Dual‐micropillar‐based microfluidic platform for single embryonic stem cell‐derived neuronal differentiation

We developed the dual‐micropillar‐based microfluidic platform to direct embryonic stem (ES) cell fate. 4 × 4 dual‐micropillar‐based microfluidic platform consisted of 16 circular‐shaped outer micropillars and 8 saddle‐shaped inner micropillars in which single ES cells were cultured. We hypothesized that dual‐micropillar arrays would play an important role in controlling the shear stress and cell docking. Circular‐shaped outer micropillars minimized the shear stress, whereas saddle‐shaped inner micropillars allowed for docking of individual ES cells. We observed the effect of saddle‐shaped inner micropillars on cell docking in response to hydrodynamic resistance. We also demonstrated that ES cells cultured for 6 days within the dual‐micropillar‐based microfluidic platform differentiated into neural‐like cells. Therefore, this dual‐micropillar‐based microfluidic platform could be a potentially powerful method for screening of lineage commitments of single ES cells.     

A Phytochrome Sensory Domain Permits Receptor Activation by Red Light

Optogenetics and photopharmacology enable the spatio‐temporal control of cell and animal behavior by light. Although red light offers deep‐tissue penetration and minimal phototoxicity, very few red‐light‐sensitive optogenetic methods are currently available. We have now developed a red‐light‐induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases—the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non‐invasively in mammalian tissue and in multicolor cell‐signaling experiments. The light‐controlled dimerizer and red‐light‐activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light.     

Theoretical analysis of aggregation in block‐copolymer films: The optical signature

We focus this work on the theoretical investigation of the block‐copolymer poly[oxyoctyleneoxy‐(2,6‐dimethoxy‐1,4phenylene‐1,2‐ethinylene‐phenanthrene‐2,4diyl) named as LaPPS19, recently proposed for optoelectronic applications. We used for that a variety of methods, from molecular mechanics to quantum semiempirical techniques (AM1, ZINDO/S‐CIS). Our results show that as expected isolated LaPPS19 chains present relevant electron localization over the phenanthrene group. We found, however, that LaPPS19 could assemble in a π‐stacked form, leading to impressive interchain interaction; the stacking induces electronic delocalization between neighbor chains and introduces new states below the phenanthrene‐related absorption; these results allowed us to associate the red‐shift of the absorption edge, seen in the experimental results, to spontaneous π‐stack aggregation of the chains. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

Π‐Stacking Functionalization of Carbon Nanotubes through Micelle Swelling

We report on a new, original and efficient method for π‐stacking functionalization of single‐wall carbon nanotubes. This method is applied to the synthesis of a high‐yield light‐harvesting system combining single‐wall carbon nanotubes and porphyrin molecules. We developed a micelle‐swelling technique that leads to controlled and stable complexes presenting an efficient energy transfer. We demonstrate the key role of the organic solvent in the functionalization mechanism. By swelling the micelles, the solvent helps the non‐water‐soluble porphyrins to reach the micelle core and allows a strong enhancement of the interaction between porphyrins and nanotubes. This technique opens new avenues for the functionalization of carbon nanostructures.     

Incorporation of Non‐canonical Amino Acids into 2,5‐Diketopiperazines by Cyclodipeptide Synthases

The manipulation of natural product biosynthetic pathways is a powerful means of expanding the chemical diversity of bioactive molecules. 2,5‐diketopiperazines (2,5‐DKPs) have been widely developed by medicinal chemists, but their biological production is yet to be exploited. We introduce an in vivo method for incorporating non‐canonical amino acids (ncAAs) into 2,5‐DKPs using cyclodipeptide synthases (CDPSs), the enzymes responsible for scaffold assembly in many 2,5‐DKP biosynthetic pathways. CDPSs use aminoacyl‐tRNAs as substrates. We exploited the natural ability of aminoacyl‐tRNA synthetases to load ncAAs onto tRNAs. We found 26 ncAAs to be usable as substrates by CDPSs, leading to the enzymatic production of approximately 200 non‐canonical cyclodipeptides. CDPSs constitute an efficient enzymatic tool for the synthesis of highly diverse 2,5‐DKPs. Such diversity could be further expanded, for example, by using various cyclodipeptide‐tailoring enzymes found in 2,5‐DKP biosynthetic pathways.     

Cryogenic OrbiSIMS Localizes Semi‐Volatile Molecules in Biological Tissues

OrbiSIMS is a recently developed instrument for label‐free imaging of chemicals with micron spatial resolution and high mass resolution. We report a cryogenic workflow for OrbiSIMS (Cryo‐OrbiSIMS) that improves chemical detection of lipids and other biomolecules in tissues. Cryo‐OrbiSIMS boosts ionization yield and decreases ion‐beam induced fragmentation, greatly improving the detection of biomolecules such as triacylglycerides. It also increases chemical coverage to include molecules with intermediate or high vapor pressures, such as free fatty acids and semi‐volatile organic compounds (SVOCs). We find that Cryo‐OrbiSIMS reveals the hitherto unknown localization patterns of SVOCs with high spatial and chemical resolution in diverse plant, animal, and human tissues. We also show that Cryo‐OrbiSIMS can be combined with genetic analysis to identify enzymes regulating SVOC metabolism. Cryo‐OrbiSIMS is applicable to high resolution imaging of a wide variety of non‐volatile and semi‐volatile molecules across many areas of biomedicine.     

In Vitro Selection of pH‐Activated DNA Nanostructures

We report the first in vitro selection of DNA nanostructures that switch their conformation when triggered by change in pH. Previously, most pH‐active nanostructures were designed using known pH‐active motifs, such as the i‐motif or the triplex structure. In contrast, we performed de novo selections starting from a random library and generated nanostructures that can sequester and release Mipomersen, a clinically approved antisense DNA drug, in response to pH change. We demonstrate extraordinary pH‐selectivity, releasing up to 714‐fold more Mipomersen at pH 5.2 compared to pH 7.5. Interestingly, none of our nanostructures showed significant sequence similarity to known pH‐sensitive motifs, suggesting that they may operate via novel structure‐switching mechanisms. We believe our selection scheme is general and could be adopted for generating DNA nanostructures for many applications including drug delivery, sensors and pH‐active surfaces.     

Isolation and desalting with cation‐exchange chromatography for compound‐specific nitrogen isotope analysis of amino acids: application to biogeochemical samples

We have established a procedure for removing interfering materials from extracts of geological and biological samples, in order to determine precise compound‐specific nitrogen isotopic compositions of amino acids. We employed cation‐exchange chromatography of protein and non‐protein amino acids prior to derivatization for gas chromatographic separation. The average recovery of a standard amino acid solution was better than 94%, without nitrogen isotope fractionation during the cation‐exchange chromatography. We applied the procedure to various environmental samples including ‘hard’ (calcareous, siliceous, rock and sediment samples) and ‘soft’ materials (aggregated microbial samples and biological soft tissue samples). We conclude that cation‐exchange chromatography is a pre‐treatment procedure which should be widely useful for the determination of compound‐specific nitrogen isotopic compositions of amino acids. Copyright © 2010 John Wiley & Sons, Ltd.     

Automated,Accelerated Nanoscale Synthesis of Iminopyrrolidines

Miniaturization and acceleration of synthetic chemistry is an emerging area in pharmaceutical, agrochemical, and materials research and development. Herein, we describe the synthesis of iminopyrrolidine‐2‐carboxylic acid derivatives using chiral glutamine, oxo components, and isocyanide building blocks in an unprecedented Ugi‐3‐component reaction. We used I‐DOT, a positive‐pressure‐based low‐volume and non‐contact dispensing technology to prepare more than 1000 different derivatives in a fully automated fashion. In general, the reaction is stereoselective, proceeds in good yields, and tolerates a wide variety of functional groups. We exemplify a pipeline of fast and efficient nanomole‐scale scouting to millimole‐scale synthesis for the discovery of a useful novel reaction with great scope.     

Self‐Healing,Expansion–Contraction,and Shape‐Memory Properties of a Preorganized Supramolecular Hydrogel through Host–Guest Interactions

Supramolecular materials cross‐linked between polymer chains by noncovalent bonds have the potential to provide dynamic functions that are not produced by covalently cross‐linked polymeric materials. We focused on the formation of supramolecular polymeric materials through host–guest interactions: a powerful method for the creation of nonconventional materials. We employed two different kinds of host–guest inclusion complexes of β‐cyclodextrin (βCD) with adamantane (Ad) and ferrocene (Fc) to bind polymers together to form a supramolecular hydrogel (βCD‐Ad‐Fc gel). The βCD‐Ad‐Fc gel showed self‐healing ability when damaged and responded to redox stimuli by expansion or contraction. Moreover, the βCD‐Ad‐Fc gel showed a redox‐responsive shape‐morphing effect. We thus succeeded in deriving three functions from the introduction of two kinds of functional units into a supramolecular material.     

Self‐Healing,Expansion–Contraction,and Shape‐Memory Properties of a Preorganized Supramolecular Hydrogel through Host–Guest Interactions

Supramolecular materials cross‐linked between polymer chains by noncovalent bonds have the potential to provide dynamic functions that are not produced by covalently cross‐linked polymeric materials. We focused on the formation of supramolecular polymeric materials through host–guest interactions: a powerful method for the creation of nonconventional materials. We employed two different kinds of host–guest inclusion complexes of β‐cyclodextrin (βCD) with adamantane (Ad) and ferrocene (Fc) to bind polymers together to form a supramolecular hydrogel (βCD‐Ad‐Fc gel). The βCD‐Ad‐Fc gel showed self‐healing ability when damaged and responded to redox stimuli by expansion or contraction. Moreover, the βCD‐Ad‐Fc gel showed a redox‐responsive shape‐morphing effect. We thus succeeded in deriving three functions from the introduction of two kinds of functional units into a supramolecular material.     

A Usual G‐Protein‐Coupled Receptor in Unusual Membranes

G‐protein‐coupled receptors (GPCRs) are the largest family of membrane‐bound receptors and constitute about 50 % of all known drug targets. They offer great potential for membrane protein nanotechnologies. We report here a charge‐interaction‐directed reconstitution mechanism that induces spontaneous insertion of bovine rhodopsin, the eukaryotic GPCR, into both lipid‐ and polymer‐based artificial membranes. We reveal a new allosteric mode of rhodopsin activation incurred by the non‐biological membranes: the cationic membrane drives a transition from the inactive MI to the activated MII state in the absence of high [H⁺] or negative spontaneous curvature. We attribute this activation to the attractive charge interaction between the membrane surface and the deprotonated Glu134 residue of the rhodopsin‐conserved ERY sequence motif that helps break the cytoplasmic “ionic lock”. This study unveils a novel design concept of non‐biological membranes to reconstitute and harness GPCR functions in synthetic systems.     

Capillary zone electrophoresis tandem mass spectrometry detects low concentration host cell impurities in monoclonal antibodies

We have evaluated CZE‐ESI‐MS/MS for detection of trace amounts of host cell protein impurities in recombinant therapeutics. Compared to previously published procedures, we have optimized the buffer pH used in the formation of a pH junction to increase injection volume. We also prepared a 5‐point calibration curve by spiking 12 standard proteins into a solution of a human mAb. A custom CZE‐MS/MS system was used to analyze the tryptic digest of this mixture without depletion of the antibody. CZE generated a ～70‐min separation window (～90‐min total analysis duration) and ～300‐peak capacity. We also analyzed the sample using ultra‐performance LC‐MS/MS. CZE‐MS/MS generated approximately five times higher base peak intensity and more peptide identifications for low‐level spiked proteins. Both methods detected all proteins spiked at ～100 ppm level with respect to the antibody.     

DNA‐Catalyzed Introduction of Azide at Tyrosine for Peptide Modification

We show that DNA enzymes (deoxyribozymes) can introduce azide functional groups at tyrosine residues in peptide substrates. Using in vitro selection, we identified deoxyribozymes that transfer the 2′‐azido‐2′‐deoxyadenosine 5′‐monophosphoryl group (2′‐Az‐dAMP) from the analogous 5′‐triphosphate (2′‐Az‐dATP) onto the tyrosine hydroxyl group of a peptide, which is either tethered to a DNA anchor or free. Some of the new deoxyribozymes are general with regard to the amino acid residues surrounding the tyrosine, while other DNA enzymes are sequence‐selective. We use one of the new deoxyribozymes to modify free peptide substrates by attaching PEG moieties and fluorescent labels.     

Harnessing Effective Molarity to Design an Electrochemical DNA‐based Platform for Clinically Relevant Antibody Detection

Easy‐to‐use platforms for rapid antibody detection are likely to improve molecular diagnostics and immunotherapy monitoring. However, current technologies require multi‐step, time‐consuming procedures that limit their applicability in these fields. Herein, we demonstrate effective molarity‐driven electrochemical DNA‐based detection of target antibodies. We show a highly selective, signal‐on DNA‐based sensor that takes advantage of antibody‐binding‐induced increase of local concentration to detect clinically relevant antibodies in blood serum. The sensing platform is modular, rapid, and versatile and allows the detection of both IgG and IgE antibodies. We also demonstrate the possible use of this strategy for the monitoring of therapeutic monoclonal antibodies in body fluids. Our approach highlights the potential of harnessing effective molarity for the design of electrochemical sensing strategies.     

N‐doped cycloparaphenylenes: Tuning electronic properties for applications in thermally activated delayed fluorescence

We theoretically characterize a series of substituted cycloparaphenylene nanohoops to study the effect of incorporating an electron‐withdrawing group into their cyclic structure. We systematically vary the nature, position, and number of nitrogen‐containing acceptor groups in both neutral (pyridine) and charged forms (pyridinium and methylpyridinium) to provide insights into how this functionalization affects the structural, electronic, and optical properties of these systems. We focus also on the singlet‐triplet energy difference, with low values found, which might pave the way to further applications in the field of devices for light‐emitting applications providing a potential class of TADF‐based emitters.     

Reagent‐Based DOS: Developing a Diastereoselective Methodology to Access Spirocyclic‐ and Fused Heterocyclic Ring Systems

Herein, we report a diversity‐oriented‐synthesis (DOS) approach for the synthesis of biologically relevant molecular scaffolds. Our methodology enables the facile synthesis of fused N‐heterocycles, spirooxoindolones, tetrahydroquinolines, and fused N‐heterocycles. The two‐step sequence starts with a chiral‐bicyclic‐lactam‐directed enolate‐addition/substitution step. This step is followed by a ring‐closure onto the built‐in scaffold electrophile, thereby leading to stereoselective carbocycle‐ and spirocycle‐formation. We used in silico tools to calibrate our compounds with respect to chemical diversity and selected drug‐like properties. We evaluated the biological significance of our scaffolds by screening them in two cancer cell‐lines. In summary, our DOS methodology affords new, diverse scaffolds, thereby resulting in compounds that may have significance in medicinal chemistry.     

Cell‐Permeable Peptides Containing Cycloalanine Residues

We present here an efficient alternative to N‐methylation for the purpose of morphing protein‐binding peptides into more serum‐stable and cell‐permeable compounds. This involves the incorporation of a cycloalanine (CyAla) into a peptide in a way that avoids difficult coupling steps. We demonstrate the utility of this chemistry in creating a cell‐permeable derivative of a high‐affinity HIV Rev protein‐binding peptide.