In Vivo Incorporation of Azide Groups into DNA by Using Membrane‐Permeable Nucleotide Triesters

Metabolic incorporation of bioorthogonal functional groups into cellular nucleic acids can be impeded by insufficient phosphorylation of nucleosides. Previous studies found that 5azidomethyl‐2′‐deoxyuridine (AmdU) was incorporated into the DNA of HeLa cells expressing a low‐fidelity thymidine kinase, but not by wild‐type HeLa cells. Here we report that membrane‐permeable phosphotriester derivatives of AmdU can exhibit enhanced incorporation into the DNA of wild‐type cells and animals. AmdU monophosphate derivatives bearing either 5′‐bispivaloyloxymethyl (POM), 5′‐bis‐(4‐acetoxybenzyl) (AB), or “Protide” protective groups were used to mask the phosphate group of AmdU prior to its entry into cells. The POM derivative “POM‐AmdU” exhibited better chemical stability, greater metabolic incorporation efficiency, and lower toxicity than “AB‐AmdU”. Remarkably, the addition of POM‐AmdU to the water of zebrafish larvae enabled the biosynthesis of azide‐modified DNA throughout the body.     

Synthesis of a Cytotoxic Amanitin for Biorthogonal Conjugation

Alpha‐amanitin is an exceedingly toxic, naturally occurring, bicyclic octapeptide that inhibits RNA polymerase and results in cellular and organismal death. Here we report the straightforward synthesis of an amanitin analogue that exhibited near‐native toxicity. A pendant alkyne was readily installed to enable copper‐catalyzed alkyne–azide cycloaddition (CuAAC) to azido‐rhodamine and two azide‐bearing versions of the RGD peptide. The fluorescent toxin analogue entered cells and provoked morphological changes consistent with cell death. The latter two conjugates are as toxic as the parent alkyne precursor, which demonstrates that conjugation does not diminish toxicity. In addition, we showed that toxicity depends on a single diastereomer of the unnatural amino acid, dihydroxyisoleucine (DHIle), at position 3. The convenient synthesis of a heptapeptide precursor now provides access to bioactive amanitin analogues that may be readily conjugated to biomolecules of interest.     

Reactive Energetic Plasticizers for Energetic Polyurethane Binders Prepared via Simultaneous Huisgen Azide‐Alkyne Cycloaddition and Polyurethane Reaction

Reactive energetic plasticizers (REPs) for use in glycidyl azido polymer (GAP) based polyurethane (PU) energetic binders were investigated. These REPs consisted of an activated terminal alkyne group that was expected to give rise to Huisgen azide‐alkyne 1,3‐dipolar cycloaddition within the specific pot life for a PU formulation to prevent the migration of plasticizers, and with a gem‐dinitro group as an energy resource. A quantitative miscibility investigation between the plasticizers and uncured GAP showed that REPs exhibited better miscibility than conventional energetic plasticizers. The plasticization effect of the REPs on the GAP prepolymer with respect to the reduction of the viscosity illustrated REPs can effectively reduce the viscosity of the GAP prepolymer from 6,015 cP to 150–240 cP at the processing temperature when 50 wt‐% of REP was added. A comparison of the click reactivity and activation energies (E_a) of REPs and GAP prepolymer elucidated that the reactivity of azide‐alkyne cycloaddition depended on the dipolarophilicity of REPs which could be controlled by adjusting the length of methylene spacer between electron‐withdrawing groups (EWG) and neighboring alkynes in REPs. Thermogravimetric analysis manifested REP/GAP‐based PU binders maintained the thermal stability of the control GAP‐based PU binder. The mechanical properties and impact insensitivity of the GAP‐based PU binders were also improved by the incorporation of REPs.     

Preparation of ‘click’ hydrogels from polyaspartamide derivatives

Lately, copper‐assisted azide–alkyne cycloaddition (CuAAC) has become a very interesting tool for synthesizing biocompatible polymer‐based materials such as hydrogels or microgels, which can be used as biomaterials for tissue engineering and drug delivery. Novel poly(2‐hydroxyethyl aspartamide)s (PHEAs) functionalized with pendent acetylene or azide groups were prepared from polysuccinimide, which is the thermal polycondensation product of aspartic acid, through successful ring‐opening reactions using propargylamine, 1‐azido‐2‐aminoethane and ethanolamine. The composition of the prepared copolymers was analyzed using ¹H NMR spectroscopy. Clickable PHEA derivatives were crosslinked by mixing together in water with a catalyst system of Cu(I) and N, N, N′, N′, N″‐pentamethyldiethylenetriamine, a type of Huisgen's 1,3‐dipolar azide‐alkyne cycloaddition. The reaction of the polymers resulted in a chemoselective coupling between alkynyl and azido functional groups with multiple formation of triazole crosslinks to give hydrogels. The triazole linkages in the hydrogels are highly stable and may also play a role in swelling behavior. PHEA‐based hydrogels were also obtained by the crosslinking of azide‐ or alkyne‐modified PHEA with a small‐molecule crosslinker. The hydrogels prepared using these two methods were characterized by their degree of swelling and the morphology of the hydrogels was confirmed using scanning electron microscopy. The approach we describe here presents a promising alternative to common chemical hydrogel preparation techniques, and these hydrogels seem to possess structures having potential for a variety of industrial and biomedical applications. © 2012 Society of Chemical Industry     

Micelle‐Enhanced Bioorthogonal Labeling of Genetically Encoded Azido Groups on the Lipid‐Embedded Surface of a GPCR

Genetically encoded p‐azido‐phenylalanine (azF) residues in G protein‐coupled receptors (GPCRs) can be targeted with dibenzocyclooctyne‐modified (DIBO‐modified) fluorescent probes by means of strain‐promoted [3+2] azide–alkyne cycloaddition (SpAAC). Here we show that azF residues situated on the transmembrane surfaces of detergent‐solubilized receptors exhibit up to 1000‐fold rate enhancement relative to azF residues on water‐exposed surfaces. We show that the amphipathic moment of the labeling reagent, consisting of hydrophobic DIBO coupled to hydrophilic Alexa dye, results in strong partitioning of the DIBO group into the hydrocarbon core of the detergent micelle and consequently high local reactant concentrations. The observed rate constant for the micelleenhanced SpAAC is comparable with those of the fastest bioorthogonal labeling reactions known. Targeting hydrophobic regions of membrane proteins by use of micelle‐enhanced SpAAC should expand the utility of bioorthogonal labeling strategies.     

One‐Step Derivatization of Reducing Oligosaccharides for Rapid and Live‐Cell‐Compatible Chelation‐Assisted CuAAC Conjugation

We report a new reagent for the functionalization of unprotected oligosaccharides with a picolyl azide group at the anomeric position for chelation‐assisted copper‐catalyzed alkyne–azide cycloaddition (CuAAC) glycoconjugation. We show that oligosaccharides functionalized with this moiety react with an apparent second‐order rate constant of 193 m ^?1 s^?1 and can be used to functionalize biomolecules bearing alkyne moieties introduced through metabolic labeling, including in live cells.     

Biocompatible Azide–Alkyne “Click” Reactions for Surface Decoration of Glyco‐Engineered Cells

Bio‐orthogonal copper (I)‐catalyzed azide–alkyne cycloaddition (CuAAC) has been widely used to modify azide‐ or alkyne‐bearing monosaccharides on metabolic glyco‐engineered mammalian cells. Here, we present a systematic study to elucidate the design space for the cytotoxic effects of the copper catalyst on NIH 3T3 fibroblasts and on HEK 293‐F cells. Monitoring membrane integrity by flow cytometry and RT‐PCR analysis with apoptotic and anti‐apoptotic markers elucidated the general feasibility of CuAAC, with exposure time of the CuAAC reaction mixture having the major influence on biocompatibility. A high labeling efficiency of HEK 293‐F cells with a fluorescent alkyne dye was rapidly achieved by CuAAC in comparison to copper free strain‐promoted azide–alkyne cycloaddition (SPAAC). The study details effective and biocompatible conditions for CuAAC‐based modification of glyco‐engineered cells in comparison to its copper free alternative.     

Chemoselective Preparation of Clickable Aryl Sulfonyl Fluoride Monomers: A Toolbox of Highly Functionalized Intermediates for Chemical Biology Probe Synthesis

Sulfonyl fluoride (SF)‐based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF‐sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)‐mediated azide–alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.     

Fluorescent DNA Labeling by N‐Mustard Analogues of S‐Adenosyl‐L‐Methionine

Azide and alkyne‐functionalized N‐mustard analogues of S‐adenosyl‐L ‐methionine have been synthesized and were demonstrated to undergo efficient methyltransferase‐dependent DNA alkylation by M.TaqI and M.HhaI. Subsequent labeling of the DNA with a fluorophore was carried out using copper‐catalyzed azide–alkyne cycloaddition chemistry and was visualized by fluorescence scanning. This work demonstrates the utility of functionalized N‐mustard analogues as biochemical tools to study biological methylation and offers a facile way to site‐selectively label substrates of DNA methyltransferases.     

Synthesis and characterization of antimicrobial polylactide via ring‐opening polymerization and click chemistry methods

Novel biodegradable polylactide (PLA) copolymers bearing pendant antimicrobial agent groups were successfully fabricated with a combination of ring‐opening copolymerization and copper(I)‐catalysed azide–alkyne cycloaddition click reaction in a two‐step reaction procedure. First, biodegradable PLA copolymers bearing azido groups were synthesized by the ring‐opening copolymerization of l ‐lactide and 2,2‐ bis(azidomethyl)trimethylene carbonate in the presence of 1‐dodecanol as protic co‐initiator and tin(II) 2‐ethylhexanoate (Sn(Oct)₂) as the catalyst. Then, alkyne functionalized quaternary ammonium salts were attached onto the azido groups of the copolymers via a Huisgen 1,3‐dipolar cycloaddition reaction to give PLA imparting antimicrobial activity. The chemical structure and composition of the copolymers were clearly confirmed using ¹H NMR and Fourier transform infrared spectroscopies and gel permeation chromatography. Thermal phase transition temperatures (T_m and T_g) and the thermal stability of the polymers were investigated by DSC and TGA experiments, respectively. The antimicrobial activity tests were carried out against Gram‐negative (Escherichia coli) and Gram‐positive (Staphylococcus aureus) bacteria by the drop plate method. It was observed that antimicrobial agents are more active in the polymeric form than in the monomeric form. Also, the activity depends on the compositional ratio and the length of the alkyl group on the ammonium salts. © 2018 Society of Chemical Industry     

Quick Click: The DNA‐Templated Ligation of 3′‐O‐Propargyl‐ and 5′‐Azide‐Modified Strands Is as Rapid as and More Selective than Ligase

The copper(I)‐mediated azide–alkyne cycloaddition (CuAAC) of 3′‐propargyl ether and 5′‐azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate–sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA‐templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5 min, resulting in a k_obs value of 1.1 min^?1, comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating C:A or C:T mismatches from the C:G match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3′‐propargyl ether and 5′‐azide‐terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.     

Copper‐Free Click Reactions with Polar Bicyclononyne Derivatives for Modulation of Cellular Imaging

The ability of cells to incorporate azidosugars metabolically is a useful tool for extracellular glycan labelling. The exposed azide moiety can covalently react with alkynes, such as bicyclo[6.1.0]nonyne (BCN), by strain‐promoted alkyne–azide cycloaddition (SPAAC). However, the use of SPAAC can be hampered by low specificity of the cycloalkyne. In this article we describe the synthesis of more polar BCN derivatives and their properties for selective cellular glycan labelling. The new polar derivatives [amino‐BCN, glutarylamino‐BCN and bis(hydroxymethyl)‐BCN] display reaction rates similar to those of BCN and are less cell‐permeable. The labelling specificity in HEK293 cells is greater than that of BCN, as determined by confocal microscopy and flow cytometry. Interestingly, amino‐BCN appears to be highly specific for the Golgi apparatus. In addition, the polar BCN derivatives label the N‐glycan of the membrane calcium channel TRPV5 in HEK293 cells with significantly enhanced signal‐to‐noise ratios.     

Highly Active Dinuclear Copper Catalysts for Homogeneous Azide–Alkyne Cycloadditions

The main feature of the herein presented class of molecularly defined catalysts for the copper‐catalyzed azide–alkyne cycloaddition reaction is the presence of two copper centres in one catalyst molecule. We report the facile three‐step synthesis of two representative bis‐NHC‐dicopper complexes as well as their catalytic performance in the azide–alkyne cycloaddition. A screening with one of these complexes has proved its wide applicability and excellent performance as homogeneous catalyst in various organic solvents and with different alkyne and azide substrates.     

One‐pot synthesis of photosensitive dendrimer‐like polystyrenes from simultaneous copper(I)‐catalyzed azide–alkyne cycloaddition and atom transfer radical polymerization

Dendrimer‐like polystyrenes (PSs) containing azobenzene moieties/core were prepared by one‐pot copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) and atom transfer radical polymerization (ATRP) in the presence of PS with terminal azide groups, styrene monomers, 2,4‐dibromoisobutyrate‐4′‐propynyloxyazobenzene, copper(I) bromide and ligand. The successful preparation of the dendrimer‐like PSs was confirmed using gel permeation chromatography and ¹H NMR and Fourier transform infrared spectroscopy. The dendrimer‐like PSs prepared from one‐pot CuAAC and ATRP exhibit a well‐defined and controlled molecular structure in both whole macromolecules and the branches, which is attributed to the controlled character of ATRP and the quantitative yield of click chemistry. The dendrimer‐like PSs prepared also exhibit good photo‐isomerization properties due to the incorporation of azobenzene units within the dendrimers. Copyright © 2012 Society of Chemical Industry     

Soybean oil based thermoset networks via photoinduced CuAAC click chemistry

A simple method based on photochemically induced copper(I)‐catalyzed azide ? alkyne cycloaddition click reaction (CuAAC ) is developed for the preparation of thermoset networks from soybean oils as renewable resources. The incorporation of clickable azide and alkyne functionalities into epoxidized soybean oils is done by simultaneous ring‐opening reactions between the epoxide group of soybean oils and sodium azide and propargyl alcohol, respectively. The obtained azide‐ and alkyne‐functionalized soybean oils are easily transformed crosslinked networks via the photoinduced CuAAC reaction in ambient conditions. The introduction of additional multifunctional monomers in the formulation not only increases the crosslinking density but also improves the mechanical properties of the thermoset material obtained. In a comparison of the two formulations, the sample containing additional multifunctional monomers has a higher glass transition temperature, storage modulus and damping properties. © 2017 Society of Chemical Industry     

1,2,4‐Triazine‐Modified 2′‐Deoxyuridine Triphosphate for Efficient Bioorthogonal Fluorescent Labeling of DNA

In order to establish the Diels–Alder reaction with inverse electron demand for postsynthetic DNA modification, a 1,2,4‐triazine‐modified 2′‐deoxyuridine triphosphate was synthesized. The bioorthogonally reactive 1,2,4‐triazine group was attached at the 5‐position of 2′‐deoxyuridine by a flexible alkyl linker to facilitate its acceptance by DNA polymerases. The screening of four DNA polymerases showed successful primer extensions, using a mixture of dATP, dGTP, dCTP, and the modified 2′‐deoxyuridine triphosphate, by using KOD XL or Vent polymerase. The triazine moiety was stable under the conditions of primer extension, which was evidenced by labeling with a BCN‐modified rhodamine at room temperature in yields of up to 82 %. Two or three modified bases could be incorporated in quantitative yields when the modification sites were separated by three base pairs. These results establish the 1,2,4‐triazene group as a bioorthogonally reactive moiety in DNA, thereby replacing the problematic 1,2,4,5‐tetrazine for postsynthetic labeling by the Diels–Alder reaction with inverse electron demand.     

A Compact and Synthetically Accessible Fluorine‐18 Labelled Cyclooctyne Prosthetic Group for Labelling of Biomolecules by Copper‐Free Click Chemistry

A new fluorine‐containing azadibenzocyclooctyne (ADIBO‐F) was designed using a synthetically accessible pathway. The fluorine‐18 prosthetic group was prepared from its toluenesulfonate precursor and isolated in 21–35 % radiochemical yield in 30 minutes of synthetic time. ADIBO‐F has been incorporated into azide‐functionalized, cancer‐targeting peptides through a strain‐promoted alkyne–azide cycloaddition with high radiochemical yields and purities. The final products are novel peptide‐based positron emission tomography (PET) imaging agents that possess high affinities for their targets, growth hormone secretagogue receptor 1a (GHSR‐1a) and gastrin‐releasing peptide receptor (GRPR), with IC₅₀ values of 9.7 and 0.50 nm , respectively. This is a new and rapid labelling option for the incorporation of fluorine‐18 into biomolecules for PET imaging.     

Generation of a mono-ubiquitinated PCNA mimic by click chemistry

Genotoxic stress results in more than 50 000 damaged DNA sites per cell per day. During DNA replication, processive high‐fidelity DNA polymerases generally stall at DNA lesions and have to be displaced by translesion synthesis DNA polymerases, which are able to bypass the lesion. This switch is mediated by mono‐ubiquitination of the processivity factor proliferating cell nuclear antigen (PCNA). To further investigate the regulation of the DNA polymerase exchange, we developed an easy and efficient method to synthesize site‐specifically mono‐ubiquitinated PCNA by click chemistry. By incorporating artificial amino acids that carry an azide (Aha) or an alkyne (Plk) in their side chains, into ubiquitin (Ub) and PCNA, respectively, we were able to link the two proteins site‐specifically by the Cu^I‐catalyzed azide–alkyne cycloaddition. Finally, we show that the synthetic PCNA–Ub is able to stimulate DNA synthesis by DNA polymerase δ, and that DNA polymerase η has a higher affinity for PCNA–Ub than to PCNA.     

Direct azidation of isotactic polypropylene and synthesis of ‘grafted to’ derivatives thereof using azide–alkyne cycloaddition chemistry

Azido‐functionalized isotactic polypropylene was prepared via the direct C? H azidation of a commercially available polymer using a stable azidoiodinane. Including imidazole or benzimidazole in the reaction mixture was found to significantly improve the yields of the post‐polymerization modification. Although chain cleavage was observed, the methodology afforded high‐molecular‐weight (M _w > 100 kDa ) functionalized polypropylene containing up to 3 mol% of azido groups and enabled access to polypropylene‐graft ‐poly(ethylene glycol) copolymers via azide–alkyne cycloaddition chemistry. © 2016 Society of Chemical Industry