoselective amino-sulfhydryl stapling on native peptides and proteins. NHS-activated acrylamides allow for a fast functionalization of N-terminal cysteines (k2 =1.54±0.18×103  M-1 s-1 ) under dilute aqueous conditions, enabling selectivity over other nucleophilic amino acids. Additionally, the versatility of these new bioconjugation handles was demonstrated in the cross-linking of in-chain or C-terminal cysteines with nearby lysine residues. NHS-activated acrylamides are compatible with the use of other cysteine selective reagents, allowing for orthogonal dual-modifications. This strategy was successfully applied to the late-stage functionalization of peptides and proteins with a PEG unit, fluorescent probe, and cytotoxic agent. The level of molecular control offered by NHS-activated acrylamides is expected to promote amino-sulfhydryl stapling technology as a powerful strategy to design functional bioconjugates.

Diazaborines Are a Versatile Platform to Develop ROS-Responsive Antibody Drug Conjugates*.

Antibody-drug conjugates (ADCs) are a new class of therapeutics that combine the lethality of potent cytotoxic drugs with the targeting ability of antibodies to selectively deliver drugs to cancer cells. In this study we show for the first time the synthesis of a reactive-oxygen-species (ROS)-responsive ADC (VL-DAB31-SN-38) that is highly selective and cytotoxic to B-cell lymphoma (CLBL-1 cell line, IC50 value of 54.1 nM). The synthesis of this ADC was possible due to the discovery that diazaborines (DABs) are a very effective ROS-responsive unit that are also very stable in buffer and in plasma. DFT calculations performed on this system revealed a favorable energetic profile (ΔGR=-74.3 kcal mol-1 ) similar to the oxidation mechanism of aromatic boronic acids. DABs' very fast formation rate and modularity enabled the construction of different ROS-responsive linkers featuring self-immolative modules, bioorthogonal functions, and bioconjugation handles. These structures were used in the site-selective functionalization of a VL antibody domain and in the construction of the homogeneous ADC.

Engineering Boron Hot Spots for the Site-Selective Installation of Iminoboronates on Peptide Chains.

Boronic acids (BAs) are a promising bioconjugation function to design dynamic materials as they can establish reversible covalent bonds with oxygen/nitrogen nucleophiles that respond to different pH, ROS, carbohydrates and glutathione levels. However, the dynamic nature of these bonds also limits the control over the stability and site-selectivity of the bioconjugation, which ultimately leads to heterogeneous conjugates with poor stability under physiological conditions. Here we disclose a new strategy to install BAs on peptide chains. In this study, a "boron hot spot" based on the 3-hydroxyquinolin-2(1H)-one scaffold was developed and upon installation on a peptide N-terminal cysteine, enables the site-selective formation of iminoboronates with 2-formyl-phenyl boronic acids (Ka of 58128±2 m-1 ). The reaction is selective in the presence of competing lysine ϵ-amino groups, and the resulting iminoboronates, displayed improved stability in buffers solutions and a cleavable profile in the presence of glutathione. Once developed, the methodology was used to prepare cleavable fluorescent conjugates with a laminin fragment, which enabled the validation of the 67LR receptor as a target to deliver cargo to cancer HT29 cells.

Cyanine-Like Boronic Acid-Derived Salicylidenehydrazone Complexes (Cy-BASHY) for Bioimaging Applications.

Boronic acid-derived salicylidenehydrazone complex (BASHY) dyes with a polymethine backbone were designed to yield efficient red-emitting and two-photon absorbing fluorophores that can be used as markers for astrocytes. The dyes are chemically stable in aqueous solution and do not undergo photodecomposition. Their photophysical properties can be electronically fine-tuned and thereby adapted to potentially different imaging situations and requirements.

Boronic acids as building blocks for the construction of therapeutically useful bioconjugates.

Bioconjugates are multifunctional constructs in which biomolecules like peptides, proteins, vitamins and nucleic acids are endowed with the properties of specific payloads. These constructs recently emerged as a new generation of high-precision therapeutics, with several representatives reaching the market. This success stimulated an intense search for new biocompatible synthetic methodologies to connect both components and to control the bioconjugate's function. Despite the remarkable advances made in this field, most of the technologies developed for the construction of bioconjugates were engineered to yield stable constructs that can endure complex physiological conditions. Because of this, the use of reversible covalent bonds in the synthesis of bioconjugates has been rather overlooked, notwithstanding the potential of this strategy to generate stimuli responsive constructs that may operate in areas like the selective delivery of drugs, live-cell imaging and new theranostic approaches. Boronic acids are a well-known class of reagents that have been widely used in modern synthesis for the formation of C-C and C-heteroatom bonds. Apart from this, boronic acids exhibit an exquisite reversible coordination profile that can be explored as a molecular construction tool featuring specific mechanisms to control the structure and biological properties of bioconjugates. In this review, the use of boronic acids in the construction of therapeutically useful bioconjugates will be discussed, focusing on the molecular mechanisms that allow the use of these reagents as bioconjugation warheads, as central pieces of linker structures and as functional payloads.

Sequence Programming with Dynamic Boronic Acid/Catechol Binary Codes.

The development of a synthetic code that enables a sequence programmable feature like DNA represents a key aspect toward intelligent molecular systems. We developed herein the well-known dynamic covalent interaction between boronic acids (BAs) and catechols (CAs) into synthetic nucleobase analogs. Along a defined peptide backbone, BA or CA residues are arranged to enable sequence recognition to their complementary strand. Dynamic strand displacement and errors were elucidated thermodynamically to show that sequences are able to specifically select their partners. Unlike DNA, the pH dependency of BA/CA binding enables the dehybridization of complementary strands at pH 5.0. In addition, we demonstrate the sequence recognition at the macromolecular level by conjugating the cytochrome c protein to a complementary polyethylene glycol chain in a site-directed fashion.

Bioconjugation with Maleimides: A Useful Tool for Chemical Biology.

Maleimide chemistry stands out in the bioconjugation toolbox by virtue of its synthetic accessibility, excellent reactivity, and practicability. The second-generation of clinically approved antibody-drug conjugates (ADC) and much of the current ADC pipeline in clinical trials contain the maleimide linkage. However, thiosuccinimide linkages are now known to be less robust than once thought, and ergo, are correlated with suboptimal pharmacodynamics, pharmacokinetics, and safety profiles in some ADC constructs. Rational design of novel generations of maleimides and maleimide-type reagents have been reported to address the shortcomings of classical maleimides, allowing for the formation of robust bioconjugate linkages. This review highlights the main strategies for rational reagent design that have allowed irreversible bioconjugations in cysteines, reversible labelling strategies and disulfide re-bridging.

The Role of Electron Transfer in the Fragmentation of Phenyl and Cyclohexyl Boronic Acids.

In this study, novel measurements of negative ion formation in neutral potassium-neutral boronic acid collisions are reported in electron transfer experiments. The fragmentation pattern of phenylboronic acid is comprehensively investigated for a wide range of collision energies, i.e., from 10 to 1000 eV in the laboratory frame, allowing some of the most relevant dissociation channels to be probed. These studies were performed in a crossed molecular beam set up using a potassium atom as an electron donor. The negative ions formed in the collision region were mass analysed with a reflectron time-of-flight mass spectrometer. In the unimolecular decomposition of the temporary negative ion, the two most relevant yields were assigned to BO- and BO2-. Moreover, the collision-induced reaction was shown to be selective, i.e., at energies below 100 eV, it mostly formed BO-, while at energies above 100 eV, it mostly formed BO2-. In order to further our knowledge on the complex internal reaction mechanisms underlying the influence of the hybridization state of the boron atom, cyclohexylboronic acid was also investigated in the same collision energy range, where the main dissociation channel yielded BO2-. The experimental results for phenyl boronic acid are supported by ab initio theoretical calculations of the lowest unoccupied molecular orbitals (LUMOs) accessed in the collision process.

N,O-Iminoboronates: Reversible Iminoboronates with Improved Stability for Cancer Cells Targeted Delivery.

Herein a new class of iminoboronates obtained from 2-acetylbenzene boronic acids and aminophenols is presented. The N,O-ligand topology enabled the formation of an additional B-O bond that locks the boron center in a tetrahedral geometry. This molecular arrangement decisively contributes to improve the construct's stability in biocompatible conditions and retaining the iminoboronate reversibility in more acidic environments. 2-Acetylbenzene boronic acid was reacted with a fluorescent amino-coumarin to yield a stable and non-fluorescent N,O-iminoboronate. This mechanism was further used to assemble a folate receptor targeting conjugate that selectively delivered the fluorescent amino-coumarin to MDA-MB-231 human breast cancer cells.

Circularly Polarized Luminescence of Boronic Acid-Derived Salicylidenehydrazone Complexes Containing Chiral Boron as Stereogenic Unit.

Racemic mixtures of boronic acid-derived salicylidenehydrazone (BASHY) complexes were enantiomerically resolved. The chiroptical properties of the stereoisomers, containing an asymmetric boron as the only stereogenic unit, are translated into mirror-imaged electronic circular dichroism spectra and circularly polarized luminescence (CPL, dissymmetry factors of 3-5 × 10-4) is observed. The spectral position of the CPL emission is determined by the push-pull character of the dye. These features expand the functional scope of the brightly emitting BASHY dye platform.