Synthesis of β-<i>C</i>-<i>galacto</i>-Pyranosides with Fluorine on the Pseudoanomeric Substituent

β-C-galacto-Pyranosides with CHF and CF2 substitutes for the glycosidic oxygen were prepared through a four-step sequence starting from a central 1-thio-1,2-O-isopropylidene acetal alcohol and different α-fluoro- and α,α-difluoro acids. The key step in the synthesis is the oxocarbenium cyclization of an intermediate enol ether-thioacetal to a C1-substituted glycal

Synthesis of β-<i>C</i>-<i>galacto</i>-Pyranosides with Fluorine on the Pseudoanomeric Substituent

β-C-galacto-Pyranosides with CHF and CF2 substitutes for the glycosidic oxygen were prepared through a four-step sequence starting from a central 1-thio-1,2-O-isopropylidene acetal alcohol and different α-fluoro- and α,α-difluoro acids. The key step in the synthesis is the oxocarbenium cyclization of an intermediate enol ether-thioacetal to a C1-substituted glycal

Synthesis and materialization of a reaction-diffusion French flag pattern

International audienceDuring embryo development, patterns of protein concentration appear in response to mor-phogen gradients. These patterns provide spatial and chemical information that directs the fate of the underlying cells. Here, we emulate this process within non-living matter and demonstrate the autonomous structuration of a synthetic material. Firstly, we use DNA-based reaction networks to synthesize a French flag, an archetypal pattern composed of three chemically-distinct zones with sharp borders whose synthetic analogue has remained elusive. A bistable network within a shallow concentration gradient creates an immobile, sharp and long-lasting concentration front through a reaction-diffusion mechanism. The combination of two bistable circuits generates a French flag pattern whose 'phenotype' can be reprogrammed by network mutation. Secondly, these concentration patterns control the macroscopic organization of DNA-decorated particles, inducing a French flag pattern of colloidal aggregation. This experimental framework could be used to test reaction-diffusion models and fabricate soft materials following an autonomous developmental program. From a chemist's perspective, biological matter has the astonishing capability of self-constructing into shapes that are predetermined, robust to varying environmental conditions and remarkably pre-

Synthesis of β-<i>C</i>-<i>galacto</i>-Pyranosides with Fluorine on the Pseudoanomeric Substituent

β-C-galacto-Pyranosides with CHF and CF2 substitutes for the glycosidic oxygen were prepared through a four-step sequence starting from a central 1-thio-1,2-O-isopropylidene acetal alcohol and different α-fluoro- and α,α-difluoro acids. The key step in the synthesis is the oxocarbenium cyclization of an intermediate enol ether-thioacetal to a C1-substituted glycal

Synthesis of β-<i>C</i>-<i>galacto</i>-Pyranosides with Fluorine on the Pseudoanomeric Substituent

β-C-galacto-Pyranosides with CHF and CF2 substitutes for the glycosidic oxygen were prepared through a four-step sequence starting from a central 1-thio-1,2-O-isopropylidene acetal alcohol and different α-fluoro- and α,α-difluoro acids. The key step in the synthesis is the oxocarbenium cyclization of an intermediate enol ether-thioacetal to a C1-substituted glycal

Sedimentation-based confinement of individual Giant Unilamellar Vesicles in microchamber arrays with a dynamically exchangeable outer medium

Giant unilamellar vesicles (GUVs) are an ideal model to study cellular membrane functions in vitro, yet difficult to manipulate due to their fragile nature, especially when subjected to dynamic change of their external microenvironment. Here, we introduce an original microfluidic concept for constrain-free confinement of individual GUVs in microchambers with a dynamically exchangeable outer medium. With this method, GUVs self-confine in an array of laterally separated microchambers by sedimentation, avoiding any mechanical constrain and membrane deformation while allowing time-resolved microscopy observation. A microfluidic channel above the chambers allows a diffusion-based exchange of the GUV outer medium that can be completed in a few seconds for fast-diffusing molecules to about one minute for large proteins in a viscous medium. We numerically establish the geometric and flow parameters optimizing medium exchange while preventing GUV from lifting out. We experimentally demonstrate that different aqueous solutions separated by air plugs can be flowed into the channel by taking advantage of a polydimethylsiloxane-based hydrophilic channel wall. We also exploit the possibility to manipulate microliter sample volumes and dynamically control the external environment of GUV for in situ observation of membrane binding protein cell-free expression. We find in particular that the membrane-targeting sequence of Bacillus subtilis MinD binds to GUVs and induces extensive membrane tubulation. This technically simple method offers a robust way to confine GUVs and dynamically control their outer medium, thus constituting an ideal platform to study the spatio-temporal response of reconstituted membranes and/or synthetic cell studies subjected to dynamic micro-environments

<i>N</i>‑Acetylgalactosamino Dendrons as Clearing Agents to Enhance Liver Targeting of Model Antibody-Fusion Protein

Dendrimer clearing agents represent a unique class of compounds for use in multistep targeting (MST) in radioimmunotherapy and imaging. These compounds were developed to facilitate the removal of excess tumor-targeting monoclonal antibody (mAb) prior to administration of the radionuclide to minimize exposure of normal tissue to radiation. Clearing agents are designed to capture the circulating mAb, and target it to the liver for metabolism. Glycodendrons are ideally suited for MST applications as these highly branched compounds are chemically well-defined, thus advantageous over heterogeneous macromolecules. Previous studies have described glycodendron 3 as a clearing agent for use in three-step MST protocols, and early in vivo assessment of 3 showed promise. However, synthetic challenges have hampered its availability for further development. In this report we describe a new sequence of chemical steps which enables the straightforward synthesis and analytical characterization of this class of dendrons. With accessibility and analytical identification solved, we sought to evaluate both lower and higher generation dendrons for hepatocyte targeting as well as clearance of a model protein. We prepared a series of clearing agents where a single biotin is connected to glycodendrons displaying four, eight, sixteen or thirty-two α-thio-N-acetylgalactosamine (α-SGalNAc) units, resulting in compounds with molecular weights ranging from 2 to 17 kDa, respectively. These compounds were fully characterized by LCMS and NMR. We then evaluated the capacity of these agents to clear a model 131I-labeled single chain variable fragment antibody-streptavidin (131I-scFv-SAv) fusion protein from blood and tissue in mice, and compared their clearing efficiencies to that of a 500 kDa dextran-biotin conjugate. Glycodendrons and dextran–biotin exhibited enhanced blood clearance of the scFv-SAv construct. Biodistribution analysis showed liver targeting/uptake of the scFv-SAv construct to be 2-fold higher for compounds 1 to 4, as well as for the 500 kDa dextran, over saline. Additionally, the data suggest the glycodendrons clear through the liver, whereas the dextran through reticuloendothelial system (RES) metabolism

Evaluation of Glycodendron and Synthetically Modified Dextran Clearing Agents for Multistep Targeting of Radioisotopes for Molecular Imaging and Radioimmunotherapy

A series of <i>N</i>-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific antitumor antibody streptavidin fusion protein (scFv₄-SA) <i>in vivo</i>, to improve tumor-to-normal tissue concentration ratios for multistep targeted (MST) radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with an average molecular weight of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogues (e.g., CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. <i>In vivo</i> studies included dynamic positron-emission tomography (PET) imaging of ¹²⁴I-labeled-scFv₄-SA clearance and dual-label biodistribution studies following MST directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a scFv₄-SA specific for an antitumor-associated glycoprotein (TAG-72); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of the ¹²⁴I-labeled-scFv₄-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e., biotin linked with an extended spacer, referred to herein as long-chain (LC)) can impact the binding to the antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor-to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv₄-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin