Reducing Product Inhibition in Nucleic Acid‐Templated Ligation Reactions: DNA‐Templated Cycligation

Programmable interactions allow nucleic acid molecules to template chemical reactions by increasing the effective molarities of appended reactive groups. DNA/RNA‐triggered reactions can proceed, in principle, with turnover in the template. The amplification provided by the formation of many product molecules per template is a valuable asset when the availability of the DNA or RNA target is limited. However, turnover is usually impeded by reaction products that block access to the template. Product inhibition is most severe in ligation reactions, where products after ligation have dramatically increased template affinities. We introduce a potentially generic approach to reduce product inhibition in nucleic acid‐programmed ligation reactions. A DNA‐triggered ligation–cyclization sequence (“cycligation”) of bifunctional peptide nucleic acid (PNA) conjugates affords cyclic ligation products. Melting experiments revealed that product cyclization is accompanied by a pronounced decrease in template affinity compared to linear ligation products. The reaction system relies upon haloacetylated PNA‐thioesters and isocysteinyl‐PNA‐cysteine conjugates, which were ligated on a DNA template according to a native chemical ligation mechanism. Dissociation of the resulting linear product‐template duplex (induced by, for example, thermal cycling) enabled product cyclization through sulfur‐halide substitution. Both ligation and cyclization are fast reactions (ligation: 86 % yield after 20 min, cyclization: quantitative after 5 min). Under thermocycling conditions, the DNA template was able to trigger the formation of new product molecules when fresh reactants were added. Furthermore, cycligation produced 2–3 times more product than a conventional ligation reaction with substoichiometric template loads (0.25–0.01 equiv). We believe that cyclization of products from DNA‐templated reactions could ultimately afford systems that completely overcome product inhibition.     

Model systems for activation of nucleic acid encoded prodrugs

The development of more selective chemotherapeutic agents for benign treatments of malicious diseases is highly desirable. In recent years model systems for the release of small molecule drugs from nucleic acid conjugates by templated chemical or photochemical reactions have been designed. Common for these systems is that the stoichiometric or catalytic drug release is controlled by the highly selective hybridization between complementary strands of nucleic acids. Herein, the concepts of the new field of nucleic acid templated release reactions are outlined.     

Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates

A novel peptide–peptide ligation strategy is introduced that has the potential to provide peptide libraries of linearly or branched coupled fragments and will be suited to introduce simultaneous protein modifications at different ligation sites. Ligation is assisted by templating peptide nucleic acid (PNA) strands, and therefore, ligation specificity is solely encoded by the PNA sequence. PNA templating, in general, allows for various kinds of covalent ligation reactions. As a proof of principle, a native chemical ligation strategy was elaborated. This PNA‐templated ligation includes easy on‐resin procedures to couple linkers and PNA to the respective peptides, and a traceless photocleavage of the linker/PNA oligomer after the ligation step. A 4,5‐dimethoxy‐2‐nitrobenzaldehyde‐based linker that allowed the photocleavable linkage of two bio‐oligomers was developed.     

Thermodynamic analysis of nylon nucleic acids

The stability and structure of nylon nucleic acid duplexes with complementary DNA and RNA strands was examined. Thermal denaturing studies of a series of oligonucleotides that contained nylon nucleic acids (1-5 amide linkages) revealed that the amide linkage significantly enhanced the binding affinity of nylon nucleic acids towards both complementary DNA (up to 26 degrees C increase in the thermal transition temperature (T(m)) for five linkages) and RNA (around 15 degrees C increase in T(m) for five linkages) compared with nonamide linked precursor strands. For both DNA and RNA complements, increasing derivatization decreased the melting temperatures of uncoupled molecules relative to unmodified strands; by contrast, increasing lengths of coupled copolymer raised T(m) from less to slightly greater than T(m) of unmodified strands. Thermodynamic data extracted from melting curves and CD spectra of nylon nucleic acid duplexes were consistent with loss of stability due to incorporation of pendent groups on the 2'-position of ribose and recovery of stability upon linkage of the side chains.     

Integrating Ligands into Nucleic Acid Systems

Signal transduction from non-nucleic acid ligands (small molecules and proteins) to structural changes of nucleic acids plays a crucial role in both biomedical analysis and cellular regulations. However, how to bridge between these two types of molecules without compromising the expandable complexity and programmability of the nucleic acid nanomachines is a critical challenge. Compared with the previously most widely applied transduction strategies, we review the latest advances of a kinetically controlled approach for ligand-oligonucleotide transduction in this Concept article. This new design works through an intrinsic conformational alteration of the nucleic acid aptamer upon the ligand binding as a governing factor for nucleic acid strand displacement reactions. The functionalities and applications of this transduction system as a ligand converter on biosensing and DNA computation are described and discussed. Furthermore, we propose some potential scenarios for utilization of this ligand transduction design to regulate gene expression through synthetic RNA switches in the cellular contexts. Finally, future perspectives regarding this ligand-oligonucleotide transduction platform are also discussed.     

Molecular thermodynamics of LNA:LNA base pairs and the hyperstabilizing effect of 5′‐proximal LNA:DNA base pairs

Locked nucleic acids (LNAs) can greatly enhance duplex DNA stability, and are therefore creating opportunities to improve therapeutics, as well as PCR‐based disease and pathogen diagnostics. Realizing the full potential of LNAs will require better understanding of their contributions to duplex stability, and the ability to predict their hydridization thermodynamics. Melting thermodynamics data for a large set of diverse duplexes containing LNAs in one or both strands are presented. Those data reveal that LNAs, when present on both strands, can stabilize a duplex not only through direct interaction with their base‐pair partner, but also through nonlocal hyperstablization effects created by LNA:LNA base pairs and/or specific patterns of oppositely oriented LNA:DNA base pairs. The data are, therefore, used to extend a thermodynamic model previously developed in our lab to permit accurate prediction of melting temperatures for duplexes bearing LNA substitutions within both strands using a classic group‐contribution approach. © 2015 American Institute of Chemical Engineers AIChE J, 61: 2711–2731, 2015     

Catalytic hydrothermal liquefaction of D. tertiolecta for the production of bio‐oil over different acid/base catalysts

In this article, two acid catalysts (ZrO₂/SO₄^2? and HZSM‐5) and two base catalysts (MgO/MCM‐41 and KtB) were used in catalytic hydrothermal liquefaction (HTL) of Dunaliella tertiolecta (D. tertiolecta) for the production of bio‐oil. The results indicated that the acid/base property of the catalyst plays a crucial role in the catalytic HTL process, and the base catalyst is conducive to the improvement of conversion and bio‐oil yield. When KtB was used as the catalyst, the maximum conversion and bio‐oil yield was 94.84 and 49.09 wt %, respectively. The detailed compositional analysis of the bio‐oil was performed using thermogravimetric analysis, elemental analysis, FT‐IR, and GC‐MS. The compositional analysis results showed that the introduction of catalyst is beneficial for reducing the fixed carbon content in the bio‐oil, and the structure of catalyst influences on the bio‐oil composition and boiling point distribution. Based on our results and previous studies, the probable catalytic HTL microalgae model over various catalysts can be described that the main chemical reactions include ketonization, decarboxylic, dehydration, ammonolysis, and so forth. with HZSM‐5 and MgO/MCM‐41 as the catalyst; the cyclodimerization, decomposition, Maillard reaction, and ketonization are the main reactions with ZrO₂/SO₄^2? as the catalyst; the dehydration, ammonolysis, Maillard reaction, and ketonization can occur with KtB as the catalyst. Therefore, a plausible reaction mechanism of the main chemical component in D. tertiolecta is proposed. © 2015 American Institute of Chemical Engineers AIChE J, 61: 1118–1128, 2015     

Impact of the guanidinium group on hybridization and cellular uptake of cationic oligonucleotides

The grafting of cationic groups to synthetic oligonucleotides (ONs) in order to reduce the charge repulsion between the negatively charged strands of a duplex or triplex, and consequently to increase a complex's stability, has been extensively studied. Guanidinium groups, which are highly basic and positively charged over a wide pH range, could be an efficient ON modification to enhance their affinity for nucleic acid targets and to improve cellular uptake. A straightforward post-synthesis method to convert amino functions attached to ONs (on sugar, nucleobase or backbone) into guanidinium tethers has been perfected. In comparison to amino groups, such cationic groups anchored to alpha-oligonucleotide phosphoramidate backbones play important roles in duplex stability, particularly with RNA targets. This high affinity could be explained by dual recognition resulting from Watson-Crick or Hoogsteen base pairing combined with cationic/anionic backbone recognition between strands involving H-bond formation and salt bridging. Molecular-dynamics simulations corroborate interactions between the cationic backbones of the alpha-ONs and the anionic backbones of the nucleic acid targets. Moreover, ONs with guanidinium modification increased cellular uptake relative to negatively charged ONs. The cellular localization of these new cationic phosphoramidate ONs is mainly cytoplasmic. The uptake of these ON analogues might occur through endocytosis.     

Solubilizing Trityl-Type Tag To Synthesize Asx/Glx-Containing Peptides

A novel solubilizing tag system for Asp/Asn/Glu/Gln-containing peptides is described. In this method, an Asp/Glu[Dbz-Cys-NH₂]-containing peptide (Dbz: 3,4-diaminobenzoic acid) is first synthesized through fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis. The solubilizing moiety containing an oligo-Lys group is then attached to the peptide in hexafluoroisopropanol through a trityl anchor to afford a hydrophilic tagged peptide. To detach the solubilizing tag, the Dbz moiety of the tagged peptide is activated with NaNO₂, and the Asp/Asn/Glu/Gln-containing peptide is obtained through hydrolysis or ammonolysis. This synthetic approach proved to be compatible with native chemical ligation, and amyloid β-protein 1–42 was successfully synthesized by the solubilizing-tag-aided native chemical ligation–desulfurization method.     

Chemical Modification of Bisphenol A Polycarbonate by Reactive Blending with Cyclic Anhydrides

Summary Reactive blending with cyclic anhydrides in the melt is a promising process to modify the chemical structure of bisphenol A polycarbonate (PC) by insertion of ester moieties leading to consistent changes in the chemical and physical properties of PC. In particular, by the reaction with succinic anhydride it is possible to insert aliphatic moieties in the polymer backbone, decreasing the melt viscosity and the glass transition temperature of PC (and thus increase its processability). The reaction, that is catalysed by Lewis acids (e. g. titanium butoxide), proceeds through the formation of carbonyl carbonate groups that undergo decarboxylation leading to ester moieties. No side reactions have been detected by NMR analysis. The addition of phosphorous acid as catalyst quencher at the end of the process gives rise to polymers with good colour and thermal stability.     

Synthesis and characterization of D-2PA-Pd(II)@SBA-15 catalyst via “click chemistry”: highly active catalyst for Suzuki coupling reactions

Copper catalysed cycloaddition reaction between azide and terminal alkyne, called as “click reaction”, was found to be modular approach for the synthesis of facile, highly efficient and recoverable D-2PA-Pd(II)@SBA-15 catalyst. In efforts to synthesize the catalyst, the cycloaddition reaction between azido-functionalized mesoporous SBA-15 and N,N-dimethyl-2-propynylamine (D-2PA) has been carried out, followed by the complexation with PdCl₂. To analyze physiochemical properties of synthesized materials, various characterization techniques such as CHN elemental analysis, X-ray diffraction, solid state ¹³C and ²⁹Si NMR spectra, FT-IR, XPS, SEM, TEM and N₂ sorption analysis, TGA and DTA, UV–Vis spectroscopy have been carried out. The synthesized catalyst, D-2PA-Pd(II)@SBA-15, exhibited excellent catalytic activities with good product yield and high TON towards Suzuki coupling reaction of various aryl halides with phenylboronic acid. The effect of solvent, base and temperature on coupling reactions has also been described. The developed protocol offers recyclability of catalyst for multiple (four) times without any appreciable loss in stability and catalytic reactivity.     

A kinetic approach to catalytic pyrolysis of tars

A kinetic model to describe the catalytic pyrolysis of tars is proposed and validated through the experimental pyrolysis of three tars of different characteristics and origin; calcined limestone (11 m²/g) was used as catalyst. Experiments were carried out in a catalytic fixed bed reactor. The model assumes that tars are composed of two pseudo-components, described as heavy and light tars, which can be experimentally determined by a split temperature. Tar pyrolysis is described by two simultaneous chemical reactions; catalyst deactivation due to carbon deposition is also considered. Expressions for product concentrations as functions of residence time, selectivity and deactivation were obtained and the kinetic parameters evaluated.     

Medium-chain fatty acid-rich glycerides by chemical and lipase-catalyzed polyester-monoester interchange reaction

Medium-chain triglycerides (MCT) that contain caprylic acid (C_8:0) and capric acid (C_10:0) have immense medicinal and nutritional importance. Coconut oil can be used as a starting raw material for the production of MCT. The process, based on the interchange reaction between triglycerides and methyl esters of medium-chain fatty acids by chemical catalyst (sodium methoxide) or lipase (Mucor miehei) catalyst, appears to be technically feasible. Coconut oils with 25–28.3% (w/w) and 22.1–25% (w/w) medium-chain fatty acids have been obtained by chemical and lipase-catalyzed interchange reactions. Coconut olein has also been modified with C_8:0 and C_10:0 fatty acids, individually as well as with their mixtures, by chemical and lipase-catalyzed interchange reactions. Coconut olein is a better raw material than coconut oil for production of mediumchain fatty acid-rich triglyceride products by both chemical and lipase-catalyzed processes.     

Influence of catalyst on transesterification between poly(lactic acid) and polycarbonate under flow field

This paper investigated the effect of catalyst on transesterification and transesterification mechanism between poly(lactic acid) (PLA) and polycarbonate (PC) under flow field. Three catalysts (zinc borate, titanium pigment and tetrabutyl titanate) were evaluated. It is found that transesterification reaction can take place without any catalyst, while three catalysts can all promote the transesterification reaction between poly(lactic acid) and polycarbonate to a greater extent. ¹H nuclear magnetic resonance spectroscopy, gel permeation chromatography and dynamic mechanical analysis revealed that structures of copolymers are not identical in the blends with and without catalyst. For pure blend, most of copolymers have relatively high molecular weight with low PC content, which implies that transesterification reaction most likely happens only once between a PLA chain and a PC chain during mixing process, and only a small amount of multiple reactions happen. However, for the catalyst systems, catalysts induce much more multiple reactions accompanying with the reducing molecular weight in copolymers and increasing PC content. Moreover, it is found that the catalysts not only affect the chain compositions of the product copolymers, but also influence the amount of polymers involved in the reaction. Tetrabutyl titanate is found to be the most effective catalyst in this study where the amount of reacted polycarbonate is more than 4 times of that in pure blend. It is found that PLA segments in copolymer are easily aligned on the interface due to its relatively high Deborah number, which increases the probability of its contact with more PC chains. Although the flow effect on the alignment of chain segment is similar in blends with and without catalysts, the acceleration of reaction due to catalyst makes it possible for multiple reactions. The match of the reaction time and contact time of chain segment of PC and PLA at interfaces is then of key importance in the interfacial transesterification reaction. The effect of flow field on the interfacial reaction is then not only from the interfacial update, but also from the change of chain conformation near the interface.     

Fe3+-montmorillonite as a cost-effective and recyclable solid acidic catalyst for the synthesis of xanthenediones

Fe³⁺-montmorillonite was first used as a solid acidic catalyst for the synthesis of xanthenedione derivatives through condensation reactions between aromatic aldehydes and dimedone. This process provides several advantages such as good yields of products, simple operation, convenient separation, and inexpensive and recyclable catalyst.     

Synthesis of a nano-crystalline solid acid catalyst from fly ash and its catalytic performance

The synthesis of nano-crystalline activated fly ash catalyst (AFAC) with crystallite size of 12 nm was carried out by chemical and thermal treatment of fly ash, a waste material generated from coal-burning power plants. Fly ash was chemically activated using sulfuric acid followed by thermal activation at 600 °C. The variation of surface and Physico-chemical properties of the fly ash by activation methods resulted in improved acidity and therefore, catalytic activity for acid catalyzed reactions. The AFAC was characterized by X-ray diffraction, FT-IR spectroscopy, N₂-adsorption–desorption isotherm, scanning electron microscopy, flame atomic absorption spectrophotometry and sulfur content by CHNS/O elemental analysis. It showed amorphous nature due to high silica content (81%) and possessed high BET surface area (120 m²/g). The catalyst was found to be highly active solid acid catalyst for liquid phase esterification of salicylic acid with acetic anhydride and methanol giving acetylsalicylic acid and methyl salicylate respectively. A maximum yield of 97% with high purity of acetylsalicylic acid (aspirin) and a very high conversion 87% of salicylic acid to methyl salicylate (oil of wintergreen) was obtained with AFAC. The surface acidity and therefore, catalytic activity in AFAC was originated by increased silica content, hydroxyl content and higher surface area as compared to fly ash. The study shows that coal generated fly ash can be converted into potential solid acid catalyst for acid catalyzed reactions. Furthermore, this catalyst may replace conventional environmentally hazardous homogeneous liquid acids making an ecofriendly; solvent free, atom efficient, solid acid based catalytic process. The application of fly ash to synthesize a solid acid catalyst finds a noble way to utilize this abundant waste material.