Hydrophilic trans‐Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling

Introduction of bioorthogonal functionalities (e.g., trans‐cyclooctene‐TCO) into a protein of interest by site‐specific genetic encoding of non‐canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine‐functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours‐long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO‐ncAAs. One derivative, DOTCO‐lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.     

Cycloadditions of Trans-Cyclooctenes and Nitrones as Tools for Bioorthogonal Labelling

Trans-cyclooctenes (TCOs) represent interesting and highly reactive dipolarophiles for organic transformations including bioorthogonal chemistry. Herein we show that TCOs react rapidly with nitrones and that these reactions are bioorthogonal. Kinetic analysis of acyclic and cyclic nitrones with strained-trans-cyclooctene (s-TCO) shows fast reactivity and demonstrates the utility of this cycloaddition reaction for bioorthogonal labelling. Labelling of the bacterial peptidoglycan layer with unnatural d -amino acids tagged with nitrones and s-TCO-Alexa488 is demonstrated. These new findings expand the bioorthogonal toolbox, and allow TCO reagents to be used in bioorthogonal applications beyond tetrazine ligations for the first time and open up new avenues for bioorthogonal ligations with diverse nitrone reactants.     

Biomimetic Synthesis of a Water‐Soluble Conducting Polymer of 3,4‐Ethylene‐dioxythiophene

A novel biomimetic route for the synthesis of a water‐soluble poly(3,4‐ethylenedioxithiophene) (PEDT) in the presence of poly(styrene sulfonate) (PSS) and using iron(III)‐tetra(p‐sulfonatophenyl)porphyrin (Fe^IIITSPP), cobalt(III)‐tetra(p‐sulfonatophenyl)porphyrin (Co^IIITSPP), manganese(III)‐tetra(p‐sulfonatophenyl)porphyrin (Mn^IIITSPP), and copper(II)‐tetra(p‐sulfonatophenyl)porphyrin (Cu^IITSPP) as effective catalysts is presented. The reactions were performed with different monomer, catalyst, template, and initiator concentrations. The absorbance of the polaron bands at various pH values indicated pH 2 as the best condition for polymerization. Precipitation or salting out was highly dependent on the mentioned factors. The formation of PEDT was confirmed by UV‐Vis and FT‐IR spectroscopy. Cyclic voltammetry proved the convenient electroactivity of the synthesized polymer. The presence of PSS that serves as a charge‐compensating dopant provides processability and water solubility and, in addition, a distinct advantage over similar reactions employing native enzymes due to higher stability and lower cost of the catalysts.     

Non‐Enzymatic Geometry‐Selective Acylation of Tri‐ and Tetrasubstituted α,α′‐Alkenediols

A highly geometry‐selective organocatalytic acylation of tri‐ and tetra‐substituted 2‐alkylidene‐1,3‐propanediols has been developed. The highly E‐selective acylation of various tetrasubstituted 2‐alkylidene‐1,3‐propanediols was achieved in 96 to >99% selectivity for the first time by a non‐enzymatic protocol.     

Coordination‐Assisted Bioorthogonal Chemistry: Orthogonal Tetrazine Ligation with Vinylboronic Acid and a Strained Alkene

Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse‐electron‐demand Diels–Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl‐s‐tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA–tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit‐selective VBA bearing a norbornene moiety.     

Ultrastructural Imaging of Salmonella–Host Interactions Using Super‐resolution Correlative Light‐Electron Microscopy of Bioorthogonal Pathogens

The imaging of intracellular pathogens inside host cells is complicated by the low resolution and sensitivity of fluorescence microscopy and by the lack of ultrastructural information to visualize the pathogens. Herein, we present a new method to visualize these pathogens during infection that circumvents these problems: by using a metabolic hijacking approach to bioorthogonally label the intracellular pathogen Salmonella Typhimurium and by using these bioorthogonal groups to introduce fluorophores compatible with stochastic optical reconstruction microscopy (STORM) and placing this in a correlative light electron microscopy (CLEM) workflow, the pathogen can be imaged within its host cell context Typhimurium with a resolution of 20 nm. This STORM‐CLEM approach thus presents a new approach to understand these pathogens during infection.     

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.     

Light‐triggered reversible solubility of α‐cyclodextrin and azobenzene moiety complexes in PDMAA‐co‐PAPA via molecular recognition

Photoresponsive polymer with azobenzene pendant group (PDMAA‐co‐PAPA) was synthesized by radical polymerization of N,N‐dimethylacrylamide (DMAA) and N‐4‐phenylazophenyl acrylamide (PAPA), and the characterization of the inclusion complexes of the PDMAA‐co‐PAPA with α‐cyclodextrin (α‐CD) were performed by FTIR, GPC, ¹H NMR, 2D NOESY, and UV–vis spectroscopy. It was found that the solubility of PDMAA‐co‐PAPA and α‐CD inclusion complexes in aqueous solution showed tunable property, which could be triggered by alternating UV–vis light irradiation at a certain temperature due to the effect of molecular recognition of α‐CD with azobenzene moiety in the polymer. After UV irradiation, the lower critical solution temperature (LCST) of the polymer aqueous solution increased slightly without α‐CD while the LCST decreased sharply at presence of α‐CD. Furthermore, UV spectroscopy showed that the photoisomerization of the polymer solution went on rapidly and reversibly, and 2D NOESY data suggested that the inclusion complexation of α‐CD with trans azobenzene moiety and the decomplexation with cis azobenzene resulted in reversible solubility behavior when objected to UV and Vis light irradiation alternately. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Synthesis and Characterization of N‐Heterocyclic Carbene Substituted Phosphine and Phosphite Rhodium Complexes and their Catalytic Properties in Hydrogenation Reactions

Mixed N‐heterocyclic carbene‐substituted phosphine and phosphite complexes of rhodium were prepared, starting from [Rh(COE)₂Cl]₂ (COE=cyclooctene) by addition of free N‐heterocyclic carbenes (NHC) and PR₃. All new complexes were characterized by spectroscopy. In addition, the structures of trans‐chloro(1,3‐dicyclohexylimidazol‐2‐ylidene)‐bis(triphenylphosphite)rhodium(I) ( 5 ), chloro‐trans‐bis(1,3‐dicyclohexylimidazol‐2‐ylidene) (triphenylphosphine)rhodium(I) ( 6 ), and chloro(η⁴‐1,5‐cyclooctadiene)(1,3‐di‐[(1R,2S,5R)‐2‐isopropyl‐5‐menthylcyclohex‐1‐yl]imidazol‐2‐ylidene)rhodium(I) ( 8 ) were determined by single crystal X‐ray analyses. The hydrogenation of cyclohexene using molecular hydrogen has been optimized for some N‐heterocyclic carbene‐substituted phosphine and phosphite rhodium complexes by variation of the reaction conditions.     

Synthesis of Chiral N‐Sulfonyl and N‐Phosphinoyl α‐Halo Aldimine Precursors

α‐Halogenated aldimines have emerged as an important class of synthetic intermediates. The stability and reactivity of α‐halo aldimines can vary greatly depending on the nitrogen protecting group. A general synthesis of stable, chiral α‐halo‐N‐sulfonyl and N‐phosphinoyl aldimine precursors is presented (42–96% yield). The corresponding α‐halo aldimines can be isolated upon treatment with a mild base. Enantioenriched α‐chloro aldehydes can be employed to afford aldimine precursors with no erosion of optical purity. Both the enantioenriched aldimine precursor and the isolated aldimine can react with an alkynyllithium nucleophile to give trans‐β‐chloroamine products with excellent dr. Ring closure affords the enantioenriched trans‐aziridine, demonstrating the potential for this approach in complex molecule synthesis.     

PNA Molecular Beacons Assembled by Post‐Synthetic Click Chemistry Functionalization

To avoid the tedious synthesis of functionalized peptide nucleic acid (PNA) monomers for probe development, we proposed a simple approach to modify PNA oligomers by post‐synthetic on‐resin click chemistry. PNA molecular beacons (MBs) were prepared by incorporation of azide‐containing monomers into the oligomer by automatic solid‐phase peptide synthesis and subsequent derivatization with pyrene moieties by copper‐catalyzed azide–alkyne cycloaddition. Two pyrene‐based quencher‐free PNA molecular beacons, a stemless MB and one possessing a stem–loop structure, targeting a portion of the cystic fibrosis gene, were successfully synthesized by using this method. Fluorescence studies showed that the stem–loop MB exhibited better discrimination of changes in excimer/monomer ratios as compared to the stemless MB construct.     

Flow Photochemical Syntheses of trans-Cyclooctenes and trans-Cycloheptenes Driven by Metal Complexation

trans-Cyclooctenes and trans-cycloheptenes have long been the subject of physical organic study, but the broader application had been limited by synthetic accessibility. This account describes the development of a general, flow photochemical method for the preparative synthesis of trans-cycloalkene derivatives. Here, photoisomerization takes place in a closed-loop flow reactor where the reaction mixture is continuously cycled through Ag(I) on silicagel. Selective complexation of the trans-isomer by Ag(I) during flow drives an otherwise unfavorable isomeric ratio toward the trans-isomer. Analogous photoreactions under batch-conditions are low yielding, and flow chemistry is necessary in order to obtain trans-cycloalkenes in preparatively useful yields. The applications of the method to bioorthogonal chemistry and stereospecific transannulation chemistry are described.     

Well‐Defined End‐Functionalized Conjugated Polymers/Oligomers Exhibiting Unique Emission Properties through the End Groups: The Exclusive Synthesis by Combined Olefin Metathesis with Wittig‐type Coupling

Acyclic diene metathesis polymerization using ruthenium–carbene catalysts affords defect‐free, high molecular weight poly(arylene vinylene)s containing all trans olefinic double bonds. The exclusive end‐functionalization in the resultant poly(fluorene vinylene)s or poly(phenylene vinylene)s can be attained by treating the vinyl end groups using a molybdenum–alkylidene catalyst/reagent (through olefin metathesis) followed by addition of various aldehydes (Wittig‐type coupling). Some of these end‐modified conjugated materials display unique emission properties, which are different from the original ones, through an interaction (energy transfer or structural change in the excited state) between the conjugated main chain and the end groups [oligo(thiophene)s, F‐BODIPY, etc.]. Exclusive synthesis of well‐defined, all‐trans end‐functionalized oligo(2,5‐dialkoxy‐1,4‐phenylene vinylene)s [(oligo(phenylene vinylene), alkoxy = O(CH₂)₂OSiⁱ Pr₃, up to 31 repeat units] is demonstrated by adopting a stepwise synthetic approach (olefin metathesis and the subsequent Wittig‐type cleavage). It is clearly demonstrated that their optical properties (especially the fluorescence spectra including photoluminescence quantum yields) are strongly affected by the end groups as well as the conjugation repeat units.     

Convenient Approach to 3,4‐Diarylisoxazoles Based on the Suzuki Cross‐Coupling Reaction

The Suzuki cross‐coupling reaction was found effective for rapid access to a series of 3,4‐diarylisoxazoles of pharmacological interest. The efficiency of this approach was demonstrated by the synthesis of the highly potent COX‐2‐selective inhibitor, 4‐(5‐methyl‐3‐phenyl‐4‐isoxazolyl)benzenesulfonamide (valdecoxib), and its analogues. Thus, the coupling reaction between (3‐aryl‐5‐methyl‐4‐isoxazolyl)boronic acids, prepared in situ from the corresponding bromides using triisopropyl borate, and aryl bromides containing a 4‐sulfonamide or 4‐methylsulfonyl group under the standard conditions [Pd(PPh₃)₄, Na₂CO₃, EtOH‐H₂O, reflux] yielded the target 3,4‐diarylisoxazoles in good yields.     

Fragment‐Based Phenotypic Lead Discovery: Cell‐Based Assay to Target Leishmaniasis

A rapid and practical approach for the discovery of new chemical matter for targeting pathogens and diseases is described. Fragment‐based phenotypic lead discovery (FPLD) combines aspects of traditional fragment‐based lead discovery (FBLD), which involves the screening of small‐molecule fragment libraries to target specific proteins, with phenotypic lead discovery (PLD), which typically involves the screening of drug‐like compounds in cell‐based assays. To enable FPLD, a diverse library of fragments was first designed, assembled, and curated. This library of soluble, low‐molecular‐weight compounds was then pooled to expedite screening. Axenic cultures of Leishmania promastigotes were screened, and single hits were then tested for leishmanicidal activity against intracellular amastigote forms in infected murine bone‐marrow‐derived macrophages without evidence of toxicity toward mammalian cells. These studies demonstrate that FPLD can be a rapid and effective means to discover hits that can serve as leads for further medicinal chemistry purposes or as tool compounds for identifying known or novel targets.     

Biomedical applications of tetrazine cycloadditions

Disease mechanisms are increasingly being resolved at the molecular level. Biomedical success at this scale creates synthetic opportunities for combining specifically designed orthogonal reactions in applications such as imaging, diagnostics, and therapy. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k > 10(3) M(-1) s(-1)) and high specificity. Improving kinetics would minimize both the time and amount of labeling agent required to maintain high coupling yields. In this Account, we discuss our recent efforts to design extremely rapid bioorthogonal coupling reactions between tetrazines and strained alkenes. These selective reactions were first used to covalently couple conjugated tetrazine near-infrared-emitting fluorophores to dienophile-modifed extracellular proteins on living cancer cells. Confocal fluorescence microscopy demonstrated efficient and selective labeling, and control experiments showed minimal background fluorescence. Multistep techniques were optimized to work with nanomolar concentrations of labeling agent over a time scale of minutes: the result was successful real-time imaging of covalent modification. We subsequently discovered fluorogenic probes that increase in fluorescence intensity after the chemical reaction, leading to an improved signal-to-background ratio. Fluorogenic probes were used for intracellular imaging of dienophiles. We further developed strategies to react and image chemotherapeutics, such as trans-cyclooctene taxol analogues, inside living cells. Because the coupling partners are small molecules (<300 Da), they offer unique steric advantages in multistep amplification. We also describe recent success in using tetrazine reactions to label biomarkers on cells with magneto-fluorescent nanoparticles. Two-step protocols that use bioorthogonal chemistry can significantly amplify signals over both one-step labeling procedures as well as two-step procedures that use more sterically hindered biotin-avidin interactions. Nanoparticles can be detected with fluorescence or magnetic resonance techniques. These strategies are now being routinely used on clinical samples for biomarker profiling to predict malignancy and patient outcome. Finally, we discuss recent results with tetrazine reactions used for in vivo molecular imaging applications. Rapid tetrazine cycloadditions allow modular labeling of small molecules with the most commonly used positron emission tomography isotope, (18)F. Additionally, recent work has applied this reaction directly in vivo for the pretargeted imaging of solid tumors. Future work with tetrazine cycloadditions will undoubtedly lead to optimized protocols, improved probes, and additional biomedical applications.     

An Azide‐Modified Nucleoside for Metabolic Labeling of DNA

Metabolic incorporation of azido nucleoside analogues into living cells can enable sensitive detection of DNA replication through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) and strain‐promoted azide–alkyne cycloaddition (SPAAC) “click” reactions. One major limitation to this approach is the poor chemical stability of nucleoside derivatives containing an aryl azide group. For example, 5‐azido‐2′‐deoxyuridine (AdU) exhibits a 4 h half‐life in water, and it gives little or no detectable labeling of cellular DNA. In contrast, the benzylic azide 5‐(azidomethyl)‐2′‐deoxyuridine (AmdU) is stable in solution at 37 °C, and it gives robust labeling of cellular DNA upon addition of fluorescent alkyne derivatives. In addition to providing the first examples of metabolic incorporation into and imaging of azide groups in cellular DNA, these results highlight the general importance of assessing azide group stability in bioorthogonal chemical reporter strategies.     

Crystallization Behavior of Molecular Compound in Binary Mixture System of 1,3‐Dioleoyl‐2‐Palmitoyl‐sn‐Glycerol and 1,3‐Dipalmitoyl‐2‐Oleoyl‐sn‐Glycerol

Previous studies showed that the stable β‐form of molecular compound (MC) crystals having a double‐chain‐length structure is formed in a binary mixture system of 1,3‐dioleoyl‐2‐palmitoyl‐sn‐glycerol (OPO) and 1,3‐dipalmitoyl‐2‐oleoyl‐sn‐glycerol (POP) with a 1:1 concentration ratio of OPO and POP. The use of MC crystals made of POP and OPO for edible applications, such as margarine, is advantageous due to no‐trans, low‐saturated, and high‐oleic fats. Industrial manufacturing technology involves rapid cooling processes, and the kinetic properties of crystallization of MC of OPO and POP are required. In this study, we clarified the crystallization of MC of OPO and POP under rapid cooling at rates of 1–150 °C min^?1, using synchrotron radiation time‐resolved X‐ray diffraction and differential scanning calorimetry methods. The main results are as follows: (1) POP and OPO crystallized in separate manners without the formation of MC crystals under rapid cooling (>40 °C min^?1), while MC crystals started to form with decreasing rates of cooling in addition to the POP and OPO crystals (<30 °C min^?1); (2) metastable and stable forms sub‐α, α, β′, and β of POP and OPO were formed, whereas the MC crystals of β were formed during the cooling processes; and (3) the heating processes after crystallization by rapid cooling caused separate melting of the metastable and stable forms of POP and OPO crystals and the formation of MC crystals of β made of POP and OPO, as well as melting of the MC crystals alone.     

Fluorescent hydroxyapatite‐loaded biodegradable polymer nanoparticles with folate decoration for targeted imaging

The preparation of the luminescent hydroxyapatite (HAP)‐loaded biocompatible nanoparticles (NPs) for targeted imaging of cancer cells is described. Currently, cellular imaging using fluorescent probes is an important technique for the early diagnosis of cancer. Compared with the quantum dots, luminescent HAP is a new fluorescent material with many advantages such as low toxicity, biocompatibility, thermal stability, resistance to erosion, and low prices. Thus, luminescent HAP has enormous potential to be used as biological fluorescent probes. However, luminescent HAP is water‐insoluble, low sensitivity, which limit its application in the field of cellular imaging. Surface modification of NPs with targeting molecule was carried out to achieve its target function. Thus, novel fluorescent NPs with low toxicity, high sensitivity, and good photostability were prepared to be used for targeted imaging of cancer cells. This study initially explored the applications of luminescent HAP in the field of targeted cellular imaging. This NPs platform will be a promising tool for molecular imaging and medical diagnostics, especially the detection of cancer at its early stage. © 2013 American Institute of Chemical Engineers AIChE J, 59: 4494–4501, 2013     

Integration of on‐the‐fly kinetic reduction with multidimensional CFD

A reduction approach for coupling complex kinetics with engine computational fluid dynamics (CFD) code has been developed. An on‐the‐fly reduction scheme was used to reduce the reaction mechanism dynamically during the reactive flow calculation in order to couple comprehensive chemistry with flow simulations in each computational cell. KIVA‐3V code is used as the CFD framework and CHEMKIN is employed to formulate chemistry, hydrodynamics and transport. Mechanism reduction was achieved by applying element flux analysis on‐the‐fly in the context of the multidimensional CFD calculation. The results show that incorporating the on‐the‐fly reduction approach in CFD code enables the simulation of ignition and combustion process accurately compared with detailed simulations. Both species and time‐dependant information can be provided by the current model with significantly reduced CPU time. © 2009 American Institute of Chemical Engineers AIChE J, 2010