Fluorous Solid-phase Extraction Research Articles

Development of a fluorous trapping reagent for rapid detection of electrophilic reactive metabolites.

A cysteine-based fluorous trapping reagent, Rf8CYS, was developed. Rf8CYS formed adducts with soft and hard electrophilic reactive metabolites. These fluorous-tagged adducts were purified via both fluorous solid-phase extraction and the direct injection method. The highly sensitive mass spectrometric detection of an unprecedented adduct of the ticlopidine metabolite was realized.

Recyclable 2-Fluoropyridine Derivative as a Storage for Highly Electrophilic 1,1-Bis(triflyl)ethylene.

As an easy-to-handle reagent for the in situ generation of outstandingly electrophilic Tf2C=CH2 (Tf=CF3SO2), we have designed and synthesised a novel 4-substituted 2-fluoropyridinium zwitterion, in which a partially fluorinated alkyl group is attached to the pyridinium 4-position. Its zwitterionic nature has been well characterised by quantum chemical bonding analysis. By using this reagent, a wide variety of organic compounds, including commercial bioactive agents, were successfully decorated by the strongly acidic or ionic functionality. Remarkably, the 4-substituted 2-fluoropyridine derivative, which results from the zwitterion with the generation of Tf2C=CH2, can be rapidly separated and recovered from the reaction mixture appropriately using distillation, organic solvent extraction, or fluorous solid phase extraction techniques. Such multi-optionality for the purification methods favours in the isolation of the strongly acidic and/or ionic products.

Synthesis and interaction with growth factors of sulfated oligosaccharides containing an anomeric fluorinated tail

Compounds that mimic the biological properties of glycosaminoglycans (GAGs) and can be more easily prepared than the native GAG oligosaccharides are highly demanded. Here, we present the synthesis of sulfated oligosaccharides displaying a perfluorinated aliphatic tag at the reducing end as GAG mimetics. The preparation of these molecules was greatly facilitated by the presence of the fluorinated tail since the reaction intermediates were isolated by simple fluorous solid-phase extraction. Fluorescence polarization competition assays indicated that the synthesized oligosaccharides interacted with two heparin-binding growth factors, midkine (MK) and FGF-2, showing higher binding affinities than the natural oligosaccharides, and can be therefore considered as useful GAG mimetics. Moreover, NMR experiments showed that the 3D structure of these compounds is similar to that of the native sequences, in terms of sugar ring and glycosidic linkage conformations. Finally, we also demonstrated that these derivatives are able to block the MK-stimulating effect on NIH3T3 cells growth.

Querying the In Vitro Proteome Cysteine Reactivity of 8:2 Fluorotelomer Acrylate.

Despite the phase out of legacy per- and polyfluoroalkyl substances (PFAS), fluorotelomer-based polymers (FTP) have been used for many applications, notably textile surface coatings. FTPs are of a health concern due to their breakdown into legacy PFAS and the co-occurrence of fluorotelomer acrylate (FTAC) monomers, of which the latter may potentially react with cellular thiols. To evaluate this hypothesis, we employed fluorous-solid-phase extraction (FSPE), to enrich peptides covalently modified by 8:2 fluorotelomer acrylate (8:2 FTAC) and coupled it to a modified nano-liquid chromatography method for the identification of in vitro protein adducts using bottom-up data-dependent proteomics analysis. Using this method, over 100 unique peptides were detected with 8:2 FTAC modifications, although none of the modified cysteine residues were annotated active site nucleophiles. In parallel, a synthetic C6F13-iodoacetamide (F13-IAM) chemical probe was used to gauge the upper bound of PFAS-thiol reactivity. Over seven hundred peptides were detected with modifications but only 9 of 28 annotated active site cysteines in this dataset were modified by F13-IAM. Further exploration of the impacts of 8:2 FTAC adducts on protein function revealed that 8:2 FTAC modification promotes protein aggregation in vitro. These results suggest that 8:2 FTAC may exhibit significant proteome thiol reactivity and imply a more general mechanism of toxicity of PFAS-induced protein aggregation.

Tunable Fluorine-Functionalized Scholl-Coupled Microporous Polymer for the Selective Adsorption and Ultrasensitive Analysis of Environmental Liquid-Crystal Monomers.

Liquid-crystal monomers (LCMs), especially fluorinated biphenyls and analogues (FBAs), are identified to be an emerging generation of persistent organic pollutants. However, there is a dearth of information about their occurrence and distribution in environmental water and lacustrine soil samples. Herein, a series of fluorine-functionalized Scholl-coupled microporous polymers (FSMP-X, X = 1-3) were designed and synthesized for the highly efficient and selective enrichment of FABs. Their hydrophobicity, porosity, chemical stability, and adsorption performance (capacity, rate, and selectivity) were regulated preciously. The best-performing material (FSMP-2) was employed as the on-line fluorous solid-phase extraction (on-line FSPE) adsorbent owing to its high adsorption capacity (313.68 mg g-1), fast adsorption rate (1.05 g h-1), and specific selectivity for FBAs. Notably, an enrichment factor of up to 590.2 was obtained for FSMP-2, outperforming commercial C18 (12.6-fold). Also, the underlying adsorption mechanism was uncovered by density functional theory calculations and experiments. Based on this, a novel and automated on-line FSPE-high-performance liquid chromatography method was developed for ultrasensitive (detection limits: 0.0004-0.0150 ng mL-1) and low matrix effect (73.79-113.3%) determination of LCMs in lake water and lacustrine soils. This study offers new insight into the highly selective quantification of LCMs and the first evidence for their occurrence and distribution in these environmental samples.

A fluorous-tag-assisted fluorescent probe for simple and selective detection of hydrogen sulfide: application for turbid dyeing solutions.

Accurate hydrogen sulfide (H2S) detection has attracted much attention because its toxicity may affect aquatic environments and human health. However, recognizing H2S levels by conventional fluorescent probes in turbid wastewater has been challenging because the opaque environment interferes with their photophysical properties. To overcome this limitation, a fluorous-tagging strategy can be used for the development of fluorescent sensors to detect H2S in turbid solutions. The use of fluorescent probe assisted with fluorous-tag allowed for easy isolation of the probe using polytetrafluoroethylene (PTFE) material, while disturbing species were eliminated through a simple aqueous wash. This approach enabled the fluorescent probe to effectively quantify H2S, even in opaque solutions containing organic dyes that could interfere with fluorescence emission.

Diversity-Oriented Synthesis of a Molecular Library of Immunomodulatory α-Galactosylceramides with Fluorous-Tag-Assisted Purification and Evaluation of Their Bioactivities in Regard to IL-2 Secretion.

Structural variants of α-galactosylceramide (α-GalCer) that stimulate invariant natural killer T (iNKT) cells constitute an emerging class of immunomodulatory agents in development for numerous biological applications. Variations in lipid chain length and/or fatty acids in these glycoceramides selectively trigger specific pro-inflammatory responses. Studies that would link a specific function to a structurally distinct α-GalCer rely heavily on the availability of homogeneous and pure materials. To address this need, we report herein a general route to the diversification of the ceramide portion of α-GalCer glycolipids. Our convergent synthesis commences from common building blocks and relies on the Julia-Kocienski olefination as a key step. A cleavable fluorous tag is introduced at the non-reducing end of the sugar that facilitates quick purification of products by standard fluorous solid-phase extraction. The strategy enabled the rapid generation of a focused library of 61 α-GalCer analogs by efficiently assembling various lipids and fatty acids. Furthermore, when compared against parent α-GalCer in murine cells, many of these glycolipid variants were found to have iNKT cell stimulating activity similar to or greater than KRN7000. ELISA assaying indicated that glycolipids carrying short fatty N-acyl chains (1fc and 1ga), an unsubstituted (1fh and 1fi) or CF3-substituted phenyl ring at the lipid tail, and a flexible, shorter fatty acyl chain with an aromatic ring (1ge, 1gf, and 1gg) strongly affected the activation of iNKT cells by the glycolipid-loaded antigen-presenting molecule, CD1d. This indicates that the method may benefit the design of structural modifications to potent iNKT cell-binding glycolipids.

P-139 - Simple and quick indirect radioiodination to antibody without HPLC purification using fluorous solid-phase extraction and click reaction

P-139 - Simple and quick indirect radioiodination to antibody without HPLC purification using fluorous solid-phase extraction and click reaction

Fluorous-Tag-Assisted Synthesis of GAG-Like Oligosaccharides.

The classic, solution-phase synthesis of glycosaminoglycan (GAG) oligosaccharides is hampered by the numerous, time-consuming chromatographic purifications required for the isolation of the glycosylation products after each coupling step between sugar building blocks. Here, we present a detailed experimental procedure for a glycosylation reaction involving a glycosyl acceptor unit that is equipped with a perfluorinated tag. The presence of this fluorous tail allows the quick purification of the desired glycosylation product by performing a simple fluorous solid-phase extraction (F-SPE). The described fluorous-tag-assisted glycosylation strategy greatly facilitates the assembly of building blocks, speeding up the preparation of biologically relevant GAG-like oligomers.

FluoroTRAQ: Quantitative Analysis of Protein S-Nitrosylation through Fluorous Solid-Phase Extraction Combining with iTRAQ by Mass Spectrometry.

S-Nitrosylation is an important post-translational modification that occurs on cysteine amino acid and regulates signal transduction in diverse cell processes. Dysregulation of protein nitrosylation has shown close association with cardiovascular and neurological diseases, thus demanding further precise and in-depth understanding. Mass spectrometry-based proteomics has been the method of choice for analyzing S-nitrosylated (SNO-) proteins. However, due to their extremely low expression level and rapid turnover rate, quantitative analysis of the S-nitrosylation at the proteomic level remains challenging. Herein, we developed a novel approach termed FluoroTRAQ, which combined the fluorous solid-phase extraction of SNO-peptides and iTRAQ labeling for the quantitative analysis of the SNO-proteome with high sensitivity and specificity. This new analytical strategy was subsequently applied to examine the dynamic SNO-proteome changes of human umbilical vein endothelial cells upon in vitro S-nitrosoglutathione induction. Our data identified a number of novel SNO-proteins and revealed their temporal modulation as validated by biotin switch assay. Our study offered a practical approach for quantitative analysis of protein S-nitrosylation.

Dual isotopic labeling combined with fluorous solid-phase extraction for simultaneous discovery of neutral/sialylated N-glycans as biomarkers for gastric cancer

Mass spectrometry analysis coupled with stable isotope labeling is a powerful strategy for comparative analysis of N-glycans for glycan biomarker discovery. Here, a novel method combining dual isotopic labeling and fluorous solid-phase extraction was developed, enabling selective enrichment, simultaneous quantitation and recognition of neutral/sialylated glycans from serum samples by mass spectrometry. The isotopic label of fluorous compound on the reducing end of glycans acts as an enrichment tag and provides mass difference between neutral glycans from different samples, while the isotopic label on the non-reducing end of glycans protects the sialic acid residue and provides additional mass difference for sialylated glycans. Therefore, the neutral/sialylated glycans could be simultaneously enriched through the fluorous solid-phase extraction (FSPE) and quantified by mass spectrometry. This method provided a good linearity (R2 > 0.99) and high reproducibility (CV < 20%) within 2 orders of magnitude in the dynamic range. Finally, this strategy was successfully applied to investigate the N-glycome alteration in serum associated with gastric cancer (GC). Bisecting GlcNAc and triantennary glycan compositions were found to be significantly changed between GC cases (n = 50) and healthy control, indicating the great potential to be novel biomarkers for GC early diagnosis.

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.

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

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.

Advancement in enrichment methods for 4-Hydroxynonenal modified proteome

4-Hydroxynonenal (HNE) is an important modification with great biological characterization and clinical significance. The low-abundance HNE modified proteins need to be enriched before mass spectrometry analysis. Here we summarize the current research state and theory of enrichment methods (four methods: immunoaffinity enrichment, biotin-avidin enrichment, fluorous solid-phase extraction, solid-phase hydrazide enrichment) for HNE modified proteomes, and describe the advantages and disadvantages of each method. Finally, we discuss the search trends of the HNE modified proteins in future.

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.

Fluorous Solid-Phase Extraction Technique Based on Nanographite Fluoride.

Fluorous solid-phase extraction (FSPE) has been employed to isolate target compounds from complex chemical or biological samples in many research areas. However, the lack of efficient and economical available perfluorinated materials impeded its development. In this study, we present a novel nanographite fluoride-based fluorous solid-phase extraction (GF-FSPE) as a replacement of commercially available cartridge-based FSPE, which showed remarkable selectivity, low LOD, high post enrichment recovery, and large enrichment capacity. To demonstrate the potency of GF-FSPE, it was designed and successfully applied to isolate cysteine-containing peptide subsets from complex protein samples to improve the proteome coverage. Additionally, since graphite fluoride was inexpensive and highly commercialized, this study was expected to promote the popularization of FSPE in both chemical and biological separations as well as encourage the synthesis and broaden the application of highly fluorinated carbon fluoride in material science.

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.

Front Cover: Synthesis of Nontoxic Fluorous Sphingolipids as Molecular Probes of Exogenous Metabolic Studies for Rapid Enrichment by Fluorous Solid Phase Extraction (Eur. J. Org. Chem. 6/2017)

Front Cover: Synthesis of Nontoxic Fluorous Sphingolipids as Molecular Probes of Exogenous Metabolic Studies for Rapid Enrichment by Fluorous Solid Phase Extraction (Eur. J. Org. Chem. 6/2017)

Synthesis of Nontoxic Fluorous Sphingolipids as Molecular Probes of Exogenous Metabolic Studies for Rapid Enrichment by Fluorous Solid Phase Extraction

Fluorous solid‐phase extraction (FSPE) is a useful technique for efficient selective enrichment of fluorous compounds from nonfluorous molecules. Sphingolipids and their metabolites, which are ubiquitous building blocks of eukaryotic and prokaryotic cell membranes, play crucial roles, for example, as signaling molecules. However, details of the functions and metabolic mechanisms of exogenous sphingolipids have remained unknown compared with those of their endogenous analogs. To better understand these unknown roles, chemical probes with appropriate biological and physicochemical properties are needed. In this study, we designed and synthesized new fluorous sphingolipids to reveal these roles. Furthermore, we confirmed that they could be efficiently and rapidly separated from normal sphingolipids by FSPE, and that they hardly showed any cytotoxic activity, similarly to normal sphingolipids at the same dose. We also showed that these fluorinated ceramides could act as metabolic substrates for sphingomyelin synthase 2 (SMS2). This demonstrates their potential for further biological studies.