Covalent Attachment Research Articles

Pharmaceutical Integrity of Lyophilized Methemoglobin-Albumin Clusters after Reconstitution.

Covalent attachment of a ferric hemoglobin (metHb) core to three human serum albumin molecules to form metHb-albumin clusters has previously been used to develop an antidote for hydrogen sulfide poisoning. Lyophilization is one of the most effective approaches to preserve protein pharmaceuticals with minimum contamination and decomposition. However, there is concern that lyophilized proteins may undergo pharmaceutical alteration on reconstitution. This study investigated the pharmaceutical integrity of metHb-albumin clusters on lyophilization and reconstitution with three clinically available reconstitution fluids, (i) sterile water for injection, (ii) 0.9% sodium chloride injection, and (iii) 5% dextrose injection. The metHb-albumin clusters retained their physicochemical properties and structural integrity on lyophilization and reconstitution with sterile water for injection or 0.9% sodium chloride injection, along with comparable hydrogen sulfide scavenging ability compared to non-lyophilized metHb-albumin clusters. The reconstituted protein completely rescued lethal hydrogen sulfide poisoning in mice. On the other hand, lyophilized metHb-albumin clusters reconstituted with 5% dextrose injection showed physicochemical changes and a higher mortality rate in mice subjected to lethal hydrogen sulfide poisoning. In conclusion, lyophilization represents a potent preservation method for metHb-albumin clusters if either sterile water for injection or 0.9% sodium chloride injection is used for reconstitution.

S-Palmitoylation during Retinoic Acid-Induced Neuronal Differentiation of SH-SY5Y Neuroblastoma Cells.

S-Palmitoylation is the covalent attachment of C14:0-C22:0 fatty acids (mainly C16:0 palmitate) to cysteines via thioester bonds. This lipid modification is highly abundant in neurons, where it plays a role in neuronal development and is implicated in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. The knowledge of S-palmitoylation in neurodevelopment is limited due to technological challenges in analyzing this highly hydrophobic protein modification. Here, we used two orthogonal methods, acyl-biotin exchange (ABE) and lipid metabolic labeling (LML), to identify S-palmitoylated proteins and sites during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. We identified 2002 putative S-palmitoylated proteins in total, of which 650 were found with both methods. Significant changes in the abundance of S-palmitoylated proteins were detected, in particular for several processes and protein classes that are known to be important for neuronal differentiation, which include proto-oncogene tyrosine-protein kinase receptor (RET) signal transduction, SNARE protein-mediated exocytosis, and neural cell adhesion molecules. Overall, S-palmitoylation profiling by employing ABE and LML in parallel during RA-induced differentiation of SH-SY5Y cells revealed a subset of high confidence bona fide S-palmitoylated proteins and suggested an important role for S-palmitoylation in neuronal differentiation.

Live-Cell Mitochondrial Targeted NIR Fluorescent Covalent Labeling of Specific Proteins Using a Dual Localization Effect.

Here, our designed water-soluble NIR fluorescent unsymmetrical Cy-5-Mal/TPP+ consists of a lipophilic cationic TPP+ subunit that can selectively target and accumulate in a live-cell inner mitochondrial matrix where a maleimide residue of the probe undergoes faster chemoselective and site-specific covalent attachment with the exposed Cys residue of mitochondrion-specific proteins. On the basis of this dual localization effect, Cy-5-Mal/TPP+ molecules remain for a longer time period even after membrane depolarization, enabling long-term live-cell mitochondrial imaging. Due to the adequate concentration of Cy-5-Mal/TPP+ reached in live-cell mitochondria, it facilitates site-selective NIR fluorescent covalent labeling with Cys-exposed proteins, which are identified by the in-gel fluorescence assay and LC-MS/MS-based proteomics and supported by a computational method. This dual targeting approach with admirable photostability, narrow NIR absorption/emission bands, bright emission, long fluorescence lifetime, and insignificant cytotoxicity has been shown to improve real-time live-cell mitochondrial tracking including dynamics and interorganelle crosstalk with multicolor imaging applications.

The E3 ligase HERC5 promotes antimycobacterial responses in macrophages by ISGylating the phosphatase PTEN.

Innate immune signaling in macrophages during viral infection is regulated by ISGylation, the covalent attachment of the ubiquitin-like protein interferon-stimulated gene 15 (ISG15) to protein targets. Here, we explored the role of ISGylation in the macrophage response to infection with Mycobacterium tuberculosis. In human and mouse macrophages, the E3 ubiquitin ligases HERC5 and mHERC6, respectively, mediated the ISGylation of the phosphatase PTEN, which promoted its degradation. The decreased abundance of PTEN led to an increase in the activity of the PI3K-AKT signaling pathway, which stimulated the synthesis of proinflammatory cytokines. Bacterial growth was increased in culture and in vivo when human or mouse macrophages were deficient in the major E3 ISG15 ligase. The findings expand the role of ISGylation in macrophages to antibacterial immunity and suggest that HERC5 signaling may be a candidate target for adjunct host-directed therapy in patients with tuberculosis.

A Label-Free Carbohydrate-Based Electrochemical Sensor to Detect Escherichia coli Pathogenic Bacteria Using D-mannose on a Glassy Carbon Electrode.

Controlling water and food contamination by pathogenic organisms requires quick, simple, and low-cost methods. Using the affinity between mannose and type I fimbriae in the cell wall of Escherichia coli (E. coli) bacteria as evaluation elements compared to the conventional plate counting technique enables a reliable sensing platform for the detection of bacteria. In this study, a simple new sensor was developed based on electrochemical impedance spectroscopy (EIS) for rapid and sensitive detection of E. coli. The biorecogniton layer of the sensor was formed by covalent attachment of p-carboxyphenylamino mannose (PCAM) to gold nanoparticles (AuNPs) electrodeposited on the surface of a glassy carbon electrode (GCE). The resultant structure of PCAM was characterized and confirmed using a Fourier Transform Infrared Spectrometer (FTIR). The developed biosensor demonstrated a linear response with a logarithm of bacterial concentration (R2 = 0.998) in the range of 1.3 × 10 1~1.3 × 106 CFU·mL-1 with the limit of detection of 2 CFU·mL-1 within 60 min. The sensor did not generate any significant signals with two non-target strains, demonstrating the high selectivity of the developed biorecognition chemistry. The selectivity of the sensor and its applicability to analysis of the real samples were investigated in tap water and low-fat milk samples. Overall, the developed sensor showed to be promising for the detection of E. coli pathogens in water and low-fat milk due to its high sensitivity, short detection time, low cost, high specificity, and user-friendliness.

Fouling Resistance of Brush-Modified Elastomers.

The most effective antifouling coatings are designed to slowly release biocides that target a broad spectrum of marine organisms. However, as biocides have a deleterious effect on marine life, there is demand for environmentally friendly coatings that resist fouling through physical interactions. We propose a simple platform for the development of such coatings based on bottlebrush-modified elastomers. The bottlebrush additives were synthesized to have side chain chemistries that are known to be fouling-resistant, and these were incorporated in a commercial elastomer through blending and/or covalent attachment. The fouling performance of these coatings was highly variable, with area coverages of hard and soft foulants ranging from 1.4% to 7.2% and 29.1% to 64.0%, respectively, across a set of eight materials. The origin of these differences was explained by examining the structure of the coating surface through chemical imaging by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and topographic imaging by atomic force microscopy (AFM). We found that fouling by certain soft and hard fouling organisms was primarily influenced by surface composition, which was controlled by both the chemistry and loading level of the bottlebrush additive, and was independent of the inherent surface roughness. While no type of coating could resist all soft and hard foulants, a formulation based on a bottlebrush copolymer additive with both siloxane and fluorinated monomers was effective against nearly all organisms encountered in the study.

Postsynthetische Photochemische Modifizierung und 2D Strukturierung von Zr‐MOF Dünnfilmen mit Benzophenon Linkermolekülen

AbstractFür die Herstellung von MOF‐basierten Bauteilen der nächsten Generation ist die Verfügbarkeit hochgradig anpassungsfähiger Materialien in geeigneten Formen entscheidend. In dieser Arbeit stellen wir Dünnfilme eines Metall‐organischen Gerüstes (MOF) mit photoreaktiven Benzophenon‐Brückenmolekülen vor. Kristalline, orientierte und poröse Filme des Zirkonium‐basierten bzpdc‐MOF (bzpdc=Benzophenon‐4‐4′‐dicarboxylat) können über direktes Wachstum auf Silizium‐ oder Glassubstraten hergestellt werden. Durch eine anschließende photochemische Modifizierung der Zr‐bzpdc‐MOF‐Filme lassen sich verschiedene Eigenschaften mittels kovalenter Anbindung von Modifizierungsreagenzien postsynthetisch einstellen. Neben der Modifizierung mit kleinen Molekülen sind auch grafting‐from Polymerisationsreaktionen möglich und in einer weiteren Ausbaustufe sogar die 2D‐Strukturierung und das Fotoschreiben definierter Strukturen; z.B. durch einen fotolithographischen Ansatz, der den Weg zu mikro‐strukturierten MOF‐Oberflächen ebnet.

Bioinspired Hierarchical Carbon Structures as Potential Scaffolds for Wound Healing and Tissue Regeneration Applications.

Engineered bio-scaffolds for wound healing provide an attractive treatment option for tissue engineering and traumatic skin injuries since they can reduce dependence on donors and promote faster repair through strategic surface engineering. Current scaffolds present limitations in handling, preparation, shelf life, and sterilization options. In this study, bio-inspired hierarchical all-carbon structures comprising carbon nanotube (CNT) carpets covalently bonded to flexible carbon fabric have been investigated as a platform for cell growth and future tissue regeneration applications. CNTs are known to provide guidance for cell growth, but loose CNTs are susceptible to intracellular uptake and are suspected to cause in vitro and in vivo cytotoxicity. This risk is suppressed in these materials due to the covalent attachment of CNTs on a larger fabric, and the synergistic benefits of nanoscale and micro-macro scale architectures, as seen in natural biological materials, can be obtained. The structural durability, biocompatibility, tunable surface architecture, and ultra-high specific surface area of these materials make them attractive candidates for wound healing. In this study, investigations of cytotoxicity, skin cell proliferation, and cell migration were performed, and results indicate promise in both biocompatibility and directed cell growth. Moreover, these scaffolds provided cytoprotection against environmental stressors such as Ultraviolet B (UVB) rays. It was seen that cell growth could also be tailored through the control of CNT carpet height and surface wettability. These results support future promise in the design of hierarchical carbon scaffolds for strategic wound healing and tissue regeneration applications.

Postsynthetic Photochemical Modification and 2D Structuring of Zr-MOF Thin Films Containing Benzophenone Linker Molecules.

For the fabrication of next-generation MOF-based devices the availability of highly adaptable materials in suitable shapes is crucial. Here, we present thin films of a metal-organic framework (MOF) containing photoreactive benzophenone units. Crystalline, oriented and porous films of the zirconium-based bzpdc-MOF (bzpdc=benzophenone-4-4'-dicarboxylate) are prepared by direct growth on silicon or glass substrates. Via a subsequent photochemical modification of the Zr-bzpdc-MOF films, various properties can be tuned postsynthetically by covalent attachment of modifying agents. Apart from the modification with small molecules, also grafting-from polymerization reactions are possible. In a further extension, 2D structuring and photo-writing of defined structures is also possible, for example by using a photolithographic approach, paving the way towards micro-patterned MOF surfaces.

Targeting of biomolecular condensates to the autophagy pathway.

Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.

Enzyme-Responsive Zr-Based Metal-Organic Frameworks for Controlled Drug Delivery: Taking Advantage of Clickable PEG-Phosphate Ligands.

We report for the first time the controlled drug release from a nanoscale Zr-based metal-organic framework (MOF), UiO-66, in the presence of the enzyme alkaline phosphatase (ALP). This unprecedented reactivity was possible thanks to the prior functionalization of the MOF with N3-PEG-PO3 ligands, which were designed for three specific aims: (1) to impart colloidal stability in phosphate-containing media; (2) to endow the MOF with multifunctionality thanks to azide groups for the covalent attachment of an imaging agent by click-chemistry; and (3) to confer stimuli-responsive properties, specifically the selective release of doxorubicin triggered by the enzymatic activity of ALP. Cell studies revealed that the functionalization of the MOF with N3-(PEG)20-PO3 ligands improved their intracellular stability and led to a sustained drug release compared to the bare MOF. More importantly, an enhanced drug release was observed in cells with higher expression of ALP genes (HeLa versus MDA-MB-231 and MCF7), confirming the ALP-responsiveness of the system inside living cells.

High-yield ligation-free assembly of DNA constructs with nucleosome positioning sequence repeats for single-molecule manipulation assays

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70± 3h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.

SUMOylation and related post-translational modifications in natural killer cell anti-cancer responses.

SUMOylation is a reversible modification that involves the covalent attachment of small ubiquitin-like modifier (SUMO) to target proteins, leading to changes in their localization, function, stability, and interactor profile. SUMOylation and additional related post-translational modifications have emerged as important modulators of various biological processes, including regulation of genomic stability and immune responses. Natural killer (NK) cells are innate immune cells that play a critical role in host defense against viral infections and tumors. NK cells can recognize and kill infected or transformed cells without prior sensitization, and their activity is tightly regulated by a balance of activating and inhibitory receptors. Expression of NK cell receptors as well as of their specific ligands on target cells is finely regulated during malignant transformation through the integration of different mechanisms including ubiquitin- and ubiquitin-like post-translational modifications. Our review summarizes the role of SUMOylation and other related pathways in the biology of NK cells with a special emphasis on the regulation of their response against cancer. The development of novel selective inhibitors as useful tools to potentiate NK-cell mediated killing of tumor cells is also briefly discussed.

Highly selective preparation of N-terminus Horseradish peroxidase-DNA conjugate with fully retained enzymatic activity: HRP-DNA structure – activity relation

Within the last decade, the field of bio-nanoengineering has achieved significant advances allowing us to generate, e.g., nanoscaled molecular machineries with arbitrary shapes. To unleash the full potential of novel methods such as DNA origami technology, it is important to functionalise complex molecules and nanostructures precisely. Thus, considerable attention has been given to site-selective modifications of proteins allowing further incorporation of various functionalities. Here, we describe a method for the covalent attachment of oligonucleotides to the glycosylated horseradish peroxidase protein (HRP) with high N-terminus selectivity and significant yield while conserving the enzymatic activity. This two-step process includes a pH-controlled metal-free diazotransfer reaction using imidazole-1-sulfonyl azide hydrogen sulfate, which at pH 8.5 results in an N-terminal azide-functionalized protein, followed by the Cu-free click SPAAC reaction to dibenzocyclooctyne- (DBCO) modified oligonucleotides. The reaction conditions were optimised to achieve maximum yield and the best performance. The resulting protein–oligonucleotide conjugates (HRP-DNA) were characterised by electrophoresis and mass spectrometry (MS). Native-PAGE experiments demonstrated different migration patterns for HRP-DNA and the azido-modified protein allowing zymogram experiments. Structure–activity relationships of novel HRP-DNA conjugates were assessed using molecular dynamics simulations, characterising the molecular interactions that define the structural and dynamical properties of the obtained protein-oligonucleotide conjugates (POC).

Porphyrin-silica gel hybrids as effective and selective copper(II) adsorbents from industrial wastewater

Porphyrins are an important class of ligands with a tremendous ability to capture metal ions closely related to the rich coordination chemistry of porphyrins. Herein we use this characteristic to develop silica gel grafted derivatives for water remediation applications. Therefore, two porphyrin derivatives, one with three and the other with four mercaptopyridyl units were grafted on silica gel functionalized with 3-aminopropyltriethoxysilane. The new adsorbents Si3PyS and Si4PyS were characterized using a suitable set of techniques confirming the covalent attachment of the porphyrins to the silica surface. Additionally, microscopy and N2 adsorption analysis confirmed the structural integrity and preservation of the mesoporous structure of Si during surface modification. The results show that both hybrid materials exhibit good chemical and thermal stability and an outstanding Cu2+ removal capability, with a chemical adsorption capacity of 176.32 mg g–1 and 184.16 mg g–1, respectively. These materials have also been used in real water and industrial wastewater samples with minimal interference in their adsorption capabilities. Density Functional Theory calculations were performed to confirm the good performance of the hybrid materials Si3PyS and Si4PyS towards metal ions. The functionalization of silica surface with porphyrin-based ligands bearing additional binding motifs drastically improves the adsorption capability of the new hybrids towards metal ions. The presence of pyridyl units brings a meaningful advantage, since both porphyrin core and appended pyridyl groups are able of binding Cu2+ ions with high affinity, contributing to the enhancement of the chelating features of the adsorbents prepared when compared with other ligands supported in silica-based materials.

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop “universal” receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

Surface Functionalization of Calcium Phosphate Nanoparticles via Click Chemistry: Covalent Attachment of Proteins and Ultrasmall Gold Nanoparticles

Calcium phosphate nanoparticles (60 nm) were stabilized with either polyethyleneimine (PEI; polycationic electrolyte) or carboxymethylcellulose (CMC; polyanionic electrolyte). Next, a silica shell was added and terminated with either azide or alkyne groups via siloxane coupling chemistry. The particles were covalently functionalized by copper-catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry) with proteins or gold nanoparticles that carried the complementary group, i.e., either alkyne or azide. The model proteins hemoglobin and bovine serum albumin (BSA) were attached as well as ultrasmall gold nanoparticles (2 nm). The number of protein molecules and gold nanoparticles attached to each calcium phosphate nanoparticle was quantitatively determined by extensive fluorescent labelling and UV–Vis spectroscopy on positively (PEI) or negatively (CMC) charged calcium phosphate nanoparticles, respectively. Depending on the cargo and the nanoparticle charge, this number was in the range of several hundreds to thousands. The functionalized calcium phosphate particles were well dispersible in water as shown by dynamic light scattering and internally amorphous as shown by X-ray powder diffraction. They were easily taken up by HeLa cells and not cytotoxic. This demonstrates that the covalent surface functionalization of calcium phosphate nanoparticles is a versatile method to create transporters with firmly attached cargo molecules into cells.

Immobilization of Phosphamide on the TiO2 Surface for Heterogeneous Phase Catalytic Appel Reaction

Global consumption of triphenylphosphine (Ph3P) for phosphorus-mediated organic synthesis and production of the dead-end triphenylphosphine oxide (Ph3PO) waste is exceptionally high. Recycling Ph3PO and/or use of it as a reaction mediator gained significant attention. On the other end, phosphamides, traditionally used as a flame redundant, are stable analogues to Ph3PO. Herein, via a low temperature condensation reaction of methyl 4-(aminomethyl)benzoate (AMB) and diphenyl phosphinic chloride (DPPC), methyl 4-((N,N-diphenylphosphinamido)methyl)benzoate (1) has been synthesized and hydrolysis of the ester functional group of 1 leads to a phosphamide with a carboxylate terminal, 4-((N,N-diphenylphosphinamido)methyl)benzoic acid (2). The presence of phosphamide functionality (NH─P═O) in 2 can be confirmed by its characteristic Raman vibration at 999 cm-1 with expected P-N and P═O bonds distances from the single-crystal X-ray structure. In-situ hydrolysis of [Ti(OiPr)4] in the presence of 2 followed by hydrothermal heating results in immobilization of 2 on a ca. 5 nm TiO2 surface (2@TiO2). The covalent attachment of 2 via coordination through the carboxylate terminal to the TiO2 nanocrystal's surface has been established via multiple spectroscopic and microscopic studies. 2@TiO2 is further used as the heterogeneous mediator for the catalytic Appel reaction, halogenation of alcohol (typically mediated by phosphine), with a fair catalytic conversion and a recorded TON up to 31. The major advantage of the heterogeneous approach studied herein is the recovery of used 2@TiO2 from the reaction mixture via centrifugation only leaving the organic product in the supernatant, which is limiting in Ph3P-mediated homogeneous catalysis. Time-resolved Raman spectroscopy confirms amino phosphine as the active species formed in-situ during the catalytic Appel reaction. Post-catalytic characterization of the material recovered after catalysis from the reaction mixture confirms the chemical integrity and that can further be utilized for another two catalytic runs. The developed reaction scheme showcases the use of a phosphamide as a reactive analogue to Ph3PO for an organic reaction in a heterogeneous approach, and the same strategy can be explored further as a general scheme for other phosphorus-mediated reactions.

Modified chitosan as a multi-functional material in eco-friendly formulation for anti-counterfeit applications

Renewable resources and waste materials can be used as viable raw materials to prepare security formulations devoid of volatile organic compounds and would essentially offer environmental protection. Therefore, chitosan (deacetylated chitin), which is a marine waste material is modified through simultaneous N-conjugation with pyrene-1-carboxaldehyde (PCA) as an aggregation caused quenching fluorophore and fluorene-2-carboxaldehyde (FCA) to obtain a smart fluorescent chitosan-based Schiff base (FCSSB). The covalent attachment could immobilize pyrene onto the chitosan, and the subsequent diluting effect of the biopolymer framework could hinder the aggregation of pyrene and, thereby, the self-quenching of fluorescence. The modified FCSSB could serve as a multi-functional material; both as a binder and a fluorophore in an eco-friendly formulation using aqueous acetic acid as the mild solvent and glycerol as the viscosity regulator. The formulation is used as a security ink to obtain single/multilayer coating/s and further prints using flexography technique. The prints that appeared invisible in daylight exhibited a blue fluorescence under 370 nm UV illumination. Moreover, the ink film presented good rub resistance and colour stability after different cycles of UV exposure. Therefore, FCSSB formulation can be readily utilized as a security ink for information decryption and could also be processed into products of diverse forms, including ink pads, coatings, printed patterns, and films. This simple, but effective approach provides additional impetus to real-life applications of biopolymers from natural/waste materials in anti-counterfeiting.

Arylidene Meldrum's Acid: A Versatile Structural Motif for the Design of Enzyme-Responsive "Covalent-Assembly" Fluorescent Probes with Tailor-Made Properties.

Latent cyclic C-nucleophiles are recently proving their value in the field of reaction-based fluorescent probes, far beyond their primary utility in organic synthesis. They are typically used to introduce a Michael acceptor moiety acting as a recognition/reaction site for analyte to be detected or as a kinetic promoter of fluorogenic cascade reactions triggered by a reactive species. C-nucleophiles bearing a further reactive handle offer an additional opportunity for tuning the physicochemical/targeting properties or providing drug-releasing capabilities to these probes, through the covalent attachment of ad hoc chemical moiety. In order to implement such strategy to fluorogenic enzyme substrates based on the "covalent-assembly" principle, we have explored the potential of some functionalized derivatives of barbituric acid, piperidine-2,4-dione and Meldrum's acid. Our investigations based on the rational design and validations of enzyme-responsive caged precursors of fluorescent pyronin dyes and 7-(diethylamino)coumarin-3-carboxylic acid, led to identify a versatile candidate suitable for this late-stage structural optimization approach. This Meldrum's acid derivative enables to either enhance water solubility or achieve the reversible conjugation of a targeting ligand, while promoting in situ formation of fluorophore upon enzymatic activation. This study opens the way to novel multifunctional fluorescence imaging probes and optically modulated small conjugate-based theranostics.