A DNA‐Encoded Library of Chemical Compounds Based on Common Scaffolding Structures Reveals the Impact of Ligand Geometry on Protein Recognition

A DNA‐encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5‐bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a K_d value of 6 nm , while compounds with the same substituents on an equidistant but flexible l ‐lysine scaffold showed 140‐fold lower affinity. A 18 nm tankyrase‐1 binder featured l ‐lysine as linking moiety, while molecules based on d ‐Lysine or (2S,4S)‐amino‐l ‐proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries.     

A Quencher-Free Linear Probe from Serinol Nucleic Acid with a Fluorescent Uracil Analogue

With the goal of developing a quencher-free probe composed of an artificial nucleic acid, the fluorescent nucleobase analogue 5-(perylenylethynyl)uracil (^PeU), which was incorporated into totally artificial serinol nucleic acid (SNA) as a substitute for thymine, has been synthesized. In the context of a 12-mer duplex with RNA, these fluorophores reduce duplex stability slightly compared with that of an SNA without ^PeU modification; thus suggesting that structural distortion is not induced by the modification. If two ^PeUs were incorporated at separate positions in an SNA, the fluorescent emission at λ≈490 nm was clearly enhanced upon hybridization with complementary RNA. A quencher-free SNA linear probe containing three ^PeUs, each separated by six nucleobases, has been designed. Detection of target RNA with high sensitivity and discrimination of a single-base mismatch has also been demonstrated.     

Pipecolic Acid Hydroxylases: A Monophyletic Clade among cis‐Selective Bacterial Proline Hydroxylases that Discriminates l‐Proline

Proline hydroxylases are iron(II)/2‐oxoglutarate‐dependent enzymes that hydroxylate l ‐proline and derivatives, such as l pipecolic acid, which is the six‐membered‐ring homologue of l ‐proline. It has been established that there is a distinct group of conserved bacterial enzymes that hydroxylate l ‐pipecolic acid and trans‐3‐ and trans‐4‐methyl‐l ‐proline, but virtually no l ‐proline. This allows the organism to produce hydroxyproline congeners without hydroxylation of the physiologically omnipresent l ‐proline. In vitro conversions showed that the substrate spectrum of the pipecolic acid hydroxylases GetF (from a Streptomyces sp.; producer of the tetrapeptide antibiotic GE81112) and PiFa (from Frankia alni) overlaps that of proline hydroxylases, except for the nonacceptance of l ‐proline and smaller homologues. Distinct and conserved residues were determined for both types of enzymes. However, site‐directed mutagenesis in GetF did not yield variants that accepted l ‐proline; this suggested a complex interaction of several residues around the active site, which resulted in delicate changes in substrate specificity. This is supported by substrate docking in a homology model of GetF, which revealed an altered orientation for l ‐proline relative to that of preferred substrates.     

Dyes derived from 3‐formyl‐2(1H)‐quinolone – synthesis,spectroscopic characterisation,and their behaviour in the presence of sulfhydryl and non‐sulfhydryl amino acids

Novel dyes based on a 3‐formyl‐2(1H)‐quinolone skeleton were synthesised and characterised using ¹H nuclear magnetic resonance spectroscopy and mass spectrometry. The spectroscopic properties of these dyes, such as their absorption spectra, emission spectra, and quantum fluorescence yields, were also examined. The behaviour of the obtained compounds at a pH of 7.4 in the absence and in the presence of thiol amino acids, such as l ‐cysteine, l ‐glutathione, and N‐acetyl‐l ‐cysteine, were studied. The spectroscopic responses of the tested dyes towards other amino acids were also investigated. A reference compound was synthesised to understand the reaction mechanism between the thiols and the obtained dyes. The experimental results show that the synthesised dyes have the potential to act as sensors for thiols.     

Application of low loading of collagen in electrospun poly[(l‐lactide)‐co‐(ε‐caprolactone)] nanofibrous scaffolds to promote cellular biocompatibility

Electrospinning of various polymers has been used to produce nanofibrous scaffolds that mimic the extracellular matrix and support cell attachment for the potential repair and engineering of nerve tissue. In the study reported here, an electrospun copolymer of l ‐lactide and ε‐caprolactone (67:33 mol%) resulted in a nanofibrous scaffold with average fibre diameter and pore size of 476 ± 88 and 253 ± 17 nm, respectively. Blending with low loadings of collagen (<2.5% w/w) significantly reduced the average diameter and pore size. The uniformity of fibre diameter distributions was supported with increasing collagen loadings. The nanofibrous scaffolds significantly promoted the attachment and proliferation of olfactory ensheathing cells compared to cells exhibiting asynchronous growth. Furthermore, analysis of cell health through mitochondrial activity, membrane leakage, cell cycle progression and apoptotic indices showed that the nanofibrous membranes promoted cell vigour, reducing necrosis. The study suggests that the use of more cost‐effective, low loadings of collagen supports morphological changes in electrospun poly[(l ‐lactide)‐co‐(ε‐caprolactone)] nanofibrous scaffolds, which also support attachment and proliferation of olfactory ensheathing cells while promoting cell health. The results here support further investigation of the electrospinning of these polymer blends as conduits for nerve repair. © 2013 Society of Chemical Industry     

Dual drug release from CO2‐infused nanofibers via hydrophobic and hydrophilic interactions

This study investigated the effects of hydrophobic–hydrophilic interactions on dual drug release from CO₂‐infused nanofibers scaffolds (PCL, PCL–gelatin, and PCL “core” PCL–gelatin “shell”) using BODIPY 493/503 and Rhodamine B fluorescent dyes as drug models. Favorable dye–scaffold interactions increased total dye loading and promoted steady, more linear release. Unfavorable dye–scaffold interactions reduced overall loading and led to a greater burst release of dye. However, when CO₂ was used to infuse dye into an unfavorable scaffold, the changes in loading and release were less pronounced. When two dyes were infused, these behaviors were accentuated due to interactions between the dissolved forms of the dyes. Core–shell composite nanofibers displayed radically different release properties versus pure PCL–gelatin fibers when treated with dyes via CO₂ infusion. Dye release from core–shell scaffolds was highly sensitive to both interactions with scaffolds and the phase of CO₂ used to infuse the compounds of interest. By using different phases of CO₂ to partition dyes into hydrophobic and hydrophilic sections of core–shell nanofibers, such interactions can be manipulated to develop a bimodal drug release system with potential application in drug delivery or tissue engineering. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 42571.     

Thermally exfoliated graphene oxide reinforced fluorinated pentablock poly(l‐lactide‐co‐ε‐caprolactone) electrospun scaffolds: Insight into antimicrobial activity and biodegradation

Three‐dimensional fluorinated pentablock poly(l ‐lactide‐co‐ε‐caprolactone)‐based scaffolds were successfully produced by the incorporation of thermally exfoliated graphene oxide (TEGO) as an antimicrobial agent with an electrospinning technique. In a ring‐opening polymerization, the fluorinated groups in the middle of polymer backbone were attached with a perfluorinated reactive stabilizer having oxygen‐carrying ability. The fiber diameter and its morphologies were optimized through changes in TEGO amount, voltage, polymer concentration, and solvent type to obtain an ideal scaffold structure. Instead of the widely used graphene oxide synthesized by Hummer's method, TEGO sheets having a low amount of oxygen produced by thermal expansion were integrated into the fiber structure to investigate the effect of the oxygen functional groups of TEGO sheets on the degradation and antimicrobial activity of the scaffolds. There was no antimicrobial activity in TEGO‐reinforced scaffolds in the in vitro tests in contrast to the literature. This study confirmed that a low number of oxygen functional groups on the surface of TEGO restricted the antimicrobial activity of the fabricated composite scaffolds. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43490.     

Surface‐modified pliable PDLLA/PCL/β‐TCP scaffolds as a promising delivery system for bone regeneration

Surface‐modified poly(d , l ‐lactide)/polycaprolactone/β‐tricalcium phosphate complex scaffold was fabricated in this study and we hypothesized that pliable and mechanical strong scaffold would be achieved by regulation of ternary compositions; while superficial modification strategy conduced to preserve and controlled‐release of bioactive growth factors. Properties of the composite scaffolds were systematically investigated, including mechanical properties, surface morphology, porosity, wettability, and releasing behavior. Moreover, the representative cytokine, recombinant human bone morphogenetic protein‐2 (rhBMP‐2), was loaded and implanted into muscular pouch of mouse to assess bone formation in vivo. Improved osteogenesis was achieved ascribed to both amplified β‐tricalcium phosphate (β‐TCP) content and retarded initial burst release. Particularly, scaffold doped with hydroxypropyl methylcellulose (HPMC) displayed optimal osteogenic capability. The results indicated that the PDLLA/PCL/β‐TCP complex scaffold along with HPMC‐coating and rhBMP‐2 loading was a promising candidate for bone regeneration. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40951.     

Evaluation of polycaprolactone − poly‐D,L‐lactide copolymer as biomaterial for breast tissue engineering

The potential of the copolymer polycaprolactone‐co‐ poly‐d ,l ‐lactic acid (PCLLA ) as a biomaterial for scaffold‐based therapy for breast tissue engineering applications was assessed. First, the synthesized PCLLA was evaluated for its processability by means of additive manufacturing (AM ). We found that the synthesized PCLLA could be fabricated into scaffolds with an overall gross morphology and porosity similar to that of polycaprolactone. The PCLLA scaffolds possessed a compressive Young's modulus (ca 46 kPa ) similar to that of native breast (0.5 ? 25 kPa ), but lacked thermal stability and underwent thermal degradation during the fabrication process. The PCLLA scaffolds underwent rapid degradation in vitro which was characterized by loss of the scaffolds' mechanical integrity and a drastic decrease in mass‐average molar mass (M _w) and number‐average molar mass (M _n) after 4 weeks of immersion in phosphate buffer solution maintained at 37 °C. The tin‐catalysed PCLLA scaffold was also found to have cytotoxic effects on cells. Although the initial mechanical properties of the PCLLA scaffolds generally showed potential for applications in breast tissue regeneration, the thermal stability of the copolymer for AM processes, biocompatibility towards cells and degradation rate is not satisfactory at this stage. Therefore, we conclude that research efforts should be geared towards fine‐tuning the copolymer synthesizing methods. © 2016 Society of Chemical Industry     

A Peptide‐Functionalized Polymer as a Minimal Scaffold Protein To Enhance Cluster Formation in Early T Cell Signal Transduction

In cellular signal transduction, scaffold proteins provide binding sites to organize signaling proteins into supramolecular complexes and act as nodes in the signaling network. Furthermore, multivalent interactions between the scaffold and other signaling proteins contribute to the formation of protein microclusters. Such microclusters are prominent in early T cell signaling. Here, we explored the minimal structural requirement for a scaffold protein by coupling multiple copies of a proline‐rich peptide corresponding to an interaction motif for the SH3 domain of the adaptor protein GADS to an N‐(2‐hydroxypropyl)methacrylamide polymer backbone. When added to GADS‐containing cell lysates, these scaffolds (but not individual peptides) promoted the binding of GADS to peptide microarrays. This can be explained by the cross‐linking of GADS into larger complexes. Furthermore, following import into Jurkat T cell leukemia cells, this synthetic scaffold enhanced the formation of microclusters of signaling proteins.     

Random and aligned PLLA : PRGF electrospun scaffolds for regenerative medicine

Random and aligned electrospun scaffolds were prepared combining poly(l ‐lactic acid) (PLLA) and activated platelet‐rich plasma (PRGF) at various proportions, with the aim of elucidating the role of nanofibers orientation and growth factors on cell attachment and proliferation. PRGF is released from scaffolds in a sustained way for at least 3 weeks, without an initial burst effect. Mesenchymal stem cells (MSCs) seeded on the random scaffolds present a polygonal and random orientation in any direction of the scaffold. On the other hand, aligned scaffolds are able to promote cell attachment and proliferation in the direction of the nanofibers. The incorporation of PRGF in the scaffolds enhances cell proliferation for at least 2 weeks. Overall, aligned electrospun PLLA : PRGF scaffolds can encapsulate growth factors at relatively large proportions and sustain their release to enhance cell attachment and proliferation as well as eliciting cell alignment. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41372.     

A General Method for Artificial Metalloenzyme Formation through Strain‐Promoted Azide–Alkyne Cycloaddition

Strain‐promoted azide–alkyne cycloaddition (SPAAC) can be used to generate artificial metalloenzymes (ArMs) from scaffold proteins containing a p‐azido‐L ‐phenylalanine (Az) residue and catalytically active bicyclononyne‐substituted metal complexes. The high efficiency of this reaction allows rapid ArM formation when using Az residues within the scaffold protein in the presence of cysteine residues or various reactive components of cellular lysate. In general, cofactor‐based ArM formation allows the use of any desired metal complex to build unique inorganic protein materials. SPAAC covalent linkage further decouples the native function of the scaffold from the installation process because it is not affected by native amino acid residues; as long as an Az residue can be incorporated, an ArM can be generated. We have demonstrated the scope of this method with respect to both the scaffold and cofactor components and established that the dirhodium ArMs generated can catalyze the decomposition of diazo compounds and both Si?H and olefin insertion reactions involving these carbene precursors.     

Recent advances and potential applications of the photo‐induced macroscopic response of self‐assembled azopolymer in aqueous media

This mini‐review highlights issues related to the photo‐triggered macroscopic response of azopolymer in supramolecular hydrogel and polymer vesicles. Cyclodextrin (CD) can form an inclusion complex with trans‐azobenzene while cis‐azobenzene is expelled out of the CD's cavity. This photo‐reversible supramolecular host ? guest interaction is used to trigger sol ? gel transitions and to induce macroscopic assembly. When substituted azobenzene is located at chain ends of linear polyethylene glycol, monodisperse vesicles are formed exhibiting a photo‐induced pulsating behavior. The macroscopic phase transition and assembly or pulsating behavior of azopolymers can be expected to have potential applications as smart biomedical materials. © 2014 Society of Chemical Industry     

Preparation and characterization of nanohydroxyapatite strengthening nanofibrous poly(L‐lactide) scaffold for bone tissue engineering

A biomimetic nanofibrous poly(L ‐lactide) scaffold strengthened by nanohydroxyapatite particles was fabricated via a thermally induced phase separation technique. Scanning electron microscopy results showed that nanohydroxyapatite particles uniformly dispersed in the nanofibrous poly(L ‐lactide) scaffold (50–500 nm in fiber diameter) with slight aggregation at a high nHA content, but showed no influence on the interconnected macroporous and nanofibrous structure of the scaffold. The nanofibrous poly(L ‐lactide) scaffold presented a specific surface area of 34.06 m² g^?1, which was much higher than that of 2.79 m² g^?1 for the poly(L ‐lactide) scaffold with platelet structure. Moreover, the specific surface area of the nanofibrous scaffold was further enhanced by incorporating nanohydroxyapatite particles. With increasing the nanohydroxyapatite content, the compressive modulus and amount of bovine serum albumin adsorbed on the surface of the nanofibrous composite scaffold were markedly improved, as opposed to the decreased crystallinity. In comparison to poly(L ‐lactide) scaffold, both the nanofibrous poly(L ‐lactide) and poly(L ‐lactide)/nanohydroxyapatite scaffolds exhibited a faster degradation rate for their much larger specific surface area. The culture of bone mesenchymal stem cell indicated that the composite nanofibrous poly(L ‐lactide) scaffold with 50 wt % nanohydroxyapatite showed the highest cells viability among various poly(L ‐lactide)‐based scaffolds. The strengthened biomimetic nanofibrous poly(L ‐lactide)/nanohydroxyapatite composite scaffold will be a potential candidate for bone tissue engineering. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Preparation and in vitro degradation of novel bioactive polylactide/wollastonite scaffolds

Composite scaffolds for applications in bone engineering from poly(D,L ‐lactide) (PDLLA) incorporated with different proportional bioactive wollastonite powders were prepared through a salt‐leaching method, using NH₄HCO₃ as porogen. The pore structures and morphology of the scaffolds were determined by scanning electron microscopy (SEM). The bioactivity of composite materials was evaluated by examining its ability to initiate the formation of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂)(HAp) on its surface when immersed in simulated body fluids (SBF). The in vitro degradation behaviors of these scaffolds were systematically monitored at varying time periods of 1, 2, 4, 6, 8, 11, 14, 17, 20, 24, and 28 weeks postimmersion in SBF at 37°C. FT‐IR, XPS, XRD, and SEM measurements revealed that hydroxyapatite commenced to form on the surface of the scaffolds after 7 days of immersion in SBF. The measurements of weight loss, pH, and molecular weight of the samples indicated that PDLLA/wollastonite composite scaffolds degraded slower than the pure PDLLA scaffolds do. Addition of wollastonite enhanced the mechanical property of the composite scaffolds. The in vitro osteoblast culture experiment confirmed the biocompatibility of the scaffold for the growth of osteo‐blasts. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009.     

Cationic peptides that increase the thermal stabilities of 2'-O-MeRNA/RNA duplexes but do not affect DNA/DNA melting

Several different cationic nonapeptides have been synthesized and investigated with respect to how they can influence the thermal melting of 2′‐O‐methylRNA/RNA and DNA/DNA duplexes. Each peptide has a C‐terminal L ‐phenylalanine unit and is otherwise uniformly composed of a sequence of a specific basic D ‐amino acid that in most cases will be largely charged at neutral pH. These N‐terminal octamer stretches are composed variously of the amino acids D ‐lysine, D ‐diaminobutyric acid (D ‐Dab), D ‐diaminopropionic acid (D ‐Dap), or D ‐histidine. None of the peptides substantially affected the thermal melting of DNA/DNA duplexes, which was in sharp contrast with their effects on 2′‐O‐methylRNA/RNA duplexes. In particular, the peptides based on diaminopropionic and diaminobutyric acid units had strong positive effects on the melting temperatures of the 2′‐O‐methylRNA duplexes (up to 16 °C higher with 1 equivalent of peptide) at pH 7, whereas at pH 6 the effect was even more drastic (ΔT_m up to +25 °C). The shorter R groups of the Dap and Dab groups appear to have a better length than lysine for enhancement of the thermal melting of the 2′‐O‐methylRNA/RNA duplex, an effect that is more pronounced at lower pH but substantial even at pH 7, although the Dap derivative is not likely to be fully protonated. The dramatic difference between the influence, or lack thereof, on the 2′‐O‐methylRNA/RNA and the DNA/DNA thermal meltings suggest that, although electrostatic interactions probably play a role, there is another major and structurally dependent component influencing the properties of the duplexes. This is also seen in the observation that the oligo‐Dap and oligo‐Dab peptides give greater melting point enhancements than both the lysine peptide (with a longer side chain) and a β‐linked Dap peptide with a shorter side chain and a longer backbone.     

Ionomeric polyurethanes of pyridinium type with side azobenzene groups

New polyurethane cationomers synthesized by a two‐step substitution postreaction of urethane hydrogen atoms with nitroazobenzene groups were studied. As a starting polymer, a polyurethane based on poly(tetramethylene oxide)diol, isophorone diisocyanate, and 2,6‐bis(hydroximethyl)pyridine was used. After a preliminary metalation of the above polymer with natrium hydride, by reaction of polyurethane N‐sodate with 4‐nitro‐4′(β–iodoethylurethane)azobenzene, chromophoric groups between 2.85 and 10.53 wt % could be incorporated instead of hydrogen. Such polymers partially functionalized with azobenzene and further quaternized with methyl iodide led to the formation of pyridinium polyurethane cationomers N‐modified with nitroazo groups. The photosensible properties of the azobenzene chromophore in a polymer solution and film state indicated important differences in their photoresponse. In the polymer solution, the trans–cis photoisomerization of the chromophore is accompanied by an irreversible photobleaching effect, while under the same UV irradiation conditions, the ionomeric films exhibited an enhanced photostability. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 1240–1247, 2002     

Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers

Xeno nucleic acids (XNAs) are analogues of DNA and RNA that have a non-ribose artificial scaffold. XNAs are possible prebiotic genetic carriers as well as alternative genetic systems in artificial life. In addition, XNA oligomers can be used as biological tools. Acyclic XNAs, which do not have cyclic scaffolds, are attractive due to facile their synthesis and remarkably high nuclease resistance. To maximize the performance of XNAs, a negatively charged backbone is preferable to provide sufficient water solubility; however, acyclic XNAs containing polyanionic backbones suffer from high entropy cost upon duplex formation, because of the high flexibility of the acyclic nature. Herein, we review the relationships between the structure and duplex hybridization properties of various acyclic XNA oligomers with polyanion backbones.     

Preparation,photoisomerization, and microfabrication with two‐photon polymerization of crosslinked azo‐polymers

We report the preparation, photoisomerization properties, and three‐dimensional (3D) microstructure fabrication with two‐photon polymerization of crosslinked azo‐polymers. A series of bi‐acrylate‐substituted azobenzene derivatives were designed and synthesized as the monomers and/or crosslinkers of the crosslinked azo‐polymers. The doping concentration of the derivatives in pre‐polymer resins was significantly increased due to the introduction of bulky tert‐butyl and flexible alkyl chains. The double‐exponential dynamics of trans‐to‐cis photoisomerization of the azo‐polymers indicated the coexistence of different processes for the azobenzene moieties in the polymeric crosslinked networks. The crosslinked azo‐polymers exhibited ideal “on–off” switching performance in the highly reversible trans–cis–trans isomerization cycles. Furthermore, we prepared a photoresist containing the azobenzene derivative for 3D microstructure fabrication based on two‐photon polymerization. A woodpile photonic crystal with a photonic bandgap at telecommunication wavelength region was successfully fabricated with the azobenzene‐containing photoresist, which would open the way for the design and manufacturing of miniature optical communication devices. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 2947–2956, 2013     

Ultrasensitive Molecular Beacon Designed with Totally Serinol Nucleic Acid (SNA) for Monitoring mRNA in Cells

An artificial nucleic acid based on acyclic serinol building blocks and termed “serinol nucleic acid” (SNA) was used to construct a fluorescent probe for RNA visualization in cells. The molecular beacon (MB) composed of only SNA with a fluorophore at one terminus and a quencher at the other was resistant to enzymatic digestion, due to its unnatural acyclic scaffold. The SNA‐MB could detect its complementary RNA with extremely high sensitivity; the signal‐to‐background (S/B) ratio was as high as 930 when perylene and anthraquinone were used as the fluorophore and quencher pair. A high S/B ratio was also achieved with SNA‐MB tethering the conventional Cy3 fluorophore, and this probe enabled selective visualization of target mRNA in fixed cells. Thus, SNA‐MB has potential for use as a biological tool capable of visualizing RNA in living cells.