Discovery of a pathway for terminal-alkyne amino acid biosynthesis.

Living systems can generate an enormous range of cellular functions, from mechanical infrastructure and signalling networks to enzymatic catalysis and information storage, using a notably limited set of chemical functional groups. This observation is especially notable when compared to the breadth of functional groups used as the basis for similar functions in synthetically derived small molecules and materials. The relatively small cross-section between biological and synthetic reactivity space forms the foundation for the development of bioorthogonal chemistry, in which the absence of a pair of reactive functional groups within the cell allows for a selective in situ reaction1-4. However, biologically 'rare' functional groups, such as the fluoro5, chloro6,7, bromo7,8, phosphonate9, enediyne10,11, cyano12, diazo13, alkene14 and alkyne15-17 groups, continue to be discovered in natural products made by plants, fungi and microorganisms, which offers a potential route to genetically encode the endogenous biosynthesis of bioorthogonal reagents within living organisms. In particular, the terminal alkyne has found broad utility via the Cu(I)-catalysed azide-alkyne cycloaddition 'click' reaction18. Here we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid in the bacterium Streptomyces cattleya. We found that L-lysine undergoes an unexpected reaction sequence that includes halogenation, oxidative C-C bond cleavage and triple bond formation through a putative allene intermediate. This pathway offers the potential for de novo cellular production of halo-, alkene- and alkyne-labelled proteins and natural products from glucose for a variety of downstream applications.

MOFs Modulate Copper Trafficking in Tumor Cells for Bioorthogonal Therapy.

In situ drug synthesis using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has attracted considerable attention in tumor therapy because of its satisfactory effectiveness and reduced side-effects. However, the exogenous addition of copper catalysts can cause cytotoxicity and has hampered biomedical applications in vivo. Here, we design and synthesize a metal-organic framework (MOF) to mimic copper chaperone, which can selectively modulate copper trafficking for bioorthogonal synthesis with no need of exogenous addition of copper catalysts. Like copper chaperones, the prepared ZIF-8 copper chaperone mimics specifically bind copper ions through the formation of coordination bonds. Moreover, the copper is unloaded under the acidic environment due to the dissipation of the coordination interactions between metal ions and ligands. In this way, the cancer cell-targeted copper chaperone mimics can selectively transport copper ions into cells. Regulation of intracellular copper trafficking may inspire constructing bioorthogonal catalysis system with reduced metal cytotoxicity in live cells.

Synthesis, in vitro Anti-HIV-1RT evaluation, molecular modeling, DFT and acute oral toxicity studies of some benzotriazole derivatives.

This study synthesized and evaluated a series of benzotriazole derivatives denoted 3(a-j) and 6(a-j) for their anti-HIV-1 RT activities compared to the standard drug efavirenz. Notably, compound 3 h, followed closely by 6 h, exhibited significant anti-HIV-1 RT efficacy relative to the standard drug. In vivo oral toxicity studies were conducted for the most active compound 3 h, confirming its nontoxic nature to ascertain the safety profile. By employing molecular docking techniques, we explored the potential interactions between the synthesized compounds (ligands) and a target biomolecule (protein)(PDB ID 1RT2) at the molecular level. We undertook the molecular dynamics study of 3 h, the most active compound, within the active binding pocket of the cocrystallized structure of HIV-1 RT (PDB ID 1RT2). We aimed to learn more about how biomolecular systems behave, interact, and change at the atomic or molecular level over time. Finally, the DFT-derived HOMO and LUMO orbitals, as well as analysis of the molecular electrostatic potential map, aid in discerning the reactivity characteristics of our molecule.

Visualization of Modified Bisarylbutadiyne-Tagged Small Molecules in Live-Cell Nuclei by Stimulated Raman Scattering Microscopy.

Visualizing the distribution of small-molecule drugs in living cells is an important strategy for developing specific, effective, and minimally toxic drugs. As an alternative to fluorescence imaging using bulky fluorophores or cell fixation, stimulated Raman scattering (SRS) imaging combined with bisarylbutadiyne (BADY) tagging enables the observation of small molecules closer to their native intracellular state. However, there is evidence that the physicochemical properties of BADY-tagged analogues of small-molecule drugs differ significantly from those of their parent drugs, potentially affecting their intracellular distribution. Herein, we developed a modified BADY to reduce deviations in physicochemical properties (in particular, lipophilicity and membrane permeability) between tagged and parent drugs, while maintaining high Raman activity in live-cell SRS imaging. We highlight the practical application of this approach by revealing the nuclear distribution of a modified BADY-tagged analogue of JQ1, a bromodomain and extra-terminal motif inhibitor with applications in targeted cancer therapy, in living HeLa cells. The modified BADY, methoxypyridazyl pyrimidyl butadiyne (MPDY), revealed intranuclear JQ1, while BADY-tagged JQ1 did not show a clear nuclear signal. We anticipate that the present approach combining MPDY tagging with live-cell SRS imaging provides important insight into the behavior of intracellular drugs and represents a promising avenue for improving drug development.

Visual Detection of LPS at the Femtomolar Level Based on Click Chemistry-Induced Gold Nanoparticles Electrokinetic Accumulation.

Lipopolysaccharide (LPS) presents a significant threat to human health. Herein, a novel method for detecting LPS was developed by coupling hybridization chain reaction (HCR), gold nanoparticles (AuNPs) agglutination (AA) triggered by a Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry (CuAAC), and electrokinetic accumulation (EA) in a microfluidic chip, termed the HCR-AA-EA method. Thereinto, the LPS-binding aptamer (LBA) was coupled with the AuNP-coated Fe3O4 nanoparticle, which was connected with the polymer of H1 capped on CuO (H1-CuO) and H2-CuO. Upon LPS recognition by LBA, the polymers of H1- and H2-CuO were released into the solution, creating a "one LPS-multiple CuO" effect. Under ascorbic acid reduction, CuAAC was initiated between the alkyne and azide groups on the AuNPs' surface; then, the product was observed visually in the microchannel by EA. Finally, LPS was quantified by the integrated density of AuNP aggregates. The limit of detections were 29.9 and 127.2 fM for water samples and serum samples, respectively. The levels of LPS in the injections and serum samples by our method had a good correlation with those from the limulus amebocyte lysate test (r = 0.99), indicating high accuracy. Remarkably, to popularize our method, a low-cost, wall-power-free portable device was developed, enabling point-of-care testing.

Conjugating Hemoglobin and Albumin by Strain-Promoted Azide- Alkyne Cycloaddition.

A one-to-one conjugate of cross-linked human hemoglobin and human serum albumin results from a strain-promoted alkyne-azide cycloaddition (SPAAC) of the modified proteins. Additions of a strained alkyne-substituted maleimide to the Cys-34 thiol of human serum albumin and an azide-containing cross-link between the amino groups of each ß-unit at Lys-82 of human hemoglobin provide sites for coupling by the SPAAC process. The coupled hemoglobin-albumin conjugate can be readily purified from unreacted hemoglobin. The oxygen binding properties of the two-protein bioconjugate demonstrate oxygen affinity and cooperativity that are suitable for use in an acellular oxygen carrier.

Halo-1,2,3-triazoles: Valuable Compounds to Access Biologically Relevant Molecules.

1,2,3-triazole is an important building block in organic chemistry. It is now well known as a bioisostere for various functions, such as the amide or the ester bond, positioning it as a key pharmacophore in medicinal chemistry and it has found applications in various fields including life sciences. Attention was first focused on the synthesis of 1,4-disubstituted 1,2,3-triazole molecules however 1,4,5-trisubstituted 1,2,3-triazoles have now emerged as valuable molecules due to the possibility to expand the structural modularity. In the last decade, methods mainly derived from the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction have been developed to access halo-triazole compounds and have been applied to nucleosides, carbohydrates, peptides and proteins. In addition, late-stage modification of halo-triazole derivatives by metal-mediated cross-coupling or halo-exchange reactions offer the possibility to access highly functionalized molecules that can be used as tools for chemical biology. This review summarizes the synthesis, the functionalization, and the applications of 1,4,5-trisubstituted halo-1,2,3-triazoles in biologically relevant molecules.

3-Acyl-4-Pyranone as a Lysine Residue-Selective Bioconjugation Reagent for Peptide and Protein Modification.

Chemoselective protein modification plays extremely important roles in various biological, medical, and pharmaceutical investigations. Mimicking the mechanism of the chemoselective reaction between natural azaphilones and primary amines, this work successfully simplified the azaphilone scaffold into much simpler 3-acyl-4-pyranones. Examinations confirmed that these slim-size mimics perfectly kept the unique reactivity for selective conjugation with the primary amines including lysine residues of peptides and proteins. The newly developed pyranone tool presents remarkably increased aqueous solubility and compatible second-order rate constant by comparison with the original azaphilone. Additional advantages also include the ease of biorthogonal combinative use with a copper-catalyzed azide-alkyne Click reaction, which was conveniently applied to decorate lysozyme with neutral-, positive- and negative-charged functionalities in parallel. Moderate-degree modification of lysozyme with positively charged quaternary ammoniums was revealed to increase the enzymatic activities.

Incorporation of Intracellular NanoSIMS Tracers to Oligonucleotide Conjugates via Strain Promoted Sydnone-Alkyne Cycloaddition.

Extensive efforts have been dedicated to developing cell-specific targeting ligands that can be conjugated to therapeutic cargo, offering a promising yet still challenging strategy to deliver oligonucleotide therapeutics beyond the liver. Indeed, while the cargo and the ligand are crucial, the third component, the linker, is integral but is often overlooked. Here, we present strain-promoted sydnone-alkyne cycloaddition as a versatile linker chemistry for oligonucleotide synthesis, expanding the choices for bioconjugation of therapeutics while enabling subcellular detection of the linker and payload using nanoscale secondary ion mass spectrometry (NanoSIMS) imaging. This strategy was successfully applied to peptide and lipid ligands and profiled using the well characterized N-acetylgalactosamine (GalNAc) targeting ligand. The linker did not affect the expected activity of the conjugate and was detectable and distinguishable from the labeled cargo. Finally, this work not only offers a practical bioconjugation method but also enables the assessment of the linker's subcellular behavior, facilitating NanoSIMS imaging to monitor the three key components of therapeutic conjugates.

Development of Biocompatible Cu(I)-Microdevices for Bioorthogonal Uncaging and Click Reactions.

Transition-metal-catalyzed bioorthogonal reactions emerged a decade ago as a novel strategy to implement spatiotemporal control over enzymatic functions and pharmacological interventions. The use of this methodology in experimental therapy is driven by the ambition of improving the tolerability and PK properties of clinically-used therapeutic agents. The preclinical potential of bioorthogonal catalysis has been validated in vitro and in vivo with the in situ generation of a broad range of drugs, including cytotoxic agents, anti-inflammatory drugs and anxiolytics. In this article, we report our investigations towards the preparation of solid-supported Cu(I)-microdevices and their application in bioorthogonal uncaging and click reactions. A range of ligand-functionalized polymeric devices and off-on Cu(I)-sensitive sensors were developed and tested under conditions compatible with life. Last, we present a preliminary exploration of their use for the synthesis of PROTACs through CuAAC assembly of two heterofunctional mating units.

Utilization of Spontaneous Alkyne-Gold Self-Assembly Chemistry as an Alternative Method for Fabricating Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (E-AB) sensors are a promising class of biosensors which use structure-switching redox-labeled oligonucleotides (aptamers) codeposited with passivating alkanethiol monolayers on electrode surfaces to specifically bind and detect target analytes. Signaling in E-AB sensors is an outcome of aptamer conformational changes upon target binding, with the sequence of the aptamer imparting specificity toward the analyte of interest. The change in conformation translates to a change in electron transfer between the redox label attached to the aptamer and the underlying electrode and is related to analyte concentration, allowing specific electrochemical detection of nonelectroactive analytes. E-AB sensor measurements are reagentless with time resolutions of seconds or less and may be miniaturized into the submicron range. Traditionally these sensors are fabricated using thiol-on-gold chemistry. Here we present an alternate immobilization chemistry, gold-alkyne binding, which results in an increase in sensor lifetimes under ideal conditions by up to ∼100%. We find that gold-alkyne binding is spontaneous and supports efficient E-AB sensor signaling with analytical performance characteristics similar to those of thiol generated monolayers. The surface modification differs from gold-thiol binding only in the time and aptamer concentration required to achieve similar aptamer surface coverages. In addition, alkynated aptamers differ from their thiolated analogues only by their chemical handle for surface attachment, so any existing aptamers can be easily adapted to utilize this attachment strategy.

Site-Specific Conjugation of Bottlebrush Polymers to Therapeutic Protein via Bioorthogonal Chemistry.

Achieving efficient and site-specific conjugation of therapeutic protein to polymer is crucial to augment their applicability in the realms of biomedicine by improving their stability and enzymatic activity. In this study, we exploited tetrazine bioorthogonal chemistry to achieve the site-specific conjugation of bottlebrush polymers to urate oxidase (UOX), a therapeutic protein for gout treatment. An azido-functionalized zwitterionic bottlebrush polymer (N3-ZBP) using a "grafting-from" strategy involving RAFT and ATRP methods was synthesized, and a trans-cyclooctene (TCO) moiety was introduced at the polymer end through the strain-promoted azide-alkyne click (SPAAC) reaction. The subsequent coupling between TCO-incorporated bottlebrush polymer and tetrazine-labeled UOX using a fast and safe bioorthogonal reaction, inverse electron demand Diels-Alder (IEDDA), led to the formation of UOX-ZBP conjugates with a 52% yield. Importantly, the enzymatic activity of UOX remained unaffected following polymer conjugation, suggesting a minimal change in the folded structure of UOX. Moreover, UOX-ZBP conjugates exhibited enhanced proteolytic resistance and reduced antibody binding, compared to UOX-wild type. Overall, the present findings reveal an efficient and straightforward route for synthesizing protein-bottlebrush polymer conjugates without compromising the enzymatic activity while substantially reducing proteolytic degradation and antibody binding.

Modular Synthesis of PEG-Dendritic Block Copolymers by Thermal Azide-Alkyne Cycloaddition with Internal Alkynes and Evaluation of their Self-Assembly for Drug Delivery Applications.

Linear-dendritic block copolymers assemble in solution due to differences in the solubility or charge properties of the blocks. The monodispersity and multivalency of the dendritic block provide unparalleled control for the design of drug delivery systems when incorporating poly(ethylene glycol) (PEG) as a linear block. An accelerated synthesis of PEG-dendritic block copolymers based on the click and green chemistry pillars is described. The tandem composed of the thermal azide-alkyne cycloaddition with internal alkynes and azide substitution is revealed as a flexible, reliable, atom-economical, and user-friendly strategy for the synthesis and functionalization of biodegradable (polyester) PEG-dendritic block copolymers. The high orthogonality of the sequence has been exploited for the preparation of heterolayered copolymers with terminal alkenes and alkynes, which are amenable for subsequent functionalization by thiol-ene and thiol-yne click reactions. Copolymers with tunable solubility and charge were so obtained for the preparation of various types of nanoassemblies with promising applications in drug delivery.

A Synthetic Strategy toward S-, N-, and O-Heterocyclooctynes Facilitates Bioconjugation Using Multifunctional Handles.

Over the past two decades, the introduction of bioorthogonal reactions has transformed the ways in which chemoselective labeling, isolation, imaging, and drug delivery are carried out in a complex biological milieu. A key feature of a good bioorthogonal probe is the ease with which it can be attached to a target compound through bioconjugation. This paper describes the expansion of the utility of a class of unique S-, N-, and O-containing heterocyclooctynes (SNO-OCTs), which show chemoselective reactivity with type I and type II dipoles and divergent reactivities in response to electronic tuning of the alkyne. Currently, bioconjugation of SNO-OCTs to a desired target is achieved through an inconvenient aryl or amide linker at the sulfamate nitrogen. Herein, a new synthetic approach toward general SNO-OCT scaffolds is demonstrated that enables the installation of functional handles at both propargylic carbons of the heterocycloalkyne. This capability increases the utility of SNO-OCTs as labeling reagents through the design of bifunctional bioorthogonal probes with expanded capabilities. NMR kinetics also revealed up to sixfold improvement in cycloaddition rates of new analogues compared to first-generation SNO-OCTs.

Biomacromolecule-Assisted Screening for Reaction Discovery and Catalyst Optimization.

Reaction discovery and catalyst screening lie at the heart of synthetic organic chemistry. While there are efforts at de novo catalyst design using computation/artificial intelligence, at its core, synthetic chemistry is an experimental science. This review overviews biomacromolecule-assisted screening methods and the follow-on elaboration of chemistry so discovered. All three types of biomacromolecules discussed─enzymes, antibodies, and nucleic acids─have been used as "sensors" to provide a readout on product chirality exploiting their native chirality. Enzymatic sensing methods yield both UV-spectrophotometric and visible, colorimetric readouts. Antibody sensors provide direct fluorescent readout upon analyte binding in some cases or provide for cat-ELISA (Enzyme-Linked ImmunoSorbent Assay)-type readouts. DNA biomacromolecule-assisted screening allows for templation to facilitate reaction discovery, driving bimolecular reactions into a pseudo-unimolecular format. In addition, the ability to use DNA-encoded libraries permits the barcoding of reactants. All three types of biomacromolecule-based screens afford high sensitivity and selectivity. Among the chemical transformations discovered by enzymatic screening methods are the first Ni(0)-mediated asymmetric allylic amination and a new thiocyanopalladation/carbocyclization transformation in which both C-SCN and C-C bonds are fashioned sequentially. Cat-ELISA screening has identified new classes of sydnone-alkyne cycloadditions, and DNA-encoded screening has been exploited to uncover interesting oxidative Pd-mediated amido-alkyne/alkene coupling reactions.

1,2,3-Triazole and Its Analogues: New Surrogates for Diazo Compounds.

Readily accessible and shelf-stable 1,2,3-triazole and its analogues such as pyridotriazole, triazoloindole, benzotriazole, and thiadiazole exist in equilibrium with their ring-opened isomers, viz., diazo compounds. These ring-opened isomers could be trapped by various metal catalysts (e.g., Rh, Pd, Cu, Co, Ag, etc.) to generate the corresponding metal carbenoids with extrusion of nitrogen. As a consequence, these unique N-heterocycles facilitate access to a realm of N-containing complex structural motifs of biological importance through denitrogenative transformations such as transannulations, insertions, ylide formation, and rearrangements by trapping of the metal carbenoids with a diverse range of coupling partners (e.g., alkenes, alkynes, nitriles, carbo/heterocycles, X-H/C-X bonds, etc.). Hence, suitably substituted triazole derivatives have emerged as efficient surrogates of diazo compounds for the generation of reactive metal carbenoids during the past decades. In this comprehensive review, we aim to discuss in detail the remarkable advancement in their synthesis and synthetic applications.

1,3,4,5-Tetrasubstituted Poly(1,2,3-triazolium) Obtained through Metal-Free AA+BB Polyaddition of a Diazide and an Activated Internal Dialkyne.

A tetra(ethylene glycol)-based 1,3,4,5-tetrasubstituted poly(1,2,3-triazolium) is synthesized in two steps including: i) the catalyst-free polyaddition of a diazide and an activated internal dialkyne and ii) the N-alkylation of the resulting 1,2,3-triazole groups. In order to provide detailed structure/properties correlations different analogs are also synthesized. First, parent poly(1,2,3-triazole)s are obtained via AA+BB polyaddition using copper(I)-catalyzed alkyne-azide cycloaddition or metal-free thermal alkyne-azide cycloaddition (TAAC). Poly(1,2,3-triazole)s with higher molar masses are obtained in higher yields by TAAC polyaddition. A 1,3,4-trisubstituted poly(1,2,3-triazolium) structural analog obtained by TAAC polyaddition using a terminal activated dialkyne and subsequent N-alkylation of the 1,2,3-triazole groups enables discussing the influence of the methyl group in the C-4 or C-5 position on thermal and ion conducting properties. Obtained polymers are characterized by 1H, 13C, and 19F NMR spectroscopy, differential scanning calorimetry, thermogravimetric analysis, size exclusion chromatography, and broadband dielectric spectroscopy. The targeted 1,3,4,5-tetrasubstituted poly(1,2,3-triazolium) exhibits a glass transition temperature of -23 °C and a direct current ionic conductivity of 2.0 × 10-6 S cm-1 at 30 °C under anhydrous conditions. The developed strategy offers opportunities to further tune the electron delocalization of the 1,2,3-triazolium cation and the properties of poly(1,2,3-triazolium)s using this additional substituent as structural handle.

Clickable Polymerization-Induced Emission Luminogens Toward Color-Tunable Modification of Non-Traditional Intrinsic Luminescent Polymers.

Non-traditional intrinsic luminescent (NTIL) polymer is an emerging field, and its color-tunable modification is highly desirable but still rarely investigated. Here, a click chemistry approach for the color-tunable modifications of NTIL polymers by introducing clickable polymerization-induced emission luminogen (PIEgen), is demonstrated. Through Cu-catalyzed azide-alkyne cycloaddition click chemistry, a series of PIEgens is successful prepared, which is further polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Interestingly, after clickable modification, these monomers are nonemissive in both solution and aggregation states; while, the corresponding polymers exhibit intriguing aggregation-induced emission (AIE) characteristics, confirming their PIEgen characteristics. By varying alkynyl substitutions, color-tunable NTIL polymers are achieved with emission wavelength varying from 448 to 498 nm, revealing a series of PIEgens and verifying the importance of modification of NTIL polymers. Further luminescence energy transfer application is carried out as well. This work therefore designs a series of clickable PIEgens and opens a new avenue for the modification of NTIL polymers via click chemistry, which may cause inspirations to the research fields including luminescent polymer, NTIL, click chemistry, AIE and modification.

Click chemistry in the synthesis of antibody-drug conjugates.

Antibody-Drug Conjugates (ADC) are a new class of anticancer therapeutics with immense potential. They have been rapidly advancing in the last two decades. This fast speed of development has become possible due to several new technologies and methods. One of them is Click Chemistry, an approach that was created only two decades ago, but already is actively utilized for bioconjugation, material science and drug discovery. In this review, we researched the impact of Click Chemistry reactions on the synthesis and development of ADCs. The information about the most frequently utilized reactions, such as Michael's addition, Copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC), Strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC), oxime bond formation, hydrazine-iso-Pictet-Spengler Ligation (HIPS), Diels-Alder reactions have been summarized. The implementation of thiol-maleimide Click Chemistry reaction in the synthesis of numerous FDA-approved Antibody-Drug Conjugates has been reported. The data amassed in the present review provides better understanding of the importance of Click Chemistry in the synthesis, development and improvement of the Antibody-Drug Conjugates and it will be helpful for further researches related to ADCs.

A CuSO4/Bicinchoninic acid/Reducing sugar based stable and non-ROS catalyst system for the CuAAC reaction in bioanalysis.

The limitations of commonly used sodium ascorbate-based catalyst system for copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction include excess production of reactive oxygen species and rapid catalyst deactivation. In this study instead of using a highly active reducing agent, such as, sodium ascorbate, we chose reducing sugar as a mild reducing agent to build up the catalyst system for CuAAC reaction. Interestingly, the bicinchoninic acid (BCA) assay system containing reducing sugar satisfies the essential elements of the catalyst system for CuAAC reaction. We found that CuSO4/BCA/Reducing sugar system can catalyze the CuAAC reaction but with low yield. Rational analyses of various parameters in CuSO4/BCA/Glucose catalyst system suggested storage at room temperature might enhance the catalytic activity, which was proven to be the case. Importantly, the system remains stable at room temperature and minimal H2O2 was detected. Notably, our study showed that the coordination between the slow reduction of Cu(I) by reducing sugar and the selective chelation of Cu(I) by BCA is key to developing this system. The CuSO4/BCA/Reducing sugar catalyst system was successfully applied to various CuAAC reaction based bioanalyses, and it is suitable for the CuAAC reaction based bioanalyses that are sensitive to ROS or request long reaction time.