Biotin Derivatives Research Articles

Pnictogen-Bonding Enzymes.

The objective of this study was to create artificial enzymes that capitalize on pnictogen bonding, a σ-hole interaction that is essentially absent in biocatalysis. For this purpose, stibine catalysts were equipped with a biotin derivative and combined with streptavidin mutants to identify an efficient transfer hydrogenation catalyst for the reduction of a fluorogenic quinoline substrate. Increased catalytic activity from wild-type streptavidin to the best mutants coincides with the depth of the σ hole on the Sb(V) center, and the emergence of saturation kinetic behavior. Michaelis-Menten analysis reveals transition-state recognition in the low micromolar range, more than three orders of magnitude stronger than the millimolar substrate recognition. Carboxylates preferred by the best mutants contribute to transition-state recognition by hydrogen-bonded ion pairing and anion-π interactions with the emerging pyridinium product. The emergence of challenging stereoselectivity in aqueous systems further emphasizes compatibility of pnictogen bonding with higher order systems catalysis.

Surface Functionalization of Gold Nanoparticles Using Alkyne Derivatives: Applications in Chemical Sensing.

Colloidal gold nanoparticles (AuNPs) are important nanomaterials for chemical sensing and therapeutics. For their application, it is vital to develop a reliable and robust surface functionalization method that can be applied to diverse functional molecules and offer better stability under harsh biological conditions. Currently, thiol (SH) is the most commonly used functional group for forming stable covalent bonds with AuNPs. However, thiolated molecules typically require complicated preparation procedures, are susceptible to oxidation, and are not compatible with many electrophiles and reducing groups. In this study, we report that surface functionalization of AuNPs can be achieved using alkyne derivatives, which exhibit several advantages over classical thiolation and peptide-bond methods, including straightforward preparation of alkyne derivatives, rapid and simple conjugation in buffers and complex media, higher conjugation efficiency, long-term stability, and resistance to decomposition under harsh conditions. Several alkynylated biotin and fluorescein derivatives are prepared, and the alkynylated-AuNPs are characterized using a lateral flow assay, gel electrophoresis, and spectroscopy techniques to investigate the conjugation efficiencies, size distributions, protein interaction properties, and binding mode of the Au-alkyne bond. We also demonstrate that alkynylated-AuNPs can be used for the sensitive detection of hydrogen peroxide and streptavidin proteins.

Antibacterial activity of Au(I), Pt(II), and Ir(III) biotin conjugates prepared by the iClick reaction: influence of the metal coordination sphere on the biological activity.

A series of biotin-functionalized transition metal complexes was prepared by iClick reaction from the corresponding azido complexes with a novel alkyne-functionalized biotin derivative ([Au(triazolatoR,R')(PPh3)], [Pt(dpb)(triazolatoR,R')], [Pt(triazolatoR,R')(terpy)]PF6, and [Ir(ppy)(triazolatoR,R')(terpy)]PF6 with dpb = 1,3-di(2-pyridyl)benzene, ppy = 2-phenylpyridine, and terpy = 2,2':6',2''-terpyridine and R = C6H5, R' = biotin). The complexes were compared to reference compounds lacking the biotin moiety. The binding affinity toward avidin and streptavidin was evaluated with the HABA assay as well as isothermal titration calorimetry (ITC). All compounds exhibit the same binding stoichiometry of complex-to-avidin of 4:1, but the ITC results show that the octahedral Ir(III) compound exhibits a higher binding affinity than the square-planar Pt(II) complex. The antibacterial activity of the compounds was evaluated on a series of Gram-negative and Gram-positive bacterial strains. In particular, the neutral Au(I) and Pt(II) complexes showed significant antibacterial activity against Staphylococcus aureus and Enterococcus faecium at very low micromolar concentrations. The cytotoxicity against a range of eukaryotic cell lines was studied and revealed that the octahedral Ir(III) complex was non-toxic, while the square-planar Pt(II) and linear Au(I) complexes displayed non-selective micromolar activity.

Development of Biotinylated Liposomes Encapsulating Metformin for Therapeutic Targeting of Inflammation-Based Diseases.

Inflammation is a physiological response to a damaging stimulus but sometimes can be the cause of the onset of neurodegenerative diseases, atherosclerosis, and cancer. These pathologies are characterized by the overexpression of inflammatory markers like endothelial adhesion molecules, such as Vascular Cell Adhesion Molecule-1 (VCAM-1). In the present work, the development of liposomes for therapeutic targeted delivery to inflamed endothelia is described. The idea is to exploit a three-step pretargeting system based on the biotin-avidin high-affinity interaction: the first step involves a previously described biotin derivative bearing a VCAM-1 binding peptide; in the second step, the avidin derivative NeutrAvidinTM, which strongly binds to the biotin moiety, is injected; the final step is the administration of biotinylated liposomes that would bind to NeutravidinTM immobilized onto VCAM-1 overexpressing endothelium. Stealth biotinylated liposomes, prepared via the thin film hydration method followed by extrusion and purification via size exclusion chromatography, have been thoroughly characterized for their chemico-physical and morphological features and loaded with metformin hydrochloride, a potential anti-inflammatory agent. The three-step system, tested in vitro on different cell lines via confocal microscopy, FACS analysis and metformin uptake, has proved its suitability for therapeutic applications.

Mass Spectrometry-Based Precise Identification of Citrullinated Histones via Limited Digestion and Biotin Derivative Tag Enrichment.

Histone citrullination is an essential epigenetic post-translational modification (PTM) that affects many important physiological and pathological processes, but effective tools to study histone citrullination are greatly limited due to several challenges, including the small mass shift caused by this PTM and its low abundance in biological systems. Although previous studies have reported frequent occurrences of histone citrullination, these methods failed to provide a high-throughput and site-specific strategy to detect histone citrullination. Recently, we developed a biotin thiol tag that enabled precise identification of protein citrullination coupled with mass spectrometry. However, very few histone citrullination sites were identified, likely due to the highly basic nature of these proteins. In this study, we develop a novel method utilizing limited digestion and biotin derivative tag enrichment to facilitate direct in vivo identification of citrullination sites on histones. We achieve improved coverage of histone identification via partial enzymatic digestion and lysine block by dimethylation. With biotin tag-assisted chemical derivatization and enrichment, we also achieve precise annotation of histone citrullination sites with high confidence. We further compare different fragmentation methods and find that the electron-transfer-dissociation-based approach enables the most in-depth analysis and characterization. In total, we unambiguously identify 18 unique citrullination sites on histones in human astrocytoma U87 cells, including 15 citrullinated sites being detected for the first time. Some of these citrullination sites are observed to exhibit noticeable alterations in response to DNA damage, which demonstrates the superiority of our strategy in understanding the roles of histone citrullination in critical biological processes.

Synthesis of Charge-Compensated nido-Carboranyl Derivatives of Sulfur-Containing Amino Acids and Biotin.

A new group of charge-compensated nido-carboranyl derivatives of sulfur-containing amino acids and biotin has been synthesized in which the boron atom in position 9 or 10 of carborane is attached to a positively charged sulfur atom. The possibilities of obtaining symmetrical B(10)-substituted and asymmetric B(9)-substituted nido-carboranes were studied. Using the example of (S)-methionine and D-biotin derivatives, water-soluble S-substituted charge-compensated nido-carboranes with free functional groups were prepared. The results obtained open up prospects for the development of potential boron delivery agents for BNCT as well as new bioactive compounds containing a negatively charged nido-carboranyl fragment bearing a positive charge on the sulfur atom associated with the boron cluster.

Smart bio-nano interface derived from zein protein as receptors for biotinyl moiety

Proteinaceous, tunable nanostructures of zein (prolamine of corn) were developed as biotinyl-specific receptors using a molecular imprinting technique. Sacrificial templates, such as latex beads (LB3) and anodized alumina membrane (AAM), have been used to control nanostructural patterns in biotin-imprinted zein (BMZ). Briefly, a methanolic solution of the zein-biotin complex was drop cast upon a self-organized LB3 and AAM templates on Au/quartz surfaces. Subsequent dissolution of these sacrificial templates affords highly oriented, predetermined, and uniformly grown hyperporous (300 nm) and nanowires (150 nm) motifs of zein (BMZ-LB3 and BMZ-AAM), as shown by scanning electron microscopy (SEM). Selective extraction of biotin molecular template cast-off site-selective biotin imprints within these zein nanostructures complementary to biotinyl moieties. Alternatively, biotin-imprinted zein nanoparticles (BMZ-Np) and thin film (BMZ-MeOH) were prepared by coacervation and drop casting methods, respectively. Density functional theoretical (DFT) studies reveal strong hydrogen-bonded interaction of biotin with serine and glutamine residues of zein. Quartz crystal microbalance (QCM) studies show remarkable sensitivity of the hyperporous-BMZ-LB3 and nanowires of BMZ-AAM towards biotin derivative (biotin methyl ester) by five (24.75 ± 1.34 Hz/mM) and four (18.19 ± 0.75 Hz/mM) times, respectively, higher than the BMZ-MeOH. Enhanced permeability features of the zein nanostructures, when templated with LB3, enable the QCM detection of biotin- or its derivatives down to 12.9 ng mL−1 from dairy products (Kefir). The outcome of this study shall be a key aspect in interfacing biological materials with micro-/nano-sensors and electronic devices for detecting pertinent analytes using sustainably developed biopolymer-based nanostructures.

Mirror-image streptavidin with specific binding to L-biotin, the unnatural enantiomer

The streptavidin–biotin system is known to have a very high affinity and specificity and is widely used in biochemical immunoassays and diagnostics. However, this method is affected by endogenous D-biotin in serum sample measurements (biotin interference). While several efforts using alternative high-affinity binding systems (e.g., genetically modified streptavidin and biotin derivatives) have been attempted, these efforts have all led to reduction in affinity. To solve this interference issue, the enantiomer of streptavidin was synthesized, which enabled specific binding to L-biotin. We successfully obtained a functional streptavidin molecule by peptide synthesis using D-amino acids and an in vitro folding technique. Several characterizations, including size exclusion chromatography (SEC), circular dichroism spectra (CD), and heat denaturation experiments collectively confirmed the higher-order enantiomer of natural streptavidin had been formed with comparable stability to the natural protein. L-biotin specific binding of this novel molecule enabled us to avoid biotin interference in affinity measurements using the Biacore system and enzyme-linked immunosorbent assay (ELISA). We propose the enantiomer of streptavidin as a potential candidate to replace the natural streptavidin–biotin system, even for in vivo use.

Differential Translation Activity Analysis Using Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) in Archaea.

The study of protein production and degradation in a quantitative and time-dependent manner is a major challenge to better understand cellular physiological response. Among available technologies bioorthogonal noncanonical amino acid tagging (BONCAT) is an efficient approach allowing for time-dependent labeling of proteins through the incorporation of chemically reactive noncanonical amino acids like L-azidohomoalanine (L-AHA). The azide-containing amino-acid derivative enables a highly efficient and specific reaction termed click chemistry, whereby the azide group of the L-AHA reacts with a reactive alkyne derivate, like dibenzocyclooctyne (DBCO) derivatives, using strain-promoted alkyne-azide cycloaddition (SPAAC). Moreover, available DBCO containing reagents are versatile and can be coupled to fluorophore (e.g., Cy7) or affinity tag (e.g., biotin) derivatives, for easy visualization and affinity purification, respectively.Here, we describe a step-by-step BONCAT protocol optimized for the model archaeon Haloferax volcanii , but which is also suitable to harness other biological systems. Finally, we also describe examples of downstream visualization, affinity purification of L-AHA-labeled proteins and differential expression analysis.In conclusion, the following BONCAT protocol expands the available toolkit to explore proteostasis using time-resolved semiquantitative proteomic analysis in archaea .

Lipid bilayer induces contraction of the denatured state ensemble of a helical-bundle membrane protein

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli (E. coli) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E.coli's lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

Synthesis, characterization, and vitro evaluation of new materials in vorinostat analogs containing biomolecules

Histone deacetylase inhibitors are newly emerging chemotherapeutic agents which have shown great potential in the treatment of many types of cancers. Vorinostat (SAHA) is one of the FDA-approved histone deacetylase inhibitors as a chemotherapeutic agent. However, these agents have many drawbacks which may limit their clinical application such as low oral bioavailability, and low intracellular concentration in solid tumors which may require high doses leading to side and maybe toxic effects. In silico design, synthesis and characterization of new histone deacetylase inhibitors with biomolecules (biotin and phenylalanine) as a novel moiety were achieved successfully as analogs to vorinostat, hopefully, to overcome its limitations and increase the targetability. MTT assay of compounds 2c and 3d (which are biotin and phenylalanine-linked hydroxamate derivatives) showed higher antiproliferative activity than SAHA and 5-FU against MCF7 and lower cytotoxic effect against NHF cell lines which were consistent with the docking study results. These findings are encouraging for the involvement of biomolecules in the future development of histone deacetylase inhibitors.

Proteomic dissection of large extracellular vesicle surfaceome unravels interactive surface platform.

The extracellular vesicle (EV) surface proteome (surfaceome) acts as a fundamental signalling gateway by bridging intra‐ and extracellular signalling networks, dictates EVs’ capacity to communicate and interact with their environment, and is a source of potential disease biomarkers and therapeutic targets. However, our understanding of surface protein composition of large EVs (L‐EVs, 100–800 nm, mean 310 nm, ATP5F1A, ATP5F1B, DHX9, GOT2, HSPA5, HSPD1, MDH2, STOML2), a major EV‐subtype that are distinct from small EVs (S‐EVs, 30–150 nm, mean 110 nm, CD44, CD63, CD81, CD82, CD9, PDCD6IP, SDCBP, TSG101) remains limited. Using a membrane impermeant derivative of biotin to capture surface proteins coupled to mass spectrometry analysis, we show that out of 4143 proteins identified in density‐gradient purified L‐EVs (1.07–1.11 g/mL, from multiple cancer cell lines), 961 proteins are surface accessible. The surface molecular diversity of L‐EVs include (i) bona fide plasma membrane anchored proteins (cluster of differentiation, transporters, receptors and GPI anchored proteins implicated in cell‐cell and cell‐ECM interactions); and (ii) membrane surface‐associated proteins (that are released by divalent ion chelator EDTA) implicated in actin cytoskeleton regulation, junction organization, glycolysis and platelet activation. Ligand‐receptor analysis of L‐EV surfaceome (e.g., ITGAV/ITGB1) uncovered interactome spanning 172 experimentally verified cognate binding partners (e.g., ANGPTL3, PLG, and VTN) with highest tissue enrichment for liver. Assessment of biotin inaccessible L‐EV proteome revealed enrichment for proteins belonging to COPI/II‐coated ER/Golgi‐derived vesicles and mitochondria. Additionally, despite common surface proteins identified in L‐EVs and S‐EVs, our data reveals surfaceome heterogeneity between the two EV‐subtype. Collectively, our study provides critical insights into diverse proteins operating at the interactive platform of L‐EVs and molecular leads for future studies seeking to decipher L‐EV heterogeneity and function.

Directed Nickel‐Catalyzed pseudo‐Anomeric C−H Alkynylation of Glycals as an Approach towards C‐Glycoconjugate Synthesis

AbstractThe synthesis of complex C‐glycoconjugates is presented here using a key directed nickel‐catalyzed C−H alkynylation step. Thanks to a bidentate amidoquinoline‐type directing group, the insertion of diverse alkynyl moieties onto the pseudo‐anomeric position of glycal substrates was performed on ten examples in moderate to good yields. These platforms were used as starting substrates in a click reaction with complex azides to form original C‐glycoconjugates. By this route, a C‐glycosylated amino acid, a C‐linked disaccharide and a C‐glycosylated biotin derivative were synthesized. Preliminary conditions to remove the directing group are also proposed.magnified image

Diversification of polyphosphate end-labeling viabridging molecules.

Investigation of the biological roles of inorganic polyphosphate has been facilitated by our previous development of a carbodiimide-based method for covalently coupling primary amine-containing molecules to the terminal phosphates of polyphosphate. We now extend that approach by optimizing the reaction conditions and using readily available “bridging molecules” containing a primary amine and an additional reactive moiety, including another primary amine, a thiol or a click chemistry reagent such as dibenzocyclooctyne. This two-step labeling method is used to covalently attach commercially available derivatives of biotin, peptide epitope tags, and fluorescent dyes to the terminal phosphates of polyphosphate. Additionally, we report three facile methods for purifying conjugated polyphosphate from excess reactants.

The small molecule antibody mimic SH7139 targets a family of HLA-DRs expressed by B-cell lymphomas and other solid cancers

Selective high-affinity ligands (SHALs) belong to a novel class of small-molecule cancer therapeutics that function as targeted prodrugs. SH7139, the most advanced of the SHAL drugs designed to bind to a unique β-subunit structural epitope located on HLA-DR10, has exhibited exceptional preclinical efficacy and safety profiles. A comparison of SH7139 and SH7129, a biotin derivative of the drug developed for use as a diagnostic, showed the incorporation of a biotin tag did not alter the SHALs ability to target or kill HLA-DR10 expressing Raji cells. The use of SH7129 in an immuno-histochemical type assay to stain peripheral blood mononuclear cells (PBMCs) obtained from individuals expressing specific HLA-DRB1 alleles has also revealed that in addition to HLA-DR10, seven other more commonly expressed HLA-DRs are targeted by the drug. Computational dockings of the SHAL’s recognition ligands to a number of HLA-DR structures explain, in part, why the targeting domains of SH7129 and SH7139 bind to some HLA-DRs but not others. The results also substantiate the selectivity of SH7129 and suggest it may prove useful as a companion diagnostic for pre-screening biopsy samples to identify those patients whose tumours should respond to SH7139 therapy.

Investigation of the structure of iron oxide nanoparticle assemblies in order to optimize the sensitivity of surface plasmon resonance-based sensors

Surface plasmon resonance (SPR) biosensors based on metal thin films are very attractive for detection of biomolecules. Nanoparticle assemblies were recently demonstrated to significantly enhance sensitivity. Here, we show that the fine control of the structure of nanoparticle assemblies is critical to optimize the sensitivity. Iron oxide (Fe3-δO4) nanoparticles were assembled onto a gold thin film by performing the Copper Catalyzed Alkyne-Azide cycloaddition (CuAAC) “click” reaction. A biotin derivative was grafted by CuAAC at the surface of nanoparticle assemblies in order to detect streptavidin (SA). The fine control of the size and of the density of nanoparticles allowed tuning the surface plasmon waves at the surface of the gold thin film in order to optimize the response of the sensors. The accessibility of biotin at the nanoparticle surface to SA was also investigated as a function of the interparticle distance. We show that the interplay between the volume fraction of iron oxide and the accessibility of biotin at the nanoparticle surface is critical to enhance the sensitivity of SPR sensors.

Acyl-CoA dehydrogenase long chain (ACADL) is a target protein of stylissatin A, an anti-inflammatory cyclic heptapeptide.

Stylissatin A (SA) is a cyclic heptapeptide isolated from the marine sponge Stylissa massa. SA shows anti-inflammatory activity against lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophage cells, but the detailed mechanism of action remains unclear. Here we report that D-Tyr1-tBuSA, a more potent SA derivative, inhibited production of the proinflammatory cytokines Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in LPS-stimulated RAW264.7 cells (EC50 = 1.4 and 5.9 μM, respectively). This compound also inhibited the LPS-stimulated expression of inducible nitric oxide synthase (iNOS) at 20 μM. Using a biotin derivative of SA, acyl-CoA dehydrogenase long chain (ACADL) was identified as a target protein of SA and its derivatives. It is proposed that SA and its derivatives might suppress the β-oxidation of fatty acids by ACADL, and the accumulation of fatty acids on macrophages would inhibit the nuclear factor-kappa B (NF-κB) signaling pathway and iNOS expression to show anti-inflammatory activity. Our research might provide a new mechanism of inflammation in macrophages, and contribute to the development of treatments for inflammatory diseases.

Electrochemically directed biofunctionalization of a lossy-mode resonance optical fiber sensor.

In this work, we present a direct electrochemical biofunctionalization of an indium-tin-oxide-coated lossy-mode resonance optical fiber sensor. The functionalization using a biotin derivative was performed by cyclic voltammetry in a 10 mM biotin hydrazide solution. All stages of the experiment were simultaneously verified with optical and electrochemical techniques. Performed measurements indicate the presence of a poly-biotin layer on the sensor's surface. Furthermore, dual-domain detection of 0.01 and 0.1 mg/mL of avidin confirms the sensor's viability for label-free detection.

Synthesis and biological evaluation of new antioxidant and antiproliferative chalcogenobiotin derivatives for bladder carcinoma treatment

Approximately 90% of bladder carcinomas are of the urothelial carcinoma type, which are characterized by high rates of recurrence and predisposition to progress to invasive tumors, representing one of the most costly neoplasms for health systems. Intravesical chemotherapy is a standard for the treatment of non-invasive bladder cancer. However, chemotherapy is usually aggressive and cytotoxic, which increases the death rates caused by cancer. Heterocyclic compounds which exhibit favorable pharmacokinetic and pharmacodynamic properties may enhance drug affinity for a target protein by targeting the treatment. Thus, this work presents the synthesis, characterization, and in vitro biological evaluation of new antioxidant (inhibition of lipid peroxidation, scavenging of free radical DPPH, and thiol peroxidase-like activity) and antiproliferative chalcogenobiotin derivatives and tests them against bladder carcinoma 5637 cells. A prominent response was obtained for the selected compounds, with tellurium biotin derivatives displaying effective antioxidant and antiproliferative activity. The effective compounds also demonstrated no toxicity in in vitro or in vivo studies.

Anticancer Activity of Biotin Polyoxomolybdate Bioconjugate

Objective: Polyoxometalates (POMs), polyanionic metal clusters, have been well recognized for their anticancer activity in recent decades. Despite their potential anticancer activity, normal cell toxicity is one of the pressing issues that prevent their further clinical applications. In this work, we synthesized a new bio-conjugate based on POM and biotin. Methods: We used biotin as a bio-molecule to control the cytotoxicity of POM on healthy cells and simultaneously increase the toxicity on cancerous cells. We synthesized the biotin derivative of POM via an amidation between the two molecules of biotin and amine groups on polyoxomolybdate. We approved the final structure using different spectroscopic data. We studied the cytotoxicity activity in-vitro using MTT protocol. We chose breast carcinoma cells (MCF-7) and hepatocellular carcinoma cells (HepG2) in comparison to the human umbilical vein endothelial cell (HUVEC). Results: Results showed that biotin could improve the anticancer activity of polyoxomoybdate (IC50; 0.082 mM) on MCF-7 and (IC50; 0.091 mM) on HepG2 cells. Furthermore, the biotin-polyoxomolybdate conjugate showed lower toxicity on healthy cells versus the parent polyoxomolybdate and the Cis-platin as an approved drug. Conclusion: Thus, we introduce promising novel POM bioconjugate, which can be further assessed in pre-clinical studies.