Hydrazone Chemistry Research Articles

Modularization of dual recognized CRISPR/Cas12a system for the detection of Staphylococcus aureus assisted by hydrazone chemistry

In this work, a dual recognized CRISPR/Cas12a system has been proposed, in which the activation chain is cleverly divided into two parts that can serve for precise dual target recognition, and hydrazone chemistry is introduced for the formation of a whole activation chain. It has been further explored to construct a new method for the specific and sensitive detection of Staphylococcus aureus (SA) as one of the most common pathogens in infectious diseases. In virtue of proximity effect contributed by complementary base pairing, hydrazone chemistry accelerates the formation of the whole activation strand and improves the specificity of the CRISPR/Cas12a system, serving for the accurate analysis of SA. Moreover, the temporary aggregation of CRISPR/Cas12a around SA enhances its catalytical efficiency so as to further amplify signal. With high sensitivity, stability, reproducibility and specificity, the established method has been successfully applied to detect SA in complex substrates. Meanwhile, our established method can well evaluate the inhibition effect of chlorogenic acid and congo red in comparison with flow cytometry. Environmental implicationBacterial pathogens exist widely in the environment and seriously threaten the safety of human health. Staphylococcus aureus (SA) is the most common pathogen of human suppurative infection, which can cause local suppurative infection, pneumonia, and even systemic infections such as sepsis. In this work, a dual recognized CRISPR/Cas12a system mediated by hydrazone chemistry has been proposed. With high sensitivity and low detection limit, the established method can specifically detect SA and effectively evaluate the antibacterial effect of inhibitors. This method is expected to be further developed into a detection method in different scenarios such as environmental monitoring and clinical diagnosis.

Injectable isoniazid-loaded bone cement based on hydrazone bonds achieving long-term release and decent mechanical properties.

A robust and easily manufactured high-strength and long-term release hydrazone-based isoniazid acrylic (HIA) bone cement is reported. The mechanical strength of HIA bone cement is similar to that of normal polymethyl methacrylate (PMMA) bone cement, far surpassing that of traditional isoniazid-containing antibiotic-loaded bone cement (INH bone cement). Isoniazid is connected to the bone cement through bioorthogonal hydrazone chemistry, and it possesses release properties superior to those of INH bone cement, allowing for the sustained release of isoniazid for up to 12 weeks. In vivo and in vitro studies also indicate that HIA cement exhibits better biocompatibility than INH bone cement. The results of this study not only signify progress in the realm of antimicrobial bone cement for addressing bone tuberculosis but also enhance our capacity to create and comprehend high-performing antimicrobial bone cement.

A Multifunctional Antibacterial Hydrogel with Injectable, Antifouling, and Conductive Properties

Zwitterionic hydrogels have shown promise as effective antifouling materials in wound dressing applications owing to their robust hydrate layer that offers protection against microbial infections. However, the traditional covalently crosslinked zwitterionic hydrogels may not be sufficient to adapt to irregular injury sites and fully address microbial infections within the wound. Herein, we propose an injectable zwitterionic hydrogel that utilizes dynamic hydrazone chemistry via aldehyde and hydrazide functional groups and contains N-halamine antibacterial agents. The dual dynamic exchangeable covalently and physically crosslinked structure resulting from the combination of hydrazone bonds and zwitterion interactions facilitates instantaneous gelation (within a few seconds) and extreme stability without any degradation (for over 2 months). The hydrogels exhibit remarkable fouling resistance against proteins and bacteria while demonstrating excellent antibacterial performance in vitro without compromising cytocompatibility. More importantly, we demonstrate their inherent conductivity properties and sensitivity to external pressure. The combination of the injectability, antifouling, antibacterial, cytocompatibility, and electrical conductivity properties achieved offers the potential to expand the utility of zwitterionic hydrogels in biological applications.

Hydrazides and Hydrazones: Robust Scaffolds in Neurological and Neurodegenerative Disorders

Background: Hydrazides and hydrazones are found in a variety of bioactive compounds that have a wide range of pharmacological applications. There have been significant advancements in this field in recent years, with many new aspects of hydrazone chemistry and applications emerging. Methods: This piqued researchers' interest in synthesizing a variety of hydrazone derivatives for various biological applications. Results: According to a comprehensive review of the literature, the hydrazone template possesses an immense potential to be developed for the treatment of a variety of brain-related disorders. Thus, researchers are expeditiously exploring the chemical compounds bearing hydrazone moiety to find lead molecules for the development of potential agents for the treatment of neurological and neurodegenerative disorders. Conclusion: In this review, the biological activities of various hydrazide and hydrazone analogues against various neurological and neurodegenerative disorders are summarized. Furthermore, there is a great potential for this scaffold, which medicinal chemists should look into for the development of new prospective drug candidates.

Hydrazone chemistry-mediated CRISPR/Cas12a system for bacterial analysis.

In this study, a hydrazone chemistry-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system has been proposed for the fist time and constructed. In our system, hydrazone chemistry is designed and employed to accelerate the formation of a whole activation strand by taking advantage of the proximity effect induced by complementary base pairing, thus activating the CRISPR/Cas12a system quickly and efficiently. Moreover, the introduction of hydrazone chemistry can improve the specificity of the CRISPR/Cas12a system, allowing it to effectively distinguish single-base mismatches. The established system has been further applied to analyze Pseudomonas aeruginosa by specific recognition of the probe strand with a characteristic fragment in 16S rDNA to release the hydrazine group-modified activation strand. The method shows a wide linear range from 3.8 × 102 colony-forming units (CFU)/ml to 3.8 × 106 CFU/ml, with the lowest detection limit of 24 CFU/ml. Therefore, the introduction of hydrazone chemistry may also broaden the application of the CRISPR/Cas12a system.

Copper-Catalyzed Conjugate Addition of Carbonyls as Carbanion Equivalent via Hydrazones

Copper-catalyzed conjugate addition is a classic method for forming new carbon-carbon bonds. However, copper has never showed catalytic activity for umpolung carbanions in hydrazone chemistry. Herein, we report a facile conjugate addition of hydrazone catalyzed by readily available copper complexes at room temperature. The employment of mesitylcopper(I) and electron-rich phosphine bidentate ligand is a key factor affecting reactivity. The reaction allows various aromatic hydrazones to react with diverse conjugated compounds to produce 1,4-adducts in yields of about 20 to 99%.

Copper-Catalyzed Regioselective Coupling of Tosylhydrazones and 2-Pyridones: A Strategy for the Production of N-Alkylated Compounds.

The highly regioselective N-alkylation reaction of 2-pyridones was achieved through hydrazone chemistry, especially for substrates with bulky secondary alkyl groups. Described herein is a copper-catalyzed coupling reaction of pyridone derivatives with tosylhydrazones.

Hydrazone chemistry mediated toehold strand displacement cascade and its application for 5-hydroxymethylfurfural analysis

Hydrazone chemistry has been firstly explored as capturing mode for interface supported toehold strand displacement cascade (TSDC). The method has been further established for analysis of 5-hydroxymethylfurfural (HMF) based on hydrazone chemistry-mediated TSDC. HMF containing aldehyde group can be covalently captured by hydrazine group around magnetic bead through the formation of hydrazone bond, so as to inhibit the immobilization of hybrid duplex and the occurrence of TSDC. Thereby, HMF will cause the change of the fluorescence of modified magnetic bead. With simplicity, specificity, and sensitivity, the method has been successfully applied to analyze HMF in food samples. This paper gives a new insight to explore capturing mode for interface supported TSDC and the established method can be extended for analysis of saccharic derivatives.

Hydrazone chemistry assisted DNAzyme for the analysis of double targets.

In this work, a hydrazone chemistry assisted DNAzyme has been designed and constructed. The introduction of hydrazone chemistry increases the versatility of DNAzymes. With superior catalytic capability, the hydrazone chemistry assisted DNAzyme has been successfully applied for the analysis of double targets. Taking 5-hydroxymethylfurfural (HMF) and lipopolysaccharide (LPS) as samples, the hydrazone chemistry assisted DNAzyme can be used for the detection of different combinations of targets. Moreover, because hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of DNAzymes and their multifunctionality.

Dynamic crosslinked and injectable biohydrogels as extracellular matrix mimics for the delivery of antibiotics and 3D cell culture.

Antibiotics are widely used in clinical medicine. As an important member, vancomycin often plays an irreplaceable role in some serious infections but for its use, there is still a lack of suitable carriers and effective formulations. To find a vancomycin carrier with potential for clinical applications, a new class of poly(γ-glutamic acid)/dextran-based injectable hydrogels have been constructed through dynamic covalent hydrazone linkages. Adipic dihydrazide (ADH)-grafted poly(γ-glutamic acid) (PGAADH) and sodium periodate-oxidized dextran (OD) precursors were synthesized; then, the hydrogels were formed by blending PGAADH and OD buffer solutions without any additives under physiological conditions. The newly formed precursor structures, mechanical properties, morphologies, hydrogel degradation profiles, and the interaction between the drug and precursors were investigated with FTIR spectroscopy, 1H NMR spectroscopy, rheological experiments, compression tests, SEM, and isothermal titration calorimetric (ITC) measurements. The resulting hydrogels exhibited excellent antibacterial ability and ideal variable performances. Moreover, the hydrogels exhibited different drug release kinetics and mechanisms and were applied effectively towards the controlled release of vancomycin. Significantly, benefitting from the reversibly cross-linked systems and the excellent biocompatibility, the hydrogels can work as the ideal material for HeLa cell culture, leading to encapsulated cells with higher viability and capacity that is proliferative. Therefore, the injectable PGAADH/OD hydrogels demonstrated attractive properties for future applications in pharmaceutics and tissue engineering.

MS‐Based Screening of 5‐Substituted Nipecotic Acid Derived Hydrazone Libraries as Ligands of the GABA Transporter 1

A screening of compound libraries based on nipecotic acid derivatives with lipophilic residues attached to the scarcely explored 5-position of the core structure was used for the search of new inhibitors of the γ-aminobutyric acid (GABA) transporter 1 (mGAT1). The generated compound libraries, which were based on hydrazone chemistry commonly used in dynamic combinatorial chemistry but rendered pseudostatic, were screened for their binding affinities toward mGAT1 by means of MS Binding Assays. With nipecotic acid derived hydrazone rac-16 h [rac-(3R,5S)-{5-[(E)-2-{[5-(2-phenylethynyl)thiophen-2-yl]methylidene}hydrazin-1-yl]piperidine-3-carboxylic acid}-sodium chloride (1/2)], one hit was found and evaluated displaying sub-micromolar potency (pKi =6.62±0.04) and a noncompetitive interaction mode at mGAT1. By bearing a 5-(2-phenylethynyl)thiophen-2-yl residue attached to the 5-position of nipecotic acid via a three-atom spacer, compound rac-16 h contains a structural moiety so far unprecedented for these kinds of bioactive molecules, and complements novel 5-substituted nipecotic acid derived ligands of mGAT1 revealed in a recently published screening campaign. This new class of ligands, with an inhibition mode distinct from that of benchmark mGAT1 inhibitors, could serve as research tools for investigations of mGAT1-mediated GABA transport.

Stimuli-Free and Biocompatible Hydrogel via Hydrazone Chemistry: Synthesis, Characterization, and Bioassessment

Functionalization of starch with oxidative cleavage reaction to yield the corresponding dialdehyde derivatives has been employed as an approach for gel formation via Schiff's base reaction. However, this reaction is known as reversible and hydrolysable in aqueous solutions. In this study, the potential of hydrazone chemistry to be used in the synthesis of stimuli‐free and stable hydrogels is investigated. Soluble starch is selectively oxidized to give aldehydic‐starch with different aldehyde contents 39.9, 34.6, and 22.6 aldehyde group/100 anhydroglucose unit (AGU). Adipic dihydrazide was used as an α‐effect nucleophile to react with the dialdehyde starch and obtain stable hydrazone‐based and stimuli‐free hydrogels. Hydrazone chemistry of aldehydic‐starch and adipic dihydrazide (AD) is demonstrated by spectral analysis. As well, the biocompatibility with human skin fibroblasts cells is investigated using a cytotoxicity assay. SEM images show the pore sizes vary from 575 to 4752 nm related to AD concentrations. The swelling degree recorded an increase of 900%, 500%, and 600% at pH 4, pH 7, and pH 9, respectively. Hydrogels showed varied mechanical behavior as a function of AD concentrations.

Empty Turnip yellow mosaic virus capsids as delivery vehicles to mammalian cells

Turnip yellow mosaic virus (TYMV) was able to enter animal cells when the spherical plant virus was conjugated with Tat, a cell penetrating peptide (CPP). Tat was chemically attached to the surface lysine residues of TYMV using hydrazone chemistry. Baby hamster kidney (BHK) cells were incubated with either unmodified or Tat-conjugated TYMV and examined by flow cytometry and confocal microscopic analyses. Tat conjugation was shown to be more efficient than Lipofectamine in allowing TYMV to enter the mammalian cells. Tat-assisted-transfection was also associated with less loss of cell viability than lipofection. Among the CPPs tested (Tat, R8, Pep-1 and Pen), it was observed that R8 and Pen were also effective while Pep-1 was not. We also examined if the internal space of TYMV can be used to load fluorescein dye as a model cargo. When TYMV is treated by freezing and thawing, the virus is known to convert into a structure with a 6–8 nm hole and release viral RNA. When the resultant pot-like particles were reacted with fluorescein-5-maleimide using interior sulfhydryl groups as conjugation sites, about 145 fluorescein molecules were added per particle. The fluorescein-loaded TYMV particles were conjugated with Tat and introduced into BHK cells, again with higher transfection efficiency compared to lipofection. Our studies demonstrate the potential of modified TYMV as an efficient system for therapeutic cargo delivery to mammalian cells.

Electrochemical Analysis of Enzyme Based on the Self-Assembly of Lipid Bilayer on an Electrode Surface Mediated by Hydrazone Chemistry.

In this work, a new strategy for electrochemical analysis of enzyme has been proposed based on a self-assembled lipid bilayer on an electrode surface mediated by hydrazone chemistry. Taking aldolase as an example, the enzyme can catalyze the formation of products containing carbonyl groups. These groups can react with hydrazine groups of the functional lipid derivative, resulting in the self-assembly of a lipid bilayer on a guanidinium modified electrode surface. The lipid bilayer will then prevent the movement of hydrophilic electrochemical probes. Consequently, the catalytic reaction of the enzyme may result in the change of the obtained electrochemical peak current. Experimental results reveal that aldolase activity can be analyzed over a widely linear detection range from 5 mU/L to 100 U/L with a low detection limit of 1 mU/L. Meanwhile, the method can exhibit good precision and reproducibility and it can be applied for real sample analysis. What is more, because the lipid bilayer is the universal basis for cell-membrane structure, while hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of electrochemical analysis and electrochemical biosensors.

A DFT Approach to the Mechanistic Study of Hydrozone Hydrolysis.

Hydrazone chemistry is widely utilized in biomedical field as a means of bioconjugation protocol, especially in drug delivery field due to pH labile nature of this linkage. In the light of kinetics studies, the generally accepted mechanism for the hydrolysis of hydrazones involves two main steps, namely, nucleophilic addition of water molecule to the hydrazone molecule to form carbinolamine intermediate and subsequent decomposition of this intermediate into the hydrazine and aldehyde/ketone moieties. Hydrolysis of hydrazones is catalyzed in the acidic environments and is thought to proceed through several proton transfer steps. To the best of our knowledge, in the literature no detailed theoretical study has been reported related to the mechanism of hydrolysis. In this study, we evaluated the proposed mechanism with DFT calculations with M06-2X functional at the 6-311+g(d,p) level including conductor-like polarizable continuum model solvation model. We also analyzed possible proton transfer pathways and assessed energetics of each step.

Pick-up, transport and release of a molecular cargo using a small-molecule robotic arm.

Modern-day factory assembly lines often feature robots that pick up, reposition and connect components in a programmed manner. The idea of manipulating molecular fragments in a similar way has to date only been explored using biological building blocks (specifically DNA). Here, we report on a wholly artificial small-molecule robotic arm capable of selectively transporting a molecular cargo in either direction between two spatially distinct, chemically similar, sites on a molecular platform. The arm picks up/releases a 3-mercaptopropanehydrazide cargo by formation/breakage of a disulfide bond, while dynamic hydrazone chemistry controls the cargo binding to the platform. Transport is controlled by selectively inducing conformational and configurational changes within an embedded hydrazone rotary switch that steers the robotic arm. In a three-stage operation, 79-85% of 3-mercaptopropanehydrazide molecules are transported in either (chosen) direction between the two platform sites, without the cargo at any time fully dissociating from the machine nor exchanging with other molecules in the bulk.

High-yielding and photolabile approaches to the covalent attachment of biomolecules to surfaces via hydrazone chemistry.

The development of strategies to couple biomolecules covalently to surfaces is necessary for constructing sensing arrays for biological and biomedical applications. One attractive conjugation reaction is hydrazone formation--the reaction of a hydrazine with an aldehyde or ketone--as both hydrazines and aldehydes/ketones are largely bioorthogonal, which makes this particular reaction suitable for conjugating biomolecules to a variety of substrates. We show that the mild reaction conditions afforded by hydrazone conjugation enable the conjugation of DNA and proteins to the substrate surface in significantly higher yields than can be achieved with traditional bioconjugation techniques, such as maleimide chemistry. Next, we designed and synthesized a photocaged aryl ketone that can be conjugated to a surface and photochemically activated to provide a suitable partner for subsequent hydrazone formation between the surface-anchored ketone and DNA- or protein-hydrazines. Finally, we exploit the latent functionality of the photocaged ketone and pattern multiple biomolecules on the same substrate, effectively demonstrating a strategy for designing substrates with well-defined domains of different biomolecules. We expect that this approach can be extended to the production of multiplexed assays by using an appropriate mask with sequential photoexposure and biomolecule conjugation steps.

Glycoengineered Acid α-Glucosidase With Improved Efficacy at Correcting the Metabolic Aberrations and Motor Function Deficits in a Mouse Model of Pompe Disease

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.

Molecular amplification in a dynamic system by ammonium cations

Using hydrazone chemistry, a simple dynamic system of macrocyclic pseudo-peptides was prepared which contains a cyclic structure that resembles a known receptor for ammonium cations. A series of ammonium salts were tested as templates and the expected receptor was amplified in different degrees according to the template used. Competitive non-covalent interactions with a crown ether were used to demonstrate in situ that the templated system is still dynamic. Formation of host–guest complexes was proven by ESI-MS and 1H ROESY experiments. Relative affinities of the various templates for the isolated receptor correlate well with the effect they produce on the library composition.

An Immune Complex Selective Affinity Column Utilizing Site-Specific Attachment of Bovine Conglutinin

A simple method to isolate immune complexes by column chromatography is described. The immune complex affinity column was constructed by the site-specific attachment of bovine conglutinin to agarose. Covalent attachment of conglutinin to agarose was achieved via hydrazone chemistry, which reacts aidehydes of oxidized oligosaccharides in the collagenous domain of conglutinin with hydrazide functional groups in the solid support. The constructed conglutinin affinity column captured complement-fixed model immune complexes of heat-aggregated human IgG and more classical complexes of chicken ovalbumin-antichicken ovalbumin. Neither the nonfixed immune complexes nor their individual components were retained by the column. The captured material was specifically eluted with EDTA without the use of low pH. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and protein sequence analysis of the eluates revealed the presence of the expected individual components, verifying that both antibody and antigen used to prepare the soluble immune complexes wore recovered from the conglutinin column. The advantages of this approach over traditional methods of immune complex isolation and characterization are discussed.