Triazole Linker Research Articles

Application and Structural Analysis of Triazole-Bridged Disulfide Mimetics in Cyclic Peptides.

Ruthenium-catalysed azide-alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics.

Fluorophore-Assisted Click Chemistry through Copper(I) Complexation.

The copper-catalyzed alkyne-azide cycloaddition (CuAAC) is one of the most powerful chemical strategies for selective fluorescent labeling of biomolecules in in vitro or biological systems. In order to accelerate the ligation process and ensure efficient formation of conjugates under diluted conditions, external copper(I) ligands or sophisticated copper(I)-chelating azides are used. This latter strategy, however, increases the bulkiness of the triazole linkage, thus perturbing the biological function or dynamic behavior of the conjugates. In a proof-of-concept study, we investigated the use of an extremely compact fluorophore-based copper(I) chelating azide in order to accelerate the CuAAC with concomitant fluorescence labeling; in our strategy, the fluorophore is able to complex copper(I) species while retaining its photophysical properties. It is believed that this unprecedented approach which was applied for the labeling of a short peptide molecule and the fluorescent labeling of live cells, could be extended to other families of nitrogen-based fluorophores in order to tune both the reaction rate and photophysical characteristics.

β-Sheet to Helical-Sheet Evolution Induced by Topochemical Polymerization: Cross-α-Amyloid-like Packing in a Pseudoprotein with Gly-Phe-Gly Repeats.

Protein-mimics are of great interest for their structure, stability, and properties. We are interested in the synthesis of protein-mimics containing triazole linkages as peptide-bond surrogate by topochemical azide-alkyne cycloaddition (TAAC) polymerization of azide- and alkyne-modified peptides. The rationally designed dipeptide N3 -CH2 CO-Phe-NHCH2 CCH (1) crystallized in a parallel β-sheet arrangement and are head-to-tail aligned in a direction perpendicular to the β-sheet-direction. Upon heating, crystals of 1 underwent single-crystal-to-single-crystal polymerization forming a triazole-linked pseudoprotein with Gly-Phe-Gly repeats. During TAAC polymerization, the pseudoprotein evolved as helical chains. These helical chains are laterally assembled by backbone hydrogen bonding in a direction perpendicular to the helical axis to form helical sheets. This interesting helical-sheet orientation in the crystal resembles the cross-α-amyloids, where α-helices are arranged laterally as sheets.

β‐Sheet to Helical‐Sheet Evolution Induced by Topochemical Polymerization: Cross‐α‐Amyloid‐like Packing in a Pseudoprotein with Gly‐Phe‐Gly Repeats

AbstractProtein‐mimics are of great interest for their structure, stability, and properties. We are interested in the synthesis of protein‐mimics containing triazole linkages as peptide‐bond surrogate by topochemical azide‐alkyne cycloaddition (TAAC) polymerization of azide‐ and alkyne‐modified peptides. The rationally designed dipeptide N3‐CH2CO‐Phe‐NHCH2CCH (1) crystallized in a parallel β‐sheet arrangement and are head‐to‐tail aligned in a direction perpendicular to the β‐sheet‐direction. Upon heating, crystals of 1 underwent single‐crystal‐to‐single‐crystal polymerization forming a triazole‐linked pseudoprotein with Gly‐Phe‐Gly repeats. During TAAC polymerization, the pseudoprotein evolved as helical chains. These helical chains are laterally assembled by backbone hydrogen bonding in a direction perpendicular to the helical axis to form helical sheets. This interesting helical‐sheet orientation in the crystal resembles the cross‐α‐amyloids, where α‐helices are arranged laterally as sheets.

A Study of Plant Coumarins. 18. Conjugates of Coumarins with Lupane Triterpenoids and 1,2,3-Triazoles: Synthesis and Anti-Inflammatory Activity

In the reaction of coumarin peuruthenicin with α,ω-dibromoalkanes, the corresponding 7-(ω-bromoalkyloxy)coumarins have been synthesized. The interaction of the resulting compounds with sodium azide has yielded 7-(ω-azidoalkyl)-substituted peuruthenicin derivatives, which exhibited high activity in the Cu(I)-catalyzed Huisgen cycloaddition reaction with the propargyl ester of betulonic acid. As a result of the reaction, 28-O-(chromenoalkyl-triazolylmethyl)lupenones have been synthesized. The reaction of betulonic acid propargyl ester with 2-azidooreoselones in the presence of aqueous copper sulfate and sodium ascorbate has led to the corresponding (furochromen-triazolyl)-20(29)-lupen-3-ones. The newly synthesized hybrid compound triterpenoid furocoumarin containing a triazole linker exhibits anti-inflammatory activity in a model of histamine inflammation.

Searching for the ideal triazole: Investigating the 1,5-triazole as a charge neutral DNA backbone mimic

A novel triazole linkage that mimics the phosphodiester backbone in DNA was designed, synthesised and evaluated. Unlike previous work which utilised copper to form a 1,4 triazole linkage in the DNA backbone, a ruthenium catalyst was used to yield a 1,5 triazole. The artificial linkage was incorporated into a DNA backbone via a phosphoramidite building block using solid phase synthesis. The biophysical properties of DNA with a 1,5 triazole linkage in the backbone were evaluated by UV melting and circular dichroism and compared to DNA modified with previously reported 1,4 triazole linkages of various lengths.

Efficient DNA Click Reaction Replaces Enzymatic Ligation.

We report a chemical DNA-DNA ligation method based on copper-catalyzed azide-alkyne cycloaddition (CuAAC). We demonstrate that ion addition dramatically influences the efficiency of the so-called click reaction. Even without any further additions, such as typically splint oligonucleotides for preorganization, the "click ligation" yields up to ∼83% product without any byproducts. Additionally, purification of the desired product is straightforward. In comparison to enzymatic ligation methods used to introduce adapters into, e.g., mRNA library preparation, this targeted chemical ligation method exhibits several advantages: increased ligated product and no adapter or cDNA oligomers byproducts. The advantages of the click ligation method were demonstrated by incorporation of azide modified nucleotides by several enzymes as well as broad polymerase acceptance of the obtained triazole linkage in PCR.

Triazole-Containing [FeFe] Hydrogenase Mimics: Synthesis and Electrocatalytic Behavior.

Through a Cu-catalyzed Huisgen cycloaddition between terminal alkynes and azides (CuAAC) reaction, azide [(μ-SCH2)2N(4-N3C6H4)Fe2(CO)6] has demonstrated to be a robust and versatile reagent able to incorporate the [(μ-SR)2Fe2(CO)6] fragment on a wide range of substrates, ranging from aromatic compounds to nucleosides, metallocenes, or redox and luminescent markers. The [FeIFeI]/[Fe0FeI] and [Fe0FeI]/[Fe0Fe0] reduction potentials of the triazole derivatives prepared are comparable to those of other aminodithiolate (adt) Fe-Fe hydrogenase mimics. The presence of the triazole linker influences the electrochemical behavior of these complexes depending on the strength of the acid employed.

Triazol: a privileged scaffold for proteolysis targeting chimeras.

Current traditional drugs such as enzyme inhibitors and receptor agonists/antagonists present inherent limitations due to occupancy-driven pharmacology as the mode of action. Proteolysis targeting chimeras (PROTACs) are composed of an E3 ligand, a connecting linker and a target protein ligand, and are an attractive approach to specifically knockdown-targeted proteins utilizing an event-driven mode of action. The length, hydrophilicity and rigidity of connecting linkers play important role in creating a successful PROTAC. Some PROTACs with a triazole linker have displayed promising anticancer activity. This review provides an overview of PROTACs with a triazole scaffold and discusses its structure-activity relationship. Important milestones in the development of PROTACs are addressed and a critical analysis of this drug discovery strategy is also presented.

An Azide‐Substituted Triarylborane: A Key Compound for the Facile Synthesis of Fluorescent Triarylboranes Bearing Triazole Moieties as Connectable π‐Conjugated System Linkages

Tris(4‐azido‐2,6‐dimethylphenyl)borane was readily synthesized from an amino‐substituted triarylborane using tert‐butyl nitrite and trimethylsilylazide. This compound can easily be converted into triarylboranes bearing various π‐conjugated moieties with triazole ring π‐linkers through a Huisgen cycloaddition. UV/Vis absorption spectra and DFT calculations suggested that constructing a triazole linker successfully extended the π‐conjugated system of triarylboranes. Triarylboranes obtained using this method maintained their fluorescent nature. In particular, the triarylborane linking N,N‐dimethylaminophenyl groups with triazole rings exhibited notable visible solvatofluorochromism and fluorescence color change upon addition of a fluoride anion.

Generation of Microsecond Charge-Separated Excited States in Rhenium(I) Diimine Complexes: Driving Force Is the Dominant Factor in Controlling Lifetime.

A transition-metal-based donor-(linker)-acceptor system can produce long-lived charge transfer excited states using visible excitation wavelengths. The ground- and excited-state photophysical properties of a series of [ReCl(CO)3(dppz-(linker)-TPA)] complexes, with varying donor and acceptor energies, have been systematically studied using spectroscopic techniques (both vibrational and electronic) supported by computational chemistry. The long-lived excited state is 3ILCT in nature for all complexes studied, characterized through transient absorption and emission, transient resonance Raman (TR2), and time-resolved infrared (TRIR) spectroscopy and TDDFT calculations. Modulation of the donor and acceptor energies results in changes of the 3ILCT lifetime by 1 order of magnitude, ranging from 6.1(±1) μs when a diphenylamine donor is used to 0.6(±0.2) μs when a triazole linker and triphenylamine donor is used. The excited-state lifetime may be rationalized by consideration of the driving force within the framework of Marcus theory and appears insensitive to the nature of the linker.

Artificially Linked Ubiquitin Dimers Characterised Structurally and Dynamically by NMR Spectroscopy.

As one of the most prevalent post‐translational modifications in eukaryotic cells, ubiquitylation plays vital roles in many cellular processes, such as protein degradation, DNA metabolism, and cell differentiation. Substrate proteins can be tagged by distinct types of polymeric ubiquitin (Ub) chains, which determine the eventual fate of the modified protein. A facile, click chemistry based approach for the efficient generation of linkage‐defined Ub chains, including Ub dimers, was recently established. Within these chains, individual Ub moieties are connected through a triazole linkage, rather than the natural isopeptide bond. Herein, it is reported that the conformation of an artificially K48‐linked Ub dimer resembles that of the natively linked dimer, with respect to structural and dynamic characteristics, as demonstrated by means of high‐resolution NMR spectroscopy. Thus, it is proposed that artificially linked Ub dimers, as generated by this approach, represent potent tools for studying the inherently different properties and functions of distinct Ub chains.

Self-Assembly of Diphenylalanine-Peptide Nucleic Acid Conjugates.

The synthesis and self-assembled nanostructures of a series of nucleopeptides (NPs) derived from the dipeptide Phe–Phe and the peptide nucleic acid unit which are covalently attached through an amide or a triazole linker are described. Depending on the variables such as protecting groups, linkers, and nucleobases, spherical nanoparticles were observed through scanning electron microscopy and high-resolution transmission electron microscopy images, and the porous nature of representative NPs was corroborated by carboxyfluorescein entrapment. Hydrophobic substituents on different sites of NPs and solvents employed for peptide self-assembly played a crucial role for corresponding morphologies. The stability of nanoparticles was also probed under external stimuli such as pH, temperature, and enzymatic hydrolysis using proteolytic enzymes. The semiconducting nature of the NP-modified carbon electrodes suggested their potential use as a new capacitor material.

Soluble Antigen Arrays Displaying Mimotopes Direct the Response of Diabetogenic T Cells.

Type 1 diabetes (T1D) is an autoimmune disorder which develops when insulin-producing, pancreatic beta cells are destroyed by an aberrant immune response. Current therapies for T1D either treat symptoms or cause global immunosuppression, which leave patients at risk of developing long-term complications or vulnerable to foreign pathogens. Antigen-specific immunotherapies have emerged as a selective approach for autoimmune diseases by inducing tolerance while mitigating global immunosuppression. We previously reported SAgAs with multiple copies of a multiple sclerosis (MS) autoantigen grafted onto hyaluronic acid (HA) as an efficacious therapy in experimental autoimmune encephalomyelitis. While the immune response of MS is distinct from that of T1D, the mechanism of SAgAs was hypothesized to be similar and via induction of immune tolerance to diabetes antigens. We synthesized SAgAs composed of HA polymer backbone conjugated with multiple copies of the T1D autoantigen mimotope p79 using aminooxy chemistry (SAgAp79) or using copper-catalyzed alkyne-azide cycloaddition (cSAgAp79) chemistry. SAgAs constructed using the hydrolyzable aminooxy linkage, thus capable of releasing p79, exhibited physicochemical properties similar to the triazole linkage. Both SAgAp79 versions showed high specificity and efficacy in stimulating epitope-specific T cells. SAgAs can be taken up by most immune cell populations but do not induce their maturation, and conventional dendritic cells are responsible for the brunt of antigen presentation within splenocytes. cSAgAp79 was more stimulatory than SAgAp79 both in vitro and in vivo, an effect that was ascribed to the peptide modification rather than the type of linkage. In summary, we provide here the first proof-of-principle that SAgA therapy could also be applicable to T1D.

Universal Synthetic Strategy for the Construction of Topological Polystyrenesulfonates: The Importance of Linkage Stability during Sulfonation.

Polystyrenesulfonate (PSS), as one of the most important categories of polyelectrolytes, has received increasing attention due to its great potential in the applications of energy- and biomedical-related fields. However, most of the previous studies only focused on linear PSS and its derivatives, but little attention was paid to nonlinear topological PSSs. So far, the synthesis of nonlinear PSSs with well-defined structures is still a challenging task, and the main obstacle lies in the stability issue of functional chemical linkages during the sulfonation process of polystyrene (PS) precursors, such as the carbon-oxygen-containing linkages. Herein, by rationally designing the chemical structure of the functional linkage, we introduce a versatile and efficient strategy for the preparation of topological PSSs. Specifically, by embedding firm triazole linkages (without carbon-oxygen linkages) into the backbone structure of cyclic and hyperbranched PS precursors, the backbone and functional linkages are found to present excellent chemical stability under certain sulfonation conditions, which eventually lead to the successful preparation of cyclic and hyperbranched PSSs. By using two sets of PSS samples with varied molar masses, the scaling relations between the number of repeating units and the sedimentation coefficient are established for both linear and cyclic PSSs. We believe that our proposed synthetic strategy is universal and could be extended to the synthesis of other types of topological PSSs.

Proteomic Strategy for Identification of Proteins Responding to Cisplatin-Damaged DNA.

A new proteomic strategy combining functionalized magnetic nanoparticle affinity probes with mass spectrometry was developed to capture and identify proteins specifically responding to 1,2-d(GpG) intrastrand cisplatin-cross-linked DNA, the major DNA lesion caused by cisplatin and thought to induce apoptosis. A 16-mer oligodeoxynucleotide (ODN) duplex and its cisplatin-cross-linked adduct were immobilized on magnetic nanoparticles via click reaction, respectively, to fabricate negative and positive affinity probes which were very stable in cellular protein extracts due to the excellent bio-orthogonality of click chemistry and the inertness of covalent triazole linker. Quantitative mass spectrometry results unambiguously revealed the predominant binding of HMGB1 and HMGB2, the well-established specific binders of 1,2-cisplatin-cross-linked DNA, to the cisplatin-cross-linked ODN, thus validating the accuracy and reliability of our strategy. Furthermore, 5 RNA or single-stranded DNA binding proteins, namely, hnRNP A/B, RRP44, RL30, RL13, and NCL, were demonstrated to recognize specifically the cisplatinated ODN, indicating the significantly unwound ODN duplex by cisplatin cross-linking. In contrast, the binding of a transcription factor TFIIFa to DNA was retarded due to cisplatin damage, implying that the cisplatin lesion stalls DNA transcription. These findings promote understanding in the cellular responses to cisplatin-damaged DNA and inspire further precise elucidation of the action mechanism of cisplatin.

An artificial triazole backbone linkage provides a split-and-click strategy to bioactive chemically modified CRISPR sgRNA

As the applications of CRISPR-Cas9 technology diversify and spread beyond the laboratory to diagnostic and therapeutic use, the demands of gRNA synthesis have increased and access to tailored gRNAs is now restrictive. Enzymatic routes are time-consuming, difficult to scale-up and suffer from polymerase-bias while existing chemical routes are inefficient. Here, we describe a split-and-click convergent chemical route to individual or pools of sgRNAs. The synthetic burden is reduced by splitting the sgRNA into a variable DNA/genome-targeting 20-mer, produced on-demand and in high purity, and a fixed Cas9-binding chemically-modified 79-mer, produced cost-effectively on large-scale, a strategy that provides access to site-specific modifications that enhance sgRNA activity and in vivo stability. Click ligation of the two components generates an artificial triazole linkage that is tolerated in functionally critical regions of the sgRNA and allows efficient DNA cleavage in vitro as well as gene-editing in cells with no unexpected off-target effects.

Selective Inhibitor Design Toward CA IX For Breast Cancer Treatment Using Carbohydrate‐Based Compounds

Human carbonic anhydrase IX (CA IX) is upregulated in neoplastic tissues and has been studied as a drug target for anticancer chemotherapy. Inhibition of CA IX has been shown to be therapeutically favorable in terms of reducing tumor growth. Recently, it been observed that sweeteners bind and selectively inhibit CA IX over other CA isoforms. It was found that cyclic sulfonamides, such as saccharin, inhibits CA IX with 60‐fold isoform selectivity, while disaccharides, such as sucrose, have been found bound at the opening of the active site. As fragment‐based studies show, the most potent inhibitors result from the combination of small molecules. A chemical linker would provide the spacial requirements to optimize interactions with residues in the selective pocket of the active site, improving selectivity for CA IX and preventing off target inhibition of the ubiquitously expressed isoform, CA II. Such compounds would simultaneously inhibit activity through two mechanisms: displacing the ZBS essential for catalysis and blocking the active site from substrate binding. The “tail‐approach” was done using sweeteners as fragments to develop inhibitors with higher CA IX selectivity by connecting saccharin (zinc‐binding group) to a β glucoside (tail) using a triazole linker. In this study, we present the X‐ray crystal structures of carbohydrate compounds in complex with the cancer‐related and off‐target CA isoforms, as well as the effect these compounds have on a panel of breast cancer cell lines. It was found that the carbohydrate compounds have different binding modes with compounds binding directly to the catalytic zinc in CA IX (mimic) and through a bridging water in CA II, which can account for the increased selectivity for CA IX (mimic) when compared to CA II. It was also found that the carbohydrate compounds decreased the metastatic potential of UFH‐001 breast cancer cells that are representative of triple negative breast cancer cells that have high CA IX expression. In summary, These studies will contribute to the knowledge of treating more aggressive and late stage breast cancers and identify new opportunities to increase the effectiveness of current cancer therapies.Support or Funding InformationABM was supported by training grant T32AI0007110 from the NIH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis of saccharin-glycoconjugates targeting carbonic anhydrase using a one-pot cyclization/deprotection strategy

Carbonic anhydrase IX (CA IX) has been identified as a biomarker and drug target for several malignant tumors due to its role in cancer cell growth and proliferation. Simple cyclic sulfonamides, like saccharin (SAC), have shown up to a 60-fold selectivity towards CA IX over other ubiquitous CA isoforms, with greater selectivity obtained applying the “tail-approach” to derivatize SAC with a methylene triazole linker that connected to a “tail” beta glucoside. These modifications of SAC led to an increased selectivity of more than 1000-fold towards CA IX, whereas clinically available CA inhibitors show little to no isoform selectivity. As part of our interest in the development of new CA inhibitors, we found the existing synthetic protocol, which relies on a N-tert-butyl saccharin intermediate, to be problematic in the final deprotection steps. We therefore describe an alternative approach to the synthesis of these compounds featuring a gentle “one pot” deprotection/cyclization as the final synthetic step, and report new galactosyl and glucosyl conjugates with low to mid nM inhibition of CA IX.

Antimicrobial alumina nanobiostructures of disulfide- and triazole-linked peptides: Synthesis, characterization, membrane interactions and biological activity.

Due to the its physical-chemical properties, alumina nanoparticles have potential applications in several areas, such as nanobiomaterials for medicinal or orthodontic implants, although the introduction of these devices poses a serious risk of microbial infection. One convenient strategy to circumvent this problem is to associate the nanomaterials to antimicrobial peptides with broad-spectrum of activities. In this study we present two novel synthesis approaches to obtain fibrous type alumina nanoparticles covalently bound to antimicrobial peptides. In the first strategy, thiol functionalized alumina nanoparticles were linked via disulfide bond formation to a cysteine residue of an analog of the peptide BP100 containing a four amino acid spacer (Cys-Ala-Ala-Ala). In the second strategy, alumina nanoparticles were functionalized with azide groups and then bound to alkyne-decorated analogs of the peptides BP100 and DD K through a triazole linkage obtained via a copper(I)-catalyzed cycloaddition reaction. The complete physical-chemical characterization of the intermediates and final materials is presented along with in vitro biological assays and membrane interaction studies, which confirmed the activity of the obtained nanobiostructures against both bacteria and fungi. To our knowledge, this is the first report of aluminum nanoparticles covalently bound to triazole-peptides and to a disulfide bound antimicrobial peptide with high potential for biotechnological applications.