Nucleotidyl transferase assisted DNA labeling with different click chemistries

Here, we present a simple, modular and efficient strategy that allows the 3′-terminal labeling of DNA, regardless of whether it has been chemically or enzymatically synthesized or isolated from natural sources. We first incorporate a range of modified nucleotides at the 3′-terminus, using terminal deoxynucleotidyl transferase. In the second step, we convert the incorporated nucleotides, using either of four highly efficient click chemistry-type reactions, namely copper-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, Staudinger ligation or Diels-Alder reaction with inverse electron demand. Moreover, we create internal modifications, making use of either ligation or primer extension, after the nucleotidyl transferase step, prior to the click reaction. We further study the influence of linker variants on the reactivity of azides in different click reactions. We find that different click reactions exhibit distinct substrate preferences, a fact that is often overlooked, but should be considered when labeling oligonucleotides or other biomolecules with click chemistry. Finally, our findings allowed us to extend our previously published RNA labeling strategy to the use of a different copper-free click chemistry, namely the Staudinger ligation.     

"Clickable" nanoparticles for targeted imaging

Nanomaterials functionalized with targeting ligands are increasingly recognized as useful materials for molecular imaging and drug delivery. Here we describe the development and validation of azide-alkyne reactions ("click chemistry") for the rapid, site-specific modification of nanoparticles with small molecules. The facile preparation of stable nanoparticles bearing azido or alkyne groups capable of reaction with their corresponding counterpart functionalized small molecules is demonstrated. The Cu(I)-catalyzed cycloaddition of azides and alkynes is shown to be a highly efficient and selective method for point functionalization of magnetic nanoparticles. Derivatized nanoparticles bearing biotin, fluorochrome, or steroid moieties are stable for several months. Nanoparticle click chemistry will be useful for other nanomaterials, design of novel sensors, and drug delivery vehicles.     

Fluorogenic ‘click-on’ dendrimer reporter for rapid profiling of cell proliferation

The application of small molecule fluorescent reporters to monitor biological systems is limited by their poor water solubility and background fluorescence of these reporters. Herein, we describe the synthesis and testing of a fluorogenic ‘click’ dendrimer reporter to monitor cellular processes. The reporter system consists of a polyamidoamine (PAMAM) dendrimer conjugated with 3-azido-7-hydroxy coumarin. After the copper(I)-catalyzed azide–alkyne cycloaddition reaction (‘click’ reaction) with alkyne-derivatized target molecules, the natively non-fluorescent construct has a strong enhancement in fluorescence. This fluorogenic dendrimer reporter can be used to efficiently monitor biological processes and the specificity afforded by the ‘click’ reaction greatly reduces background noise and enhances assay flexibility. We used this fluorogenic dendrimer reporter to monitor incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into newly synthesized DNA, as a surrogate marker of cellular proliferation. We anticipate that this new class of fluorogenic reporter can be used to monitor a wide array of molecules and lends itself to high-throughput profiling of biological systems.     

"Clickable" agarose for affinity chromatography

Successful purification of biological molecules by affinity chromatography requires the attachment of desired ligands to biocompatible chromatographic supports. The Cu(I)-catalyzed cycloaddition of azides and alkynes-the premier example of "click chemistry"-is an efficient way to make covalent connections among diverse molecules and materials. Both azide and alkyne units are highly selective in their reactivity, being inert to most chemical functionalities and stable to wide ranges of solvent, temperature, and pH. We show that agarose beads bearing alkyne and azide groups can be easily made and are practical precursors to functionalized agarose materials for affinity chromatography.     

Carbohydrate and protein immobilization onto solid surfaces by sequential Diels-Alder and azide-alkyne cycloadditions

We demonstrate the applicability of sequential Diels-Alder and azide-alkyne [3 + 2] cycloaddition reactions (click chemistry) for the immobilization of carbohydrates and proteins onto a solid surface. An alpha,omega-poly(ethylene glycol) (PEG) linker carrying alkyne and cyclodiene terminal groups was synthesized and immobilized onto an N-(epsilon-maleimidocaproyl) (EMC)-functionalized glass slide via an aqueous Diels-Alder reaction. In the process, an alkyne-terminated PEGylated surface was provided for the conjugation of azide-containing biomolecules via click chemistry, which proceeded to completion at low temperature and in aqueous solvent. As anticipated, alkyne, azide, cyclodiene, and EMC are independently stable and do not react with common organic reagents nor functional groups in biomolecules. Given an appropriate PEG linker, sequential Diels-Alder and azide-alkyne [3 + 2] cycloaddition reactions provide an effective strategy for the immobilization of a wide range of functionally complex substances onto solid surfaces.     

Influence of azide incorporation on binding affinity by small papain inhibitors

In order to develop affinity-based biosensor platforms, appropriate ligands with a functional handle for immobilization onto a biosensor surface are required. To this end, a library of papain inhibitors was designed and synthesized, containing different azide linkers for subsequent immobilization by ‘click’ chemistry, in this particular case by copper-free, strain-promoted azide–alkyne cycloaddition (SPAAC). Furthermore, a molecular docking study was performed to obtain a better insight as to at which position such azide handles could be tolerated without affecting binding affinity. Although the azide moiety is small, in some cases its introduction strongly influenced the binding affinity. For one class of inhibitors a swapped binding mode was proposed to explain the results. In addition, a specific site for linker introduction was identified, which did not significantly affect the binding affinity.     

Synthesis and micellar mimic properties of bile acid trimers

Two fan-shaped bile acid trimers have been synthesized via Cu^I-catalyzed azide–alkyne cycloaddition (CuAAC) ‘click chemistry’, and their extraction experiments of cresol red sodium (CR) and pyrene were investigated in the polar and non-polar solvents, respectively. The transmission electron microscopy (TEM) results showed that the homogenous hollow capsules formed with the diameter size range of 40–70 nm in a solution of water and acetone. Thus the amphiphilicity of fan-shaped bile acid trimers might be used as the promising candidate in biological and drug delivery applications.     

Tyrosine specific sequential labeling of proteins

We report (a) on the synthesis of a long-wavelength fluorescent coumarin containing an allyloxy acetate moiety, (b) the synthesis of two linkers containing an allyloxy acetate and an alkyne or azide function, respectively, and (c) the selective modification human serum albumin by a sequential method involving Pd(II) catalyzed modification of the phenolic side chain of tyrosine residues with an alkyne bearing linker and a subsequent azide–alkyne click reaction with an azide functionalized long-wavelength emitting coumarin dye. The method is likely to be applicable to various kinds of azido-modified fluorophores, and the Pd(II)-catalyzed modification of the tyrosines may also be used to introduce other kinds of tags. With these reagents, tyrosine specific modulation of proteins and peptides becomes possible either directly or in a sequential manner.     

Stereoselective synthesis of bio-hybrid amphiphiles of coumarin derivatives by Ugi-Mannich triazole randomization using copper catalyzed alkyne azide click chemistry

An efficient synthesis of ester-triazole-amide amphiphiles of coumarin derivatives by triazole randomization based on click approach is described. Twenty-five small peptide azides were synthesized using Ugi or alternate Mannich-type multi-component reactions. The new azides were then used for the triazole randomization of alkyne functionalized coumarin ester under CuAAC conditions. Sixty-five new peptide bio-hybrids are obtained in near quantitative yield with high regio and stereoselectivity.     

Strain-promoted "click" chemistry for terminal labeling of DNA

1,3-Dipolar [3 + 2] cycloaddition between azides and alkynes--an archetypal "click" chemistry--has been used increasingly for the functionalization of nucleic acids. Copper(I)-catalyzed 1,3-dipolar cycloaddition reactions between alkyne-tagged DNA molecules and azides work well, but they require optimization of multiple reagents, and Cu ions are known to mediate DNA cleavage. For many applications, it would be preferable to eliminate the Cu(I) catalyst from these reactions. Here, we describe the solid-phase synthesis and characterization of 5'-dibenzocyclooctyne (DIBO)-modified oligonucleotides, using a new DIBO phosphoramidite, which react with azides via copper-free, strain-promoted alkyne-azide cycloaddition (SPAAC). We found that the DIBO group not only survived the standard acidic and oxidative reactions of solid-phase oligonucleotide synthesis (SPOS), but that it also survived the thermal cycling and standard conditions of the polymerase chain reaction (PCR). As a result, PCR with DIBO-modified primers yielded "clickable" amplicons that could be tagged with azide-modified fluorophores or immobilized on azide-modified surfaces. Given its simplicity, SPAAC on DNA could streamline the bioconjugate chemistry of nucleic acids in a number of modern biotechnologies.     

Semi-synthesis of biologically active nisin hybrids composed of the native lanthionine ABC-fragment and a cross-stapled synthetic DE-fragment

The antimicrobial peptide nisin is a promising template for designing novel peptide-based antibiotics to improve its drug-like properties. First steps in that direction represent the synthesis of hybrid nisin derivatives that contain a native nisin ABC-part and synthesized cross-stapled DE-ring fragments and are described here. The biological activity of the newly synthesized nisin derivatives was evaluated in order to compare the bioactivity of the synthetic DE-ring containing mimic and native lanthionine-bridged DE-ring containing nisin. The native nisin ABC-ring system was obtained via chymotrypsin digestion of full-length nisin, and was subsequently functionalized at the C-terminal carboxylate with two different amino alkyne moieties. Next, nisin hybrids were successfully prepared using Cu(I)-catalyzed azide alkyne cycloaddition ‘click’ chemistry by chemo-selective ligation of the ABC-alkyne with the N-terminal azido functionalized dicarba-DE ring mimic. The newly synthesized compounds were active as potent lipid II binders and retained antimicrobial activity in a growth inhibition assay. However, pore formation was not observed, possibly either due to the different character of the ‘staples’ as compared to the parent sulfides, or due to the triazole moiety as a sub-optimal amide bond isostere.     

Synthesis of C-4 and C-7 triazole analogs of zanamivir as multivalent sialic acid containing scaffolds

The relative reactivities of C-4 and C-7 azides derived from zanamivir were compared in cycloaddition reactions with a panel of alkynes. All of the reactions proceeded efficiently with no observable differences between primary and secondary azides. Significant rate differences were observed between several members of the alkyne panel. Most notably, a trialkyne derived from a 1,3,5-triazine core underwent complete reaction within 4 h, whereas an analogous trialkyne with an all carbon aromatic core required 18 h. These results suggest that the triazine core serves as an internal catalyst.     

Preventing thiol-yne addition improves the specificity of strain-promoted azide-alkyne cycloaddition

The 1,3-dipolar cycloaddition of azides with ring-strained alkynes is one of the few bioorthogonal reactions suitable for specific biomolecule labeling in complex biological systems. Nevertheless, azide-independent labeling of proteins by strained alkynes can occur to a varying extent, thereby limiting the sensitivity of assays based on strain-promoted azide-alkyne cycloaddition (SPAAC). In this study, a subset of three cyclooctynes, dibenzocyclooctyne (DIBO), azadibenzocyclooctyne (DIBAC), and bicyclo[6.1.0]nonyne (BCN), was used to evaluate the azide-independent labeling of proteins in vitro. For all three cyclooctynes, we show that thiol-yne addition with reduced peptidylcysteines is responsible for most of the azide-independent polypeptide labeling. The identity of the reaction product was confirmed by LC-MS and NMR analysis. Moreover, we show that undesired thiol-yne reactions can be prevented by alkylating peptidylcysteine thiols with iodoacetamide (IAM). Since IAM is compatible with SPAAC, a more specific azide-dependent labeling is achieved by preincubating proteins containing reduced cysteines with IAM.     

1,2,3-Triazole-containing hybrids as leads in medicinal chemistry: A recent overview

The 1,2,3-triazole ring is a major pharmacophore system among nitrogen-containing heterocycles. These five-membered heterocyclic motifs with three nitrogen heteroatoms can be prepared easily using ‘click’ chemistry with copper- or ruthenium-catalysed azide-alkyne cycloaddition reactions. Recently, the ‘linker’ property of 1,2,3-triazoles was demonstrated, and a novel class of 1,2,3-triazole-containing hybrids and conjugates was synthesised and evaluated as lead compounds for diverse biological targets. These lead compounds have been demonstrated as anticancer, antimicrobial, anti-tubercular, antiviral, antidiabetic, antimalarial, anti-leishmanial, and neuroprotective agents. The present review summarises advances in lead compounds of 1,2,3-triazole-containing hybrids, conjugates, and their related heterocycles in medicinal chemistry published in 2018. This review will be useful to scientists in research fields of organic synthesis, medicinal chemistry, phytochemistry, and pharmacology.     

Facile synthesis of cyclopeptide-centered multivalent glycoclusters with ‘click chemistry’ and molecular recognition study by surface plasmon resonance

A facile synthesis of cyclopeptide-centered multivalent glycoclusters using Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes, so called ‘click chemistry’, has been developed. The affinities of mannose-specific protein Concanavalin A (Con A) toward two synthetic glycoclusters respectively bearing divalent or tetravalent mannoses were investigated by surface plasmon resonance (SPR). It is founded that the tetravalent glycocluster has 3.0-fold increase in binding affinity relative to the divalent glycoluster (valency-corrected values), which indicates the potential of this system in investigating carbohydrate–protein interactions.     

Modular assembly of dimeric HIV fusion inhibitor peptides with enhanced antiviral potency

The HIV-1 envelope gp120/gp41 glycoprotein complex plays a critical role in virus-host cell membrane fusion and has been a focus for the development of HIV fusion inhibitors. In this Letter, we present the synthesis of dimers of HIV fusion inhibitor peptides C37H6 and CP32M, which target the trimeric gp41 in the pre-hairpin intermediate state to inhibit membrane fusion. Reactive peptide modules were synthesized using native chemical ligation and then assembled into dimers with varying linker lengths using Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) ‘click’ chemistry. Cell–cell fusion inhibition assays demonstrated that dimers with a (PEG)₇ linker showed enhanced antiviral potency over the corresponding monomers. Moreover, the bio-orthogonal nature of the CuAAC ‘click’ reaction provides a practical way to assemble heterodimers of HIV fusion inhibitors. Heterodimers consisting of the T20-sensitive strain inhibitor C37H6 and the T20-resistant strain inhibitor CP32M were produced that may have broader spectrum activities against both T20-sensitive and T20-resistant strains.     

Parallel synthesis of potent dopaminergic N-phenyltriazole carboxamides applying a novel click chemistry based phenol linker

Taking advantage of our click chemistry based methodology to construct novel SPOS (solid phase organic synthesis) resins, the triazolylmethyl linked catechol 6a was discovered, which is readily available via copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) of azidomethyl substituted polystyrene with O-propargylcatechol and can be applied for the parallel synthesis of N-phenyltriazole carboxamides. As a proof-of-concept, a ‘catch-and-release’ strategy could be successfully applied for a parallel synthesis of dopaminergic phenyltriazoles of type 2. A focused model library of 20 test compounds revealing three points of diversity was generated by a three-step SPOS approach. Product purification was performed employing a solid-supported carboxylic acid anhydride as a scavenger. GPCR-ligand binding screening revealed dopamine D3 receptor ligands with K_i values in the single digit nanomolar range.     

Metal-free click and bioorthogonal reactions of aggregation-induced emission probes for lighting up living systems

In 2002, two transformative research paradigms emerged: ‘click chemistry’ and ‘aggregation-induced emission (AIE),’ both leaving significant impacts on early 21st-century academia. Click chemistry, which describes the straightforward and reliable reactions for linking two building blocks, has simplified complex molecular syntheses and functionalization, propelling advancements in polymer, material, and life science. In particular, nontoxic, metal-free click reactions involving abiotic functional groups have matured into bioorthogonal reactions. These are organic ligations capable of selective and efficient operations even in congested living systems, therefore enabling in vitro to in vivo biomolecular labelling. Concurrently, AIE, a fluorogenic phenomenon of twisted π-conjugated compounds upon aggregation, has offered profound insight into solid-state photophysics and promoted the creation of aggregate materials. The inherent fluorogenicity and aggregate-emission properties of AIE luminogens have found extensive application in biological imaging, characterized by their high-contrast and photostable fluorescent signals. As such, the convergence of these two domains to yield efficient labelling with excellent fluorescence images is an anticipated progression in recent life science research. In this review, we intend to showcase the synergetic applications of AIE probes and metal-free click or bioorthogonal reactions, highlighting both the achievements and the unexplored avenues in this promising field.     

Tandem photoaffinity labeling–bioorthogonal conjugation in medicinal chemistry

Photoaffinity labeling has a longstanding history as a powerful biochemical technique. However, photoaffinity labeling has significantly evolved over the past decade principally due to its coupling with bioorthogonal/click chemistry reactions. This review aims to highlight tandem photoaffinity labeling–bioorthogonal conjugation as a chemical approach in medicinal chemistry and chemical biology. In particular, recent examples of using this strategy for affinity-based protein profiling (AfBPP), drug target identification, binding ensemble profiling, studying endogenous biological molecules, and imaging applications will be presented. Additionally, recent advances in the development of ‘all-in-one’ compact moieties possessing a photoreactive group and clickable handle will be discussed.