Identification of Sequence Specificity of 5‐Methylcytosine Oxidation by Tet1 Protein with High‐Throughput Sequencing

Tet (ten‐eleven translocation) family proteins have the ability to oxidize 5‐methylcytosine (mC) to 5‐hydroxymethylcytosine (hmC), 5‐formylcytosine (fC), and 5‐carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic‐level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high‐throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10⁴‐member mC‐containing random DNA‐sequence library was constructed. The library was subjected to Tet‐reactive pulldown followed by high‐throughput sequencing. Analysis of the obtained sequence data identified the Tet1‐reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein.     

Electrosyntheses and characterization of poly(5‐bromoindole) in boron trifluoride diethyl etherate

Poly(5‐bromoindole) (PBrI) films were synthesized electrochemically by direct oxidation of 5‐bromoindole in pure boron trifluoride diethyl etherate. The oxidation potential of 5‐bromoindole in this medium was measured to be only 0.97 V vs. saturated calomel electrode, which was lower than that determined in acetonitrile + 0.1 mol L^?1 Bu₄NBF₄ (1.08 V). PBrI films obtained from this medium showed good electrochemical behavior and good thermal stability. Structural studies showed that the polymerization of 5‐bromoindole ring occurred at 2,3 position. As‐formed PBrI films were thoroughly soluble in strong polar solvent dimethylsulfoxide and partly soluble in tetrahydrofuran. Fluorescent spectral studies indicated that PBrI was a good blue‐light emitter. The excitation and emission spectra of PBrI showed a significant shift to longer wavelength compared with that of the monomer, consistent with the greater extent of electron delocalization. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 539–547, 2006     

5‐Amino‐3,4‐dinitropyrazole as a Promising Energetic Material

The nitrogen‐rich energetic compound 5‐amino‐3,4‐dinitropyrazole (5‐ADP) was investigated using complementary experimental techniques. X‐ray diffraction indicates the strong intermolecular hydrogen bonding in 5‐ADP crystals. Compound exhibits low impact sensitivity (23 J) and insensitivity to friction. The activation energy of thermolysis determined to be 230±5 kJ mol⁻¹ from DSC measurements. Accelerating rate calorimetry indicates the lower thermal stability (173 °C) of 5‐ADP than that of RDX, which is probably the main concern about using this compound. 5‐ADP also exhibits good compatibility with common energetic materials (viz. TNT, RDX, ammonium perchlorate), including an active binder. The burning rate of 5‐ADP monopropellant is higher than that of benchmark HMX, while the pressure exponent 0.51±0.04 is surprisingly low. Addition of ammonium perchlorate does not affect the pressure exponent of 5‐ADP, while the burning rate increases. The 5‐amino‐3,4‐dinitropyrazole exhibits a notable combination of combustion performance, low sensitivity, and good compatibility, which renders it as a promising energetic material.     

Synthesis,characterization and (electro)chemical polymerization of triphenylamine‐end‐functionalized poly(ε‐caprolactone)

Triphenylamine‐based oligomers and polymers with linear, hyperbranched, star‐shaped or dendrimer architectures have been synthesized and studied due to their interesting electro‐optical properties. In many cases insoluble materials are obtained. In this study, we report the synthesis of grafted polytriphenylamine by chemical and electrochemical polymerization of triphenylamine‐end‐functionalized poly(ε‐caprolactone). Functionalized ε‐caprolactone oligomers were obtained by ring‐opening polymerization of ε‐caprolactone initiated by 4‐hydroxymethyltriphenylamine/stannous octanoate (tin 2‐ethylhexanoate). The ring‐opening polymerization of ε‐caprolactone using 4‐hydroxymethyltriphenylamine/stannous octanoate as initiating system provided ε‐caprolactone oligomers, with well‐defined molecular weights, containing a triphenylamine terminal group. Chemical and electrochemical coupling oxidation of the triphenylamine ends allowed the formulation of polyarylamines with ε‐caprolactone oligomers as grafts. Graft copolymers with an aryleneamine backbone and short poly(ε‐caprolactone) grafts were obtained by (electro)chemical oxidation of oligomers containing triphenylamine terminal groups. Copyright © 2009 Society of Chemical Industry     

An Efficient Lanthanide‐Dependent DNAzyme Cleaving 2′–5′‐Linked RNA

RNA can form two types of linkage. In addition to the predominant 3′–5′ linkage, 2′–5′‐linked RNA is also important in biology, medicine, and prebiotic studies. Here, in vitro selection was used to isolate a DNAzyme that specifically cleaves 2′–5′ RNA by using Ce³⁺ as the metal cofactor, but leaves the 3′–5′ counterpart intact. This Ce5 DNAzyme requires trivalent light lanthanide ions and shows a rate of 0.16 min^?1 in the presence of 10 μm Ce³⁺; the activity decreases with heavier lanthanide ions. This is the fastest DNAzyme reported for this reaction, and it might enable applications in chemical biology. As a proof‐of‐concept, using this DNAzyme, the reactions between phosphorothioate‐modified RNA and strongly thiophilic metals (Hg²⁺ and Tl³⁺) were studied as a function of pH.     

Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

Synthetic ways towards uridine 5′‐diphosphate (UDP)‐xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP‐glucose 6‐dehydrogenase (hUGDH) and UDP‐xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP‐α‐xylose from UDP‐glucose. In a mimic of the natural biosynthetic route, UDP‐glucose is converted to UDP‐glucuronic acid by hUGDH, followed by subsequent formation of UDP‐xylose by hUXS. The nicotinamide adenine dinucleotide (NAD⁺) required in the hUGDH reaction is continuously regenerated in a three‐step chemo‐enzymatic cascade. In the first step, reduced NAD⁺ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10‐phenanthrenequinone (PQ). Radical chemical re‐oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H₂O₂) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo‐enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP‐xylose as well as an adequate oxygen supply for PQ re‐oxidation as major bottlenecks of effective performance of the overall multi‐step reaction system. Net oxidation of UDP‐glucose to UDP‐xylose by hydrogen peroxide (H₂O₂) could thus be achieved when using an in situ oxygen supply through periodic external feed of H₂O₂ during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g L ⁻¹) UDP‐α‐xylose. After two‐step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi‐step redox cascades in the synthesis of nucleotide sugar products.

     

Copper(I)‐Catalysed Multicomponent Reaction: Straightforward Access to 5‐Hydroxy‐1H‐pyrrol‐2(5H)‐ones

A copper‐catalysed multicomponent coupling reaction between readily available (Z)‐3‐iodoacrylic acids, terminal alkynes, and primary amines was developed to smoothly access a small library of 5‐hydroxy‐1H‐pyrrol‐2(5H)‐ones in good yields. This practical and general process was applied to a short‐steps synthesis of the natural product pulchellalactam.

     

Toward environment‐friendly composites of poly(ε‐caprolactone) reinforced with stereocomplex‐type poly(l‐lactide)/poly(d‐lactide)

In this work, stereocomplex‐poly(l ‐ and d ‐lactide) (sc‐PLA) was incorporated into poly(ε‐caprolactone) (PCL) to fabricate a novel biodegradable polymer composite. PCL/sc‐PLA composites were prepared by solution casting at sc‐PLA loadings of 5–30 wt %. Differential scanning calorimetry (DSC) and wide‐angle X‐ray diffraction (WAXD) demonstrated the formation of the stereocomplex in the blends. DSC and WAXD curves also indicated that the addition of sc‐PLA did not alter the crystal structure of PCL. Rheology and mechanical properties of neat PCL and the PCL/sc‐PLA composites were investigated in detail. Rheological measurements indicated that the composites exhibited evident solid‐like response in the low frequency region as the sc‐PLA loadings reached up to 20 wt %. Moreover, the long‐range motion of PCL chains was highly restrained. Dynamic mechanical analysis showed that the storage modulus (E′) of PCL in the composites was improved and the glass transition temperature values were hardly changed after the addition of sc‐PLA. Tensile tests showed that the Young's modulus, and yield strength of the composites were enhanced by the addition of sc‐PLA while the tensile strength and elongation at break were reduced. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40208.     

6‐Substituted 2‐Aminopurine‐2′‐deoxyribonucleoside 5′‐Triphosphates that Trace Cytosine Methylation

Gene expression is extensively regulated by the occurrence and distribution of the epigenetic marker 2′‐deoxy 5‐methylcytosine (5mC) in genomic DNA. Because of its effects on tumorigenesis there is an important link to human health. In addition, detection of 5mC can serve as an outstanding biomarker for diagnostics as well as for disease therapy. Our previous studies have already shown that, by processing O⁶‐alkylated 2′‐deoxyguanosine triphosphate (dGTP) analogues, DNA polymerases are able to sense the presence of a single 5mC unit in a template. Here we present the synthesis and evaluation of an extended toolbox of 6‐substituted 2‐aminopurine‐2′‐deoxyribonucleoside 5′‐triphosphates modified at position 6 with various functionalities. We found that sensing of 5‐methylation by this class of nucleotides is more general, not being restricted to O⁶‐alkyl modification of dGTP but also applying to other functionalities.     

Kinetics of homogeneous 5‐hydroxymethylfurfural oxidation to 2,5‐furandicarboxylic acid with Co/Mn/Br catalyst

2,5‐furandicarboxylic acid (FDCA) is a potential non‐phthalate based bio‐renewable substitute for terephthalic acid‐based plastics. Herein, we present an investigation of the oxidation rate of 5‐hydroxymethylfurfural (HMF) to FDCA in acetic acid medium using Co/Mn/Br catalyst. Transient concentration profiles of the reactant (HMF), intermediates [2,5‐diformylfuran (DFF), 5‐formyl‐2‐furancarboxylic acid (FFCA)], and the desired product (FDCA) were obtained for this relatively fast reaction in a stirred semi‐batch reactor using rapid in‐line sampling. Comparison of the effective rate constants for the series oxidation steps with predicted gas–liquid mass transfer coefficients reveals that except for the FFCA → FDCA step, the first two oxidation steps are subject to gas–liquid mass transfer limitations even at high stirrer speeds. Novel reactor configurations, such as a reactor in which the reaction mixture is dispersed as fine droplets into a gas phase containing oxygen, are required to overcome oxygen starvation in the liquid phase and further intensify FDCA production. © 2016 American Institute of Chemical Engineers AIChE J, 63: 162–171, 2017     

Synthesis of functionalizable and biodegradable polymers via ring‐opening polymerization of 5‐benzyloxy‐trimethylene carbonate and ε‐caprolactone

The biomedical applications of poly(ε‐caprolactone) (PCL) were limited for its high hydrophobicity and crystallinity. In this study, we copolymerized CL with amorphous 5‐hydroxyl‐trimethylene carbonate (HTMC) to solve the problem. The 5‐benzyloxy‐trimethylene carbonate (BTMC) was synthesized to copolymerize with CL, then hydrogenolyzed to obtain hydroxyl pendant groups. A serial of copolymers with different BTMC molar ratio were synthesized and their chemical structures and thermal properties were thoroughly studied with NMR, FT‐IR, GPC, XRD, DSC, and TGA. Finally we examined the water contact angle of the copolymers. DSC and XRD results showed that the PCL segments in the copolymers crystallized below 16.8%. BTMC molar content and the crystallinity of the copolymers increased after hydrolysis. With the introduced hydroxyl pendant groups, the deprotected copolymers improved their hydrophilic property significantly, and the copolymer with 9.3% HTMC molar content had static water contact angle as low as 36.5°. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Confined crystallization morphology of poly(ϵ‐caprolactone) block within poly(ϵ‐caprolactone)–poly(l‐lactide) copolymers

The confined crystallization of poly(?‐caprolactone) (PCL) block in poly(?‐caprolactone)–poly(l ‐lactide) (PCL‐PLLA) copolymers was investigated using differential scanning calorimetry, polarized optical microscopy, scanning electronic microscopy and atomic force microscopy. To study the effect of crystallization and molecular chain motion state of PLLA blocks in PCL‐PLLA copolymers on PCL crystallization morphology, high‐temperature annealing (180 °C) and low‐temperature annealing (80 °C) were applied to treat the samples. It was found that the crystallization morphology of PCL block in PCL‐PLLA copolymers is not only related to the ratio of block components, but also related to the thermal history. After annealing PCL‐PLLA copolymers at 180 °C, the molten PCL blocks are rejected from the front of PLLA crystal growth into the amorphous regions, which will lead to PCL and PLLA blocks exhibiting obvious fractionated crystallization and forming various morphologies depending on the length of PLLA segment. On the contrary, PCL blocks more easily form banded spherulites after PCL‐PLLA copolymers are annealed at 80 °C because the preexisting PLLA crystal template and the dangling amorphous PLLA chains on PCL segments more easily cause unequal stresses at opposite fold surfaces of PCL lamellae during the growth process. Also, it was found that the growth rate of banded spherulites is less than that of classical spherulites and the growth rate of banded spherulites decreases with decreasing band spacing. © 2019 Society of Chemical Industry     

Synthesis and Properties of Branched Oligo(2‐thienyl)‐ and Oligo(2,2′‐bithien‐5‐yl)‐Substituted Pyridine Derivatives

Starting from easily accessible precursors we describe the preparation of a series of branched oligo(2‐thienyl)‐ and oligo(2,2′‐bithienyl)‐substituted pyridine derivatives. With palladium‐catalyzed cross‐coupling reactions of pyridyl nonaflates/triflates as key steps we synthesized 2,6‐di(2‐thienyl)pyridines bridged by thiophene or benzene rings. By selective bromination of 2,6‐di(2‐thienyl)pyridine and 2,4,6‐tri(2‐thienyl)pyridine and subsequent coupling reactions an access to oligo(2,2′‐bithien‐5‐yl)‐substituted pyridine derivatives was gained. The constitution and solid state conformation of 2,6‐bis(2,2’‐bithien‐5‐yl)pyridine was determined by X‐ray analysis. This series of new pyridine‐thiophene conjugates was systematically investigated by UV/vis spectroscopy. Large Stokes shifts in the neutral and protonated form were observed. The electrochemical oxidation of two typical compounds was studied, however, these oxidations were irreversible forming a polymeric film at the anode. As a selected example, a thiophene‐bridged 2,6‐di(2‐thienyl)pyridine derivative was also investigated by scanning tunneling microscopy showing an interesting self‐assembly into a highly ordered monolayer on highly oriented pyrolytic graphite.

     

Synthesis of 4‐Aryl‐2(5H)‐furanones by Gold(I)‐Catalyzed Intramolecular Annulation

A novel method that involves intramolecular annulation and a new type of rearrangement has been developed for the synthesis of 4‐aryl‐2(5H)‐furanones. A variety of prop‐2‐ynyl 3‐oxo‐3‐phenylpropanoates undergo annulation cyclization in the presence of chloro(triphenylphosphine)gold and trifluoromethanesulfonic to afford the desired products in moderate to high yields.     

Poly(vinyl pyrrolidone‐co‐vinyl acetate)‐graft‐poly(ε‐caprolactone) as a compatibilizer for cellulose acetate/poly(ε‐caprolactone) blends

Poly(vinyl pyrrolidone‐co‐vinyl acetate)‐graft‐poly(ε‐caprolactone) (PVPVAc‐g‐PCL) was synthesized by radical copolymerization of N‐vinyl‐2‐pyrrolidone (VP)/vinyl acetate (VAc) comonomer and PCL macromonomer containing a reactive 2‐hydroxyethyl methacrylate terminal. The graft copolymer was designed in order to improve the interfacial adhesiveness of an immiscible blend system composed of cellulose acetate/poly(ε‐caprolactone) (CA/PCL). Adequate selections of preparation conditions led to successful acquisition of a series of graft copolymer samples with different values of molecular weight ( ), number of grafts (n), and segmental molecular weight of PVPVAc between adjacent grafts (M_n (between grafts)). Differential scanning calorimetry measurements gave a still immiscible indication for all of the ternary blends of CA/PCL/PVPVAc‐g‐PCL (72 : 18 : 10 in weight) that were prepared by using any of the copolymer samples as a compatibilizer. However, the incorporation enabled the CA/PCL (4 : 1) blend to be easily melt‐molded to give a visually homogeneous film sheet. This compatibilizing effect was found to be drastically enhanced when PVPVAc‐g‐PCLs of higher and M_n (between grafts) and lower n were employed. Scanning electron microscopy revealed that a uniform dispersion of the respective ingredients in the ternary blends was attainable with an assurance of the mixing scale of several hundreds of nanometers. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Preparation and drug release behaviors of 5‐fluorouracil loaded poly(glycolide‐co‐lactide‐co‐caprolactone) nanoparticles

5‐Fluorouracil (5‐Fu) loaded poly(glycolide‐co‐lactide‐co‐caprolactone) (PGLC) nanoparticles were prepared by modified spontaneous emulsification solvent diffusion method (modified‐SESD method) and characterized by dynamic light scattering, scanning electron microscopy and ¹H NMR determination. It was found that the obtained nanoparticles showed near spherical shape and was controllable with the radius range of 30–100 nm. Compared with the nanoparticles prepared by polylactide and poly (lactide‐co‐glycolide) (PLGA) under the similar preparation condition, yield of PGLC nanoparticles was the highest, which reached to about 100%. On the other hand, drug entrapment efficiency of PGLC nanoparticles was also higher than that of PLGA and PLLA nanoparticles. 5‐Fu release behavior of PGLC nanoparticles in vitro showed that 5‐Fu release of PGLC nanoparticles showed a near zero‐order release profile, and 5‐Fu release rate of PGLC nanoparticles was faster than that of PLLA and PLGA nanoparticles. According to degradation behavior of PGLC nanoparticles, it could be proposed that the kinetic of degradation controlled release played an important role in the release process of PGLC nanoparticles. It revealed that the PGLC nanoparticles could be a promising drug carrier. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci, 2007     

End‐capping double‐chain stranded polypseudorotaxanes using lengthily tunable poly(2‐hydroxyethyl methacrylate) blocks via atom transfer radical polymerization

Owing to a large cavity, γ‐cyclodextrin (γ‐CD) can form double‐chain inclusion complexes with linear polymeric chains such as poly(ethylene glycol) (PEG) and poly(ε‐caprolactone). However, to date few reports have focused on the end‐capping of these kinds of fascinating supramolecular entities. In this study, atom transfer radical polymerization (ATRP) was employed to prepare double‐chain stranded polyrotaxanes (PRs) with lengthily tunable poly(2‐hydroxyethyl methacrylate) (PHEMA) blocks as bulky end‐cappers. ATRP of HEMA was carried out using pseudopolyrotaxanes (PPRs) self‐assembled from a distal 2‐bromoisobutyryl end‐capped PEG (BriB‐PEG‐iBBr) with varying amounts of γ‐CDs as macroinitiator in the presence of Cu(I)Br/N,N,N′,N″,N″‐pentamethyldiethylenetriamine in aqueous solution at room temperature. The resulting PRs were demonstrated by the unique H‐like double‐chain stranded supramolecular architecture end‐capped with lengthily tunable PHEMA blocks. When the feed molar ratio of γ‐CD:BriB‐PEG‐iBBr varied from 5 to 60 in PPRs, the feed molar ratio in PRs was found to remain at around 20 with a higher yield. ATRP was successfully applied to prepare double‐chain stranded PRs end‐capped with lengthily tunable PHEMA blocks. This provides a protocol for the preparation of novel H‐like PR‐based block copolymers. Copyright © 2010 Society of Chemical Industry     

Fluorescent 2‐Aminopyridine Nucleobases for Triplex‐Forming Peptide Nucleic Acids

Development of new fluorescent peptide nucleic acids (PNAs) is important for fundamental research and practical applications. The goal of this study was the design of fluorogenic nucleobases for incorporation in triplex‐forming PNAs. The underlying design principle was the use of a protonation event that accompanied binding of a 2‐aminopyridine (M) nucleobase to a G‐C base pair as an on switch for a fluorescence signal. Two fluorogenic nucleobases, 3‐(1‐phenylethynyl)‐M and phenylpyrrolo‐M, were designed, synthesized and studied. The new M derivatives provided modest enhancement of fluorescence upon protonation but showed reduced RNA binding affinity and quenching of fluorescence signal upon triple‐helix formation with cognate double‐stranded RNA. Our study illustrates the principal challenges of design and provides guidelines for future improvement of fluorogenic PNA nucleobases. The 3‐(1‐phenylethynyl)‐M may be used as a fluorescent nucleobase to study PNA–RNA triple‐helix formation.     

5′‐(E)‐Vinylphosphonate: A Stable Phosphate Mimic Can Improve the RNAi Activity of siRNA–GalNAc Conjugates

Small interfering RNA (siRNA)‐mediated silencing requires siRNA loading into the RNA‐induced silencing complex (RISC). Presence of 5′‐phosphate (5′‐P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid‐domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA–GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5′‐P on the activity of siRNA–GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5′‐P. For those that do, incorporation of 5′‐(E)‐vinylphosphonate (5′‐VP), a metabolically stable phosphate mimic, results in up to 20‐fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.