Fluorous Tag Research Articles

Gold(i)-catalyzed Nicholas reaction with aromatic molecules utilizing a bifunctional propargyl dicobalt hexacarbonyl complex.

A benchtop-stable reagent for the catalytic Nicholas reaction was developed. By combining a propargyl dicobalt hexacarbonyl cluster with an ortho-alkynylbenzoate unit and a fluorous tag, introduction of a propargyl hexacarbonyl complex on various aromatic compounds having acid- or base-sensitive functional groups becomes possible by using a gold(i) catalyst. In addition, the presence of a fluorous tag facilitates convenient separation of the target products from byproducts.

Integrated Pipeline of Isotopic Labeling and Selective Enriching for Quantitative Analysis of N-Glycome by Mass Spectrometry.

Quantitative N-glycomics can reveal abnormal expression of N-glycan in diseases. However, mass spectrometry (MS)-based N-glycome quantitative analysis is still technically challenging. To achieve the quantitation of N-glycome with high accuracy and sensitivity, it is required to efficiently label the N-glycans with isotopic tags and selectively enrich N-glycans to avoid suppression from other substances. Herein, we developed an integrated pipeline that combines isotopically fluorous tag labeling and fluorous solid-phase extraction to quantitatively analyze the N-glycome by MS. In this strategy, the N-glycans were labeled with light and heavy aminoxy-functionalized fluorous tags (PFBHA and PFBHA- d2) through the oxime click reaction. Then through the fluorous solid-phase extraction, the fluorous tag labeled N-glycan could be purified from contaminants like salts and proteins for the following quantitative analysis by mass spectrometry. This new approach enables selective purification (molar ratio of glycan to protein at 1:100) and accurate ( R2 > 0.99) and reproducible (coefficient of variation (CV)) < 25%, n = 6) quantitation of N-glycans within 2 orders of magnitude. Uniquely, diagnostic ions (D and [D-221]) were generated in tandem MS analysis after fluorous tags labeling, which could be used to deduce the composition of the 6-antenna and to distinguish isomers. Finally, this strategy was successfully applied to analyze the N-glycan changes in human serum associated with hepatocellular carcinoma (HCC). Fifteen N-glycan compositions with bisecting GlcNAc, sialic acid, and core fucosylation showed significant differences in HCC serum.

Some Like It Branched: Fluorous Tags for Enhanced Solubility and Biocompatibility

Some Like It Branched: Fluorous Tags for Enhanced Solubility and Biocompatibility

A General Approach to Biocompatible Branched Fluorous Tags for Increased Solubility in Perfluorocarbon Solvents.

A modular, cost-effective route to a library of branched fluorous tags with two short, biocompatible, fluorinated chains (C6F13) is reported. These branched fluorous tags provide high fluorous content without the use of long-chain linear perfluorocarbons, which have rising health concerns due to their bioaccumulation. By attaching these tags to a porphyrin, it is demonstrated that high solubility can be achieved in fluorous solvents that are readily cleared from mammals. This work enhances the biocompatibility of perfluorocarbon nanoemulsions for photodynamic therapy.

Water‐Soluble Sulfo‐Fluorous Affinity (SOFA) Tag‐Assisted Enzymatic Synthesis of Oligosaccharides

AbstractHerein, we report a bifunctional sulfo‐fluorous affinity (SOFA) tag‐assisted enzymatic synthesis and purification strategy for the facile preparation of bioactive glycans using fluorous solid‐phase extraction (FSPE). The incorporation of a sulfonate moiety onto the heavy fluorous tag significantly increases its water solubility, which allows the broad use of the inherently hydrophobic fluorous tag in aqueous buffers. In addition, the SOFA tag contains a photocleavable linker, enabling the easy release of amino‐functionalized oligosaccharides by UV irradiation. The SOFA tag was used in the synthesis of both negatively charged and neutral glycans to demonstrate its broad utility as an acceptor toward six different glycosyltransferases, significantly improving the feasibility of the preparation of complex glycans using FSPE. All the reactions were performed in an aqueous buffer, a minimum amount of methanol was used to purify the products, and the SOFA tag was easily recovered after photo‐irradiation. Thus, the entire synthetic process is environmentally benign.magnified image

Streamlined Synthesis and Evaluation of Teichoic Acid Fragments.

Teichoic acids (TAs) are key components of the Gram‐positive bacterial cell wall that are composed of alditol phosphate repeating units, decorated with alanine or carbohydrate appendages. Because of their microhetereogeneity, pure well‐defined TAs for biological or immunological evaluation cannot be obtained from natural sources. We present here a streamlined automated solid‐phase synthesis approach for the rapid generation of well‐defined glycosylated, glycerol‐based TA oligomers. Building on the use of a “universal” linker system and fluorous tag purification strategy, a library of glycerolphosphate pentadecamers, decorated with various carbohydrate appendages, is generated. These are used to create a structurally diverse TA‐microarray, which is used to reveal, for the first time, the binding preferences of anti‐LTA (lipoteichoic acids) antibodies at the molecular level.

Fluorous-tag assisted synthesis of a glycosaminoglycan mimetic tetrasaccharide as a high-affinity FGF-2 and midkine ligand

Here, we present the preparation of a sulfated, fully protected tetrasaccharide derivative following the glycosaminoglycan (GAG)-related sequence GlcNAc-β(1 → 4)-Glc-β(1 → 3). The tetramer was efficiently assembled via an iterative glycosylation strategy using monosaccharide building blocks. A fluorous tag was attached at position 6 of the reducing end unit enabling the purification of reaction intermediates by simple fluorous solid phase extraction. Fluorescence polarization competition experiments revealed that the synthesized tetrasaccharide strongly interacts with two heparin-binding growth factors, midkine and FGF-2 (IC50 of 270 nM and 2.4 µM, respectively). Our data indicate that this type of oligosaccharide derivatives, displaying sulfates, hydrophobic protecting groups and a fluorinated tail can be considered as interesting GAG mimetics for the regulation of relevant carbohydrate-protein interactions.

Fluorous-Phase Approach to α-Hydroxytropolone Synthesis.

α-Hydroxytropolones (αHTs) are troponoids that demonstrate inhibition against an array of therapeutically significant targets, making them potential drug leads for several human diseases. We have utilized a recently discovered one-pot three-component oxidopyrylium cycloaddition in a solid-supported synthesis of αHTs. Though the procedure is time efficient and generates assay-ready molecules, the system suffers from low yields and an inability to perform reaction modifications on resin-bound intermediates. In order to combat these issues with the solid-phase platform, we incorporated fluorous tags into our synthetic route. Through the implementation of fluorous phase chemistry, we demonstrate a substantial increase in the overall yield of αHTs, as well as an ability to execute metal-catalyzed cross coupling and amide coupling on fluorous tagged intermediates. We also show that tagged molecules can be separated from nonfluorous impurities, and vice versa, by utilizing fluorous liquid-liquid and solid-phase extractions. Hence, these proof-of-principle investigations describe the viability of a fluorous phase approach to αHT synthesis and its potential to serve as a combinatorial technique to produce structurally diverse substrates.

Fabrication of a protein microarray by fluorous-fluorous interactions

Fluorous-modified surfaces have emerged as a powerful tool for the immobilization of fluorous-tagged biomolecules based on their specificity and the strength of fluorous-fluorous interactions. To fabricate a fluorous-based protein microarray, we designed two strategies for site-specific modification of proteins with a fluorous tag: attaching the fluorous tag to the C-termini of expressed proteins by native chemical ligation (NCL) or to the Fc domain of antibodies through boronic acid (BA)-diol interactions. The perfluoro-tagged proteins could be easily purified by fluorous-functionalized magnetic nanoparticles (MNPs) and immobilized on a fluorous chip with minimal non-specific adsorption. Importantly, proteins immobilized on the solid support through non-covalent fluorous-fluorous interactions were sufficiently stable to withstand continuous washing. We believe that this fluorous-fluorous immobilization strategy will be a highly valuable tool in protein microarray fabrication.

Recyclable benzyl-type fluorous tags: Preparation and application in oligosaccharide synthesis

We herein described the design, synthesis and application of two recyclable benzyl-type fluorous tags with double fluorous chains. The benzyl-type fluorous tags were prepared in 3 steps from a commercially available fluorous alcohol. The glycosylation of the benzyl-type tags with imidate donors proceeded smoothly to provide the corresponding fluorous-tagged carbohydrates in good to excellent yields, which were readily purified by fluorous solid-phase extraction (FSPE). Efficient removal of the tags from tag-tethered carbohydrates were conducted under the common catalytic hydrogenation condition and the initial benzyl-type fluorous tags could be regenerated via a 2-step simple procedure in 69%–93% yields. The utility of the new benzyl-fluorous tag was demonstrated via the FSPE-assisted synthesis of oligosaccharides Gb3.

Highly Selective and Large Scale Mass Spectrometric Analysis of 4-Hydroxynonenal Modification via Fluorous Derivatization and Fluorous Solid-Phase Extraction.

Modification of proteins with 4-hydroxynonenal (HNE) is known to alter the function of proteins and regulate the associated biological processes in eukaryotic cells. The development of mass spectrometry (MS) makes high-throughput analysis of HNE modification accessible. However, the identification of HNE modification is still hampered by the low frequency of this modification. Therefore, only a limited number of HNE modification sites have been identified. The enrichment of HNE-modified peptides is critical for the MS analysis of this modification because of its low abundance. Herein, we explored a novel strategy for specifically extracting the HNE-modified peptides using fluorous derivatization through oxime click chemistry combined with following fluorous solid-phase extraction (FSPE). This oxime click chemistry-based derivatization is highly efficient (with a yield of almost 100%) and fast (30 min). Because of the hydrophobicity of the fluorous tag, the signal of fluorous-derivatized HNE-modified peptides was greatly enhanced, making the detection of HNE-modified peptides sensitive. The FSPE further allowed the selective enrichment of fluorous-derivatized HNE-modified peptides from salt solutions and complex mixtures with specificity. Finally, 673 HNE modification sites (607 histidine sites, 60 cysteine sites, 5 lysine sites, and 1 arginine site) on 661 HNE-modified peptides mapped to 432 proteins were successfully identified using this novel approach, which presented the largest data set of HNE modification in MCF-7 cells. Three identified proteins were validated by Western blotting.

Grubbs–Hoveyda Second-Generation Catalysts Activated by the Introduction of a Light Fluorous Tag onto the Bidentate Ligands

Various novel Grubbs–Hoveyda second-generation catalysts activated by a fluorous tag on the ligands were prepared. The catalyst bearing the 1-naphthyl group on the bidentate ligand exhibited the highest catalytic activity among the studied catalysts for the ring-closing­-metathesis reaction of diethyl 2-allyl-2-(2-methylallyl)malonate.

Synthesis of Tetrasubstituted Thieno[3,2‐b]pyridin‐5(4H)‐one Derivatives as a Heterocyclic Scaffold for Multisite‐specific Fluorous Fluorescent Tagging and Fluorous Solid‐Phase Extraction

Tetrasubstituted thieno[3,2‐b]pyridin‐5(4H)‐one derivatives were selected as a highly functionalized heterocyclic scaffold for a multisite‐specific tagging process utilizing a previously devised fluorous fluorescent tag system. A suitable synthetic method was established for the 7‐alkoxy‐2,4,6‐trisubstituted‐thieno[3,2‐b]pyridin‐5(4H)‐one derivatives, and incorporating t‐butoxycarbonyl‐functionalized building blocks into the reaction sequence produced precursors that could be used in the tagging process. Fluorous solid‐phase extraction facilitated the purification of the tagged target compounds after a series of reactions, including t‐Bu deprotection/N‐hydroxysuccinimidyl ester formation/amidation.

ChemInform Abstract: Development of Advanced Macrosphelides: Potent Anticancer Agents

AbstractReview: 44 refs.

Development of Ionic Liquid-Tagged Carbohydrate Building Block for Electrochemical Glycosylation

Recently we have developed an electrochemical method for oligosaccharide synthesis based on the automated electrochemical assembly (AEA) of carbohydrate building blocks. This method enables us to access fully protected oligosaccharides rapidly in a preparative scale; however, the assembly of building blocks is one of the steps of oligosaccharide synthesis. In order to increase efficiency of the entire process, it is important to introduce a strategy which facilitates separation and purification of oligosaccharides thus-obtained. Separation tags are small molecules which are covalently attached to the substrates to make the separation step easier and faster. There are several tags including a fluorous tag, an ionic liquid tag, and others which have already been introduced into oligosaccharide synthesis. In this study, we introduced an ionic liquid tag to the building block for electrochemical glycosylation, because we presume that an ionic liquid tag works not only as a separation tag but also as an electrolyte and a reservoir of the counter anion for the reactive intermediate of glycosylation. Here we report the development of a novel ionic liquid tag and its application for electrochemical glycosylation. The developed benzyl ether-type ionic liquid tags were prepared from the corresponding precursors that can be prepared in a single step by the reaction of tertiary amines and introduced to a thioglycoside building block also in a single step with high yields. Electrochemical glycosylation of the thioglycoside equipped with the ionic liquid tag affords the corresponding disaccharides which can be purified easily by washing with water and organic solvent sequentially. In the presence of palladium carbon under the hydrogen gas atmosphere, removal of the tag and benzyl protecting groups of hydroxyl groups can be performed to obtain the fully deprotected disaccharides.

Fluorous-Tag Assisted Syntheses of Sulfated Keratan Sulfate Oligosaccharide Fragments.

The first block iteration strategy for iterative solution-phase synthesis of protected keratan sulfate (KS)-like fragments is reported. Obstacles in a strategy using galactose-glucosamine (Gal-GlcNAc) modules led to the discovery of a differentially protected GlcNAc-Gal module that could be used to synthesize KS-like fragments using a fluorous tag that maintained solubility in organic solvents for purification of all intermediates via fluorous solid-phase extraction.

Automated Solution-Phase Synthesis of Insect Glycans to Probe the Binding Affinity of Pea Enation Mosaic Virus.

Pea enation mosaic virus (PEMV)—a plant RNA virus transmitted exclusively by aphids—causes disease in multiple food crops. However, the aphid-virus interactions required for disease transmission are poorly understood. For virus transmission, PEMV binds to a heavily glycosylated receptor aminopeptidase N in the pea aphid gut and is transcytosed across the gut epithelium into the aphid body cavity prior to release in saliva as the aphid feeds. To investigate the role of glycans in PEMV–aphid interactions and explore the possibility of viral control through blocking a glycan interaction, we synthesized insect N-glycan terminal trimannosides by automated solution-phase synthesis. The route features a mannose building block with C-5 ester enforcing a β-linkage, which also provides a site for subsequent chain extension. The resulting insect N-glycan terminal trimannosides with fluorous tags were used in a fluorous microarray to analyze binding with fluorescein isothiocyanate-labeled PEMV; however, no specific binding between the insect glycan and PEMV was detected. To confirm these microarray results, we removed the fluorous tag from the trimannosides for isothermal titration calorimetry studies with unlabeled PEMV. The ITC studies confirmed the microarray results and suggested that this particular glycan–PEMV interaction is not involved in virus uptake and transport through the aphid.

Assembly of a Complex Branched Oligosaccharide by Combining Fluorous-Supported Synthesis and Stereoselective Glycosylations using Anomeric Sulfonium Ions.

There is an urgent need to develop reliable strategies for the rapid assembly of complex oligosaccharides. This paper presents a set of strategically selected orthogonal protecting groups, glycosyl donors modified by a (S)-phenylthiomethylbenzyl ether at C-2, and a glycosyl acceptor containing a fluorous tag, which makes it possible to rapidly prepare complex branched oligosaccharides of biological importance. The C-2 auxiliary controlled the 1,2-cis anomeric selectivity of the various galactosylations. The orthogonal protecting groups, 2-naphthylmethyl ether (Nap) and levulinic ester (Lev), made it possible to generate glycosyl acceptors and allowed the installation of a crowded branching point. After the glycosylations, the chiral auxiliary could be removed using acidic conditions, which was compatible with the presence of the orthogonal protecting groups Lev and Nap, thereby allowing the efficient installation of 1,2-linked glycosides. The light fluorous tag made it possible to purify the compounds by a simple filtration method using silica gel modified by fluorocarbons. The set of building blocks was successfully employed for the preparation of the carbohydrate moiety of the GPI anchor of Trypanosoma brucei, which is a parasite that causes sleeping sickness in humans and similar diseases in domestic animals.

Extraction and Quantitation of Ketones and Aldehydes from Mammalian Cells Using Fluorous Tagging and Capillary LC-MS.

The extraction and quantitation of carbonyl metabolites from cell lysate was accomplished using a carbonyl-reactive fluorous tag and capillary liquid chromatography coupled to mass spectrometry (capLC-MS). Selective fluorous tagging for ketones and aldehydes provided a 30-fold increase in sensitivity using electrospray ionization MS. Separation of fluorous tagged carbonyl resulted in good separation of all components, and tandem MS was able to differentiate structural carbonyl isomers. The average limit of detection for carbonyl standards was 37 nM (range 1.5-250 nM), with linearity of R(2) > 0.99. Reproducibility for metabolites in cell lysate averaged 9% RSD. Human aortic endothelial cells (HAECs) were exposed to varying levels of glucose, and their carbonyl metabolite levels were quantified. Significant metabolite changes were seen in glycolysis and the propanoate pathway from a glucose challenge. Using an untargeted approach, 120 carbonyl metabolites were found to change in hyperglycemic HAECs. From this list of compounds, multiple metabolites from the pentose phosphate and tryptophan metabolic pathways were discovered. This system provides excellent sensitivity and quantitation of carbonyl metabolites without the need for isotope standards or labels.

Tag and Capture Flow Hydrogen Exchange Mass Spectrometry with a Fluorous-Immobilized Probe.

Analysis of complex mixtures of proteins by hydrogen exchange (HX) mass spectrometry (MS) is limited by one's ability to resolve the protein(s) of interest from the proteins that are not of interest. One strategy for overcoming this problem is to tag the target protein(s) to allow for rapid removal from the mixture for subsequent analysis. Here we illustrate a new solution involving fluorous conjugation of a retrievable probe. The appended fluorous tag allows for facile immobilization on a fluorous surface. When a target protein is passed over the immobilized probe molecule, it can be efficiently captured and then exposed to a flowing stream of deuterated buffer for hydrogen exchange. The utility of this method is illustrated for a model system of the Elongin BC protein complex bound to a peptide from HIV Vif. Efficient capture is demonstrated, and deuteration when immobilized was identical to deuteration in conventional solution-phase hydrogen exchange MS. Protein captured from a crude bacterial cell lysate could also be deuterated without the need for separate purification steps before HX MS. The advantages and disadvantages of the method are discussed in light of miniaturization and automation.