Click‐Assembled,Oxygen‐Sensing Nanoconjugates for Depth‐Resolved,Near‐Infrared Imaging in a 3 D Cancer Model

Hypoxia is an important contributing factor to the development of drug‐resistant cancer, yet few nonperturbative tools exist for studying oxygenation in tissues. While progress has been made in the development of chemical probes for optical oxygen mapping, penetration of such molecules into poorly perfused or avascular tumor regions remains problematic. A click‐assembled oxygen‐sensing (CAOS) nanoconjugate is reported and its properties demonstrated in an in vitro 3D spheroid cancer model. The synthesis relies on the sequential click‐based ligation of poly(amidoamine)‐like subunits for rapid assembly. Near‐infrared confocal phosphorescence microscopy was used to demonstrate the ability of the CAOS nanoconjugates to penetrate hundreds of micrometers into spheroids within hours and to show their sensitivity to oxygen changes throughout the nodule. This proof‐of‐concept study demonstrates a modular approach that is readily extensible to a wide variety of oxygen and cellular sensors for depth‐resolved imaging in tissue and tissue models.     

Ortho‐Stabilized 18F‐Azido Click Agents and their Application in PET Imaging with Single‐Stranded DNA Aptamers

Azido ¹⁸F‐arenes are important and versatile building blocks for the radiolabeling of biomolecules via Huisgen cycloaddition (“click chemistry”) for positron emission tomography (PET). However, routine access to such clickable agents is challenged by inefficient and/or poorly defined multistep radiochemical approaches. A high‐yielding direct radiofluorination for azido ¹⁸F‐arenes was achieved through the development of an ortho‐oxygen‐stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido ¹⁸F‐arene clickable agent for bioconjugation reactions. A ssDNA aptamer was radiolabeled with this agent and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates that this OID approach is a convenient and highly efficient way of labeling and tracking biomolecules.     

Metal‐ and solvent‐free,clickable synthesis and postpolymerization functionalization of poly(triazole)s

In this study, a series of linear poly(triazole)s (PTAs) were successfully synthesized by the metal‐ and solvent‐free, thermal click polymerization of diazide and dialkyne (A₂ + B₂) monomers. All click polymerizations proceeded smoothly at 80 °C in an open atmosphere without protection from oxygen and moisture. After being polymerized for 36 h, the crude polymer was further fractionated into three fractions using a multistep precipitation method. By selectively choosing precipitating agents, this process produced poly(triazole) fractions with low polydispersity index (<1.30). The resulting PTAs are soluble in common organic solvents and stable at a temperature up to 320 °C. Furthermore, the methyl benzoate moieties in the main chain can serve as useful building blocks for further postpolymerization functionalization, yielding 1,2,4‐triazole derivatives. This functionalization strategy offers potential for the development of novel triazole‐based materials. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Bright, “Clickable” Porphyrins for the Visualization of Oxygenation under Ambient Light

A new group of “clickable” and brightly emissive metalloporphyrins has been developed for the visualization of oxygenation under ambient light with the naked eye. These alkynyl‐terminated compounds permit the rapid and facile synthesis of oxygen‐sensing dendrimers through azide–alkyne click chemistry. With absorption maxima overlapping with the wavelengths of common commercial laser sources, they are readily applicable to biomedical imaging of tissue oxygenation. An efficient synthetic methodology, featuring the stable trimethylacetyl (pivaloyl) protecting group, is described for their preparation. A paint‐on liquid bandage containing a new, click‐synthesized porphyrin dendrimer has been used to map oxygenation across an ex vivo porcine skin burn model.     

Luminescent,Oxygen‐Supplying,Hemoglobin‐Linked Conjugated Polymer Nanoparticles for Photodynamic Therapy

Photodynamic therapy (PDT) is a promising method for cancer treatment. Two parameters that influence the efficacy of PDT are the light source and oxygen supply. Herein, we prepared a system for PDT using hemoglobin (Hb)‐linked conjugated polymer nanoparticles (CPNs), which can luminesce and supply oxygen. Hb catalyzes the activation of luminol, the conjugated polymer poly[2‐methoxy‐5‐(2‐ethylhexyloxy)‐1,4‐phenylenevinylene] (MEH–PPV) nanoparticles can absorb the chemiluminescence of luminol through chemiluminescence resonance energy transfer (CRET) and then sensitize the oxygen supplied by Hb to produce reactive oxygen species that kill cancer cells. This system could be used for the controlled release of an anticancer prodrug. The system does not need an external light source and circumvents the insufficient level molecular oxygen under hypoxia. This work provides a proof‐of‐concept to explore smart and multifunctional nanoplatforms for phototherapy.     

Luminescent,Oxygen‐Supplying,Hemoglobin‐Linked Conjugated Polymer Nanoparticles for Photodynamic Therapy

Photodynamic therapy (PDT) is a promising method for cancer treatment. Two parameters that influence the efficacy of PDT are the light source and oxygen supply. Herein, we prepared a system for PDT using hemoglobin (Hb)‐linked conjugated polymer nanoparticles (CPNs), which can luminesce and supply oxygen. Hb catalyzes the activation of luminol, the conjugated polymer poly[2‐methoxy‐5‐(2‐ethylhexyloxy)‐1,4‐phenylenevinylene] (MEH–PPV) nanoparticles can absorb the chemiluminescence of luminol through chemiluminescence resonance energy transfer (CRET) and then sensitize the oxygen supplied by Hb to produce reactive oxygen species that kill cancer cells. This system could be used for the controlled release of an anticancer prodrug. The system does not need an external light source and circumvents the insufficient level molecular oxygen under hypoxia. This work provides a proof‐of‐concept to explore smart and multifunctional nanoplatforms for phototherapy.     

Targeted Ultrasound‐Assisted Cancer‐Selective Chemical Labeling and Subsequent Cancer Imaging using Click Chemistry

Metabolic sugar labeling followed by the use of reagent‐free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N‐azidoacetylmannosamine (Ac₄ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac₄ManAz‐loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor‐localized ultrasound pulses. The released Ac₄ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)‐Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac₄ManAz‐loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer‐selective labeling and targeted cancer therapies.     

Regioselective Conversion of Arenes to N‐aryl‐1,2,3‐triazoles Using CH Borylation

A one‐pot protocol for the synthesis of N‐aryl 1,2,3‐triazoles from arenes by an iridium‐catalyzed C?H borylation/copper catalyzed azidation/click sequence is described. 1 mol % of Cu(OTf)₂ was found to efficiently catalyze both the azidation and the click reaction. The applicability of this method is demonstrated by the late‐stage chemoselective installation of 1,2,3‐triazole moiety into unactivated molecules of pharmaceutical importance.     

Tumor‐Microenvironment‐Induced Degradation of Ultrathin Gadolinium Oxide Nanoscrolls for Magnetic‐Resonance‐Imaging‐Monitored,Activatable Cancer Chemotherapy

The development of biodegradable inorganic nanoparticles with a tumor microenvironment‐activated therapeutic mode of action is urgently needed for precision cancer medicine. Herein, the synthesis of ultrathin lanthanide nanoscrolls (Gd₂O₃ NSs) is reported, which biodegrade upon encountering the tumor microenvironment. The Gd₂O₃ NSs showed highly controlled magnetic properties, which enabled their high‐resolution magnetic resonance imaging (MRI). Importantly, Gd₂O₃ NSs degrade in a pH‐responsive manner and selectively penetrate tumor tissue, enabling the targeted release of anti‐cancer drugs. Gd₂O₃ NSs can be efficiently loaded with an anti‐cancer drug (DOX, 80 %) and significantly inhibit tumor growth with negligible cellular and tissue toxicity both in vitro and in vivo. This study may provide a novel strategy to design tumor microenvironment‐responsive inorganic nanomaterials for biocompatible bioimaging and biodegradation‐enhanced cancer therapy.     

Tumor‐Microenvironment‐Induced Degradation of Ultrathin Gadolinium Oxide Nanoscrolls for Magnetic‐Resonance‐Imaging‐Monitored,Activatable Cancer Chemotherapy

The development of biodegradable inorganic nanoparticles with a tumor microenvironment‐activated therapeutic mode of action is urgently needed for precision cancer medicine. Herein, the synthesis of ultrathin lanthanide nanoscrolls (Gd₂O₃ NSs) is reported, which biodegrade upon encountering the tumor microenvironment. The Gd₂O₃ NSs showed highly controlled magnetic properties, which enabled their high‐resolution magnetic resonance imaging (MRI). Importantly, Gd₂O₃ NSs degrade in a pH‐responsive manner and selectively penetrate tumor tissue, enabling the targeted release of anti‐cancer drugs. Gd₂O₃ NSs can be efficiently loaded with an anti‐cancer drug (DOX, 80 %) and significantly inhibit tumor growth with negligible cellular and tissue toxicity both in vitro and in vivo. This study may provide a novel strategy to design tumor microenvironment‐responsive inorganic nanomaterials for biocompatible bioimaging and biodegradation‐enhanced cancer therapy.     

Bioorthogonal Turn‐On Probe Based on Aggregation‐Induced Emission Characteristics for Cancer Cell Imaging and Ablation

Bioorthogonal turn‐on probes have been widely utilized in visualizing various biological processes. Most of the currently available bioorthogonal turn‐on probes are blue or green emissive fluorophores with azide or tetrazine as functional groups. Herein, we present an alternative strategy of designing bioorthogonal turn‐on probes based on red‐emissive fluorogens with aggregation‐induced emission characteristics (AIEgens). The probe is water soluble and non‐fluorescent due to the dissipation of energy through free molecular motion of the AIEgen, but the fluorescence is immediately turned on upon click reaction with azide‐functionalized glycans on cancer cell surface. The fluorescence turn‐on is ascribed to the restriction of molecular motion of AIEgen, which populates the radiative decay channel. Moreover, the AIEgen can generate reactive oxygen species (ROS) upon visible light (λ=400–700 nm) irradiation, demonstrating its dual role as an imaging and phototherapeutic agent.     

Click Chemistry Route to the Synthesis of Unusual Amino Acids,Peptides, Triazole‐Fused Heterocycles and Pseudodisaccharides

Conjugation of different molecular species using copper(I)‐catalyzed click reaction between azides and terminal alkynes is among the best available methods to prepare multifunctional compounds. The effectiveness of this method has provided wider acceptance to the concept of click chemistry, which is now widely employed to synthesize densely functionalized organic molecules. This article summarizes the contributions from our group in the development of new methods for the synthesis of functional molecules using copper(I)‐catalyzed click reactions. We have developed very efficient methods for the synthesis of peptides and amino acids conjugated with carbohydrates, thymidine and ferrocene. We have also developed an efficient strategy to synthesize triazole‐fused heterocycles from primary amines, amino alochols and diols. Finally, an interesting method for the synthesis of pseudodisaccharides linked through triazoles, starting from carbohydrate‐derived donor‐acceptor cyclopropanes is discussed.     

A One‐Pot Tandem Ugi Multicomponent Reaction (MCR)/Click Reaction as an Efficient Protecting‐Group‐Free Route to 1H‐1,2,3‐Triazole‐Modified α‐Amino Acid Derivatives and Peptidomimetics

By a one‐pot tandem Ugi multicomponent reaction (MCR)/click reaction sequence not requiring protecting groups, 1H‐1,2,3‐triazole‐modified Ugi‐reaction products 6a – 6n (Scheme 1 and Table 2), 7a – 7b (Table 4), and 8 (Scheme 2) were synthesized successfully. i.e., terminal, side‐chain, or both side‐chain and terminal triazole‐modified Ugi‐reaction products as potential amino acid units for peptide syntheses. Different catalyst systems for the click reaction were examined to find the optimal reaction conditions (Table 1, Scheme 1). Finally, an efficient Ugi MCR+Ugi MCR/click reaction strategy was elaborated in which two Ugi‐reaction products were coupled by a click reaction, thus incorporating the triazole fragment into the center of peptidomimetics (Scheme 3). Thus, the Ugi MCR/click reaction sequence is a convenient and simple approach to different 1H‐1,2,3‐triazole‐modified amino acid derivatives and peptidomimetics.     

Targeted Imaging and Proteomic Analysis of Tumor‐Associated Glycans in Living Animals

Although it has been well known that dynamic changes in glycosylation are associated with tumor progression, it remains challenging to selectively visualize the cancer glycome in vivo. Herein, a strategy for the targeted imaging of tumor‐associated glycans by using ligand‐targeted liposomes encapsulating azidosugars is described. The intravenously injected liposomal nanoparticles selectively bound to the cancer‐cell‐specific receptors and installed azides into the melanoma glycans in a xenograft mouse model in a tissue‐specific manner. Subsequently, a copper‐free click reaction was performed in vivo to chemoselectively conjugate the azides with a near‐infrared fluorescent dye. The glycosylation dynamics during tumor growth were monitored by in vivo fluorescence imaging. Furthermore, the newly synthesized sialylated glycoproteins were enriched during tumor growth and identified by glycoproteomics. Compared with the labeling methods using free azidosugars, this method offers improved labeling efficiency and high specificity and should facilitate the elucidation of the functional role of glycans in cancer biology.     

The [2+2] Cycloaddition‐Retroelectrocyclization (CA‐RE) Click Reaction: Facile Access to Molecular and Polymeric Push‐Pull Chromophores

The [2+2] cycloaddition‐retroelectrocyclization (CA‐RE) reaction between electron‐rich alkynes and electron‐deficient alkenes is an efficient procedure to create nonplanar donor–acceptor (D‐A) chromophores in both molecular and polymeric platforms. They feature attractive properties including intramolecular charge‐transfer (ICT) bands, nonlinear optical properties, and redox activities for use in next‐generation electronic and optoelectronic devices. This Review summarizes the development of the CA‐RE reaction, starting from the initial reports with organometallic compounds to the extension to purely organic systems. The structural requirements for rapid, high‐yielding transformations with true click chemistry character are illustrated by examples that include the broad alkyne and alkene substitution modes. The CA‐RE click reaction has been successfully applied to polymer synthesis, with the resulting polymeric push‐pull chromophores finding many interesting applications.     

A Versatile Iron–Tannin‐Framework Ink Coating Strategy to Fabricate Biomass‐Derived Iron Carbide/Fe‐N‐Carbon Catalysts for Efficient Oxygen Reduction

The conversion of biomass into valuable carbon composites as efficient non‐precious metal oxygen‐reduction electrocatalysts is attractive for the development of commercially viable polymer electrolyte membrane fuel‐cell technology. Herein, a versatile iron–tannin‐framework ink coating strategy is developed to fabricate cellulose‐derived Fe₃C/Fe‐N‐C catalysts using commercial filter paper, tissue, or cotton as a carbon source, an iron–tannin framework as an iron source, and dicyandiamide as a nitrogen source. The oxygen reduction performance of the resultant Fe₃C/Fe‐N‐C catalysts shows a high onset potential (i.e. 0.98 V vs the reversible hydrogen electrode (RHE)), and large kinetic current density normalized to both geometric electrode area and mass of catalysts (6.4 mA cm^?2 and 32 mA mg^?1 at 0.80 V vs RHE) in alkaline condition. This method can even be used to prepare efficient catalysts using waste carbon sources, such as used polyurethane foam.     

3‐miktoarm star terpolymers using triple click reactions: Diels–Alder,copper‐catalyzed azide‐alkyne cycloaddition,and nitroxide radical coupling reactions

Well‐defined linear furan‐protected maleimide‐terminated poly(ethylene glycol) (PEG‐MI), tetramethylpiperidine‐1‐oxyl‐terminated poly(ε‐caprolactone) (PCL‐TEMPO), and azide‐terminated polystyrene (PS‐N₃) or ‐poly(N‐butyl oxanorbornene imide) (PONB‐N₃) were ligated to an orthogonally functionalized core ( 1 ) in a two‐step reaction mode through triple click reactions. In a first step, Diels–Alder click reaction of PEG‐MI with 1 was performed in toluene at 110 °C for 24 h to afford α‐alkyne‐α‐bromide‐terminated PEG (PEG‐alkyne/Br). As a second step, this precursor was subsequently ligated with the PCL‐TEMPO and PS‐N₃ or PONB‐N₃ in N,N‐dimethylformamide at room temperature for 12 h catalyzed by Cu(0)/Cu(I) through copper‐catalyzed azide‐alkyne cycloaddition and nitroxide radical coupling click reactions, yield resulting ABC miktoarm star polymers in a one‐pot mode. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Epitope Targeting of Tertiary Protein Structure Enables Target‐Guided Synthesis of a Potent In‐Cell Inhibitor of Botulinum Neurotoxin

Botulinum neurotoxin (BoNT) serotype A is the most lethal known toxin and has an occluded structure, which prevents direct inhibition of its active site before it enters the cytosol. Target‐guided synthesis by in situ click chemistry is combined with synthetic epitope targeting to exploit the tertiary structure of the BoNT protein as a landscape for assembling a competitive inhibitor. A substrate‐mimicking peptide macrocycle is used as a direct inhibitor of BoNT. An epitope‐targeting in situ click screen is utilized to identify a second peptide macrocycle ligand that binds to an epitope that, in the folded BoNT structure, is active‐site‐adjacent. A second in situ click screen identifies a molecular bridge between the two macrocycles. The resulting divalent inhibitor exhibits an in vitro inhibition constant of 165 pM against the BoNT/A catalytic chain. The inhibitor is carried into cells by the intact holotoxin, and demonstrates protection and rescue of BoNT intoxication in a human neuron model.     

Regenerable‐Catalyst‐Aided,Opened to Air and Sunlight‐Driven “CuAAC&ATRP” Concurrent Reaction for Sequence‐Controlled Copolymer

An ideal stimuli‐responsive controlled/living radical polymerization should have the ability to manipulate the reaction through spatiotemporal “on/off” controls, achieving the polymerization under fully open conditions and allowing for precise control over macromolecular architecture with defined molecular weights and monomer sequence. In this contribution, the photo (sunlight)‐induced electron transfer atom transfer radical‐polymerization (PET‐ATRP) can be realized to be reversibly activated and deactivated under fully open conditions utilizing one‐component copper(II) thioxanthone carboxylate as multifunctional photocatalyst and oxygen scavenger. The polymerization behaviors are investigated, presenting controlled features with first‐order kinetics and linear relationships between molecular weights and monomer conversions. More importantly, “CuAAC&ATRP” concurrent reaction combining PET‐ATRP, photodriven deoxygenation, and photoactivated CuAAC click reaction is successfully employed to synthesize the sequence‐defined multiblock functional copolymers, in which the iterative monomer additions can be easily manipulated under fully open conditions.     

Minimal Tags for Rapid Dual‐Color Live‐Cell Labeling and Super‐Resolution Microscopy

The growing demands of advanced fluorescence and super‐resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue‐specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine‐functionalized organic dyes by the inverse‐electron‐demand Diels–Alder cycloaddition (SPIEDAC). Furthermore, we fine‐tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus‐like particles (VLPs) with dual‐color super‐resolution microscopy.