A bioorthogonally activatable photosensitiser for site-specific photodynamic therapy.

A boron dipyrromethene based photosensitiser substituted with a 1,2,4,5-tetrazine moiety has been prepared of which the photoactivity can be activated upon an inverse-electron-demand Diels-Alder reaction with trans-cyclooctene derivatives. By using a biotin-conjugated trans-cyclooctene to tag the biotin-receptor-positive HeLa cells, this photosensitiser exhibits site-specific activation through cycloaddition, leading to high photocytotoxicity.

A biotinylated ruthenium(ii) photosensitizer for tumor-targeted two-photon photodynamic therapy.

Platinum-resistant cancer cells are sensitive to changes in the levels of reactive oxidative species (ROS). Herein, we design a biotin-modified Ru(ii) complex as a photosensitizer (denoted as Ru-Biotin). Ru-Biotin can selectively target cancer cells and produce vast amounts of singlet oxygen under two-photon excitation at 820 nm leading to cell apoptosis. Ru-Biotin is therefore an excellent candidate to overcome platinum resistance via two-photon photodynamic therapy.

Synthesis of d-desthiobiotin-AI-2 as a novel chemical probe for autoinducer-2 quorum sensing receptors.

In processes regulated by quorum sensing (QS) bacteria respond to the concentration of autoinducers in the environment to engage in group behaviours. Autoinducer-2 (AI-2) is unique as it can foster interspecies communication. Currently, two AI-2 receptors are known, LuxP and LsrB, but bacteria lacking these receptors can also respond to AI-2. In this work, we present an efficient and reproducible synthesis of a novel chemical probe, d-desthiobiotin-AI-2. This probe binds both LuxP and LsrB receptors from different species of bacteria. Thus, this probe is able to bind receptors that recognise the two known biologically active forms of AI-2, presenting the plasticity essential for the identification of novel unknown AI-2 receptors. Moreover, a protocol to pull down receptors bound to d-desthiobiotin-AI-2 with anti-biotin antibodies has also been established. Altogether, this work highlights the potential of conjugating chemical signals to biotinylated derivatives to identify and tag signal receptors involved in quorum sensing or other chemical signalling processes.

Biotin and arginine modified hydroxypropyl-ß-cyclodextrin nanoparticles as novel drug delivery systems for paclitaxel.

A novel biotin and arginine modified hydroxypropyl-ß-cyclodextrin (biotin-Arg(pbf)-HP-ß-CD) was successfully synthesized. The hydroxyl groups of HP-ß-CD on the primary faces were coupled with carboxyl groups of biotin using arginine as the functional spacer. Using biotin-Arg(pbf)-HP-ß-CD as the carrier, paclitaxel (PTX)-loaded nanoparticles were developed by modified emulsion solvent evaporation method. The optimized PTX-loaded biotin-Arg(pbf)-HP-ß-CD nanoparticles had a mean diameter of 121.9 nm and zeta potential of -57.7 mV. Transmission electron microscopy (TEM) observation revealed that the nanoparticles were spherical in shape. XRD spectra confirmed the successful encapsulation of PTX. Moreover, in vitro and in vivo evaluations were performed to demonstrate the superior antitumor activity of the PTX-loaded nanoparticles. The cellular uptake study demonstrated the biotin receptor-mediated endocytosis of biotin-Arg(pbf)-HP-ß-CD nanoparticles and the increase of cellular uptake by introduction of biotin and arginine. It can be concluded that the biotin-Arg(pbf)-HP-ß-CD nanoparticles are efficient tumor-targeting drug delivery systems for PTX.

Design and Synthesis of Galactose-Biotin Lipid Materials for Liposomes to Promote the Hepatoma Cell-Targeting Effect.

A series of novel low-toxic hepatoma cell-targeting lipid materials were designed and synthesized, in which monogalactose, digalactose, and galactose-biotin were used as targeting moieties and hydrophilic heads while stearate was used as hydrophobic tail (Mono-Gal-ST, Di-Gal-ST, and Gal-Biotin-ST). The corresponding galactose-biotin-modified liposomes (Mono-Gal-LPs, Di-Gal-LPs, and Gal-Biotin-LPs) and conventional liposomes (LPs) were prepared. These galactose-biotin-modified liposomes can distinguish hepatoma cells from other tissue cells owing to the recognition of asialoglycoprotein receptor by galactose group. Moreover, the ability of liposomes to distinguish hepatoma cells from normal hepatocytes follows a trend of LPs < Mono-Gal-LPs < Di-Gal-LPs < Gal-Biotin-LPs, which is attributed to the cluster glycoside effect and the synergistic effect of galactose and biotin. In addition, the endocytosis of these galactose-biotin-modified liposomes were competitively inhibited by galactose, further confirming these liposomes entered hepatoma cells via asialoglycoprotein receptor-mediated pathway.

Probing membrane asymmetry of ABC polymersomes.

We report the sensitivity of the membrane asymmetry of ABC (PEO-b-PCL-b-PMOXA) polymersomes towards the end-group modification of a shorter C block. While a non-modified ABC polymer formed polymersomes with the A block outside and the C block inside, a mixture of ABC and ABC-biotin formed polymersomes with the C block outside.

Growth inhibition of human ovarian carcinoma by a novel AvidinOX-anchored biotinylated camptothecin derivative.

Oxidized form of avidin, named AvidinOX, provides stable fixation of biotinylated molecules in tissues thus representing a breakthrough in topical treatment of cancer. AvidinOX proved to be a stable receptor for radiolabeled biotin, biotinylated antibodies and cells. In order to expand applicability of the AvidinOX-based delivery platform, in the present study we investigated the possibility to hold biotinylated chemotherapeutics in AvidinOX-treated sites. A novel biotinylated gimatecan-derived camptothecin, coded ST8161AA1, was injected at suboptimal doses into human tumors xenografted in mice alone or pre-complexed to AvidinOX. Significantly higher growth inhibition was observed when the drug was anchored to AvidinOX suggesting the potential utility of this delivery modality for the local treatment of inoperable tumors.

Biotin-tagged fluorescent sensor to visualize 'mobile' Zn2+ in cancer cells.

A cancer cell-targeting fluorescent sensor has been developed to image mobile Zn2+ by introducing a biotin group. It shows a highly selective response to Zn2+in vitro, no toxicity in cellulo and images 'mobile' Zn2+ specifically in cancer cells. We believe this probe has the potential to help improve our understanding of the role of Zn2+ in the processes of cancer initiation and development.

Target Identification of Yaku'amide B and Its Two Distinct Activities against Mitochondrial FoF1-ATP Synthase.

Yaku'amide B (1b) is a structurally unique tetradecapeptide bearing four ß,ß-dialkylated α,ß-unsaturated amino acid residues. Growth-inhibitory profile of 1b against a panel of 39 human cancer cell lines is distinct from those of clinically used anticancer drugs, suggesting a novel mechanism of action. We achieved total syntheses of chemical probes based on 1b and elucidated the cellular target and mode of action of 1b. Fluorescent (3, 4) and biotinylated (5, 6) derivatives of 1b were prepared for cell imaging studies and pull-down assays, respectively. In addition, the unnatural enantiomer of 1b ( ent-1b) and its fluorescent probe ( ent-3) were synthesized for control experiments. Subcellular localization analysis using 3 and 4 showed that 1b selectively accumulates in the mitochondria of MCF-7 human breast cancer cells. Pull-down assays with 6 revealed FoF1-ATP synthase as the major target protein of 1b. Consistent with these findings, biochemical activity assays showed that 1b inhibits ATP production catalyzed by mitochondrial FoF1-ATP synthase. Remarkably, 1b was also found capable of enhancing the ATP hydrolytic activity of FoF1-ATP synthase. On the other hand, ent-1b inhibits ATP synthesis more weakly than does 1b and does not affect ATP hydrolysis, suggesting the stereospecific requirement for the characteristic multimodal functions of 1b. These findings corroborate that 1b causes growth arrest in MCF-7 cells by inhibiting ATP production and enhancing ATP hydrolysis, thereby depleting the cellular ATP pool. This study provides, for the first time, a structural basis for the design and development of anticancer agents exploiting the novel mode of action of 1b.

Enzymatic Fluorination of Biotin and Tetrazine Conjugates for Pretargeting Approaches to Positron Emission Tomography Imaging.

The use of radiolabelled antibodies and antibody-derived recombinant constructs has shown promise for both imaging and therapeutic use. In this context, the biotin-avidin/streptavidin pairing, along with the inverse-electron-demand Diels-Alder (iEDDA) reaction, have found application in pretargeting approaches for positron emission tomography (PET). This study reports the fluorinase-mediated transhalogenation [5'-chloro-5'-deoxyadenosine (ClDA) substrates to 5'-fluoro-5'-deoxyadenosine (FDA) products] of two antibody pretargeting tools, a FDA-PEG-tetrazine and a [18 F]FDA-PEG-biotin, and each is assessed either for its compatibility towards iEDDA ligation to trans-cyclooctene or for its affinity to avidin. A protocol to avoid radiolytically promoted oxidation of biotin during the synthesis of [18 F]FDA-PEG-biotin was developed. The study adds to the repertoire of conjugates for use in fluorinase-catalysed radiosynthesis for PET and shows that the fluorinase will accept a wide range of ClDA substrates tethered at C-2 of the adenine ring with a PEGylated cargo. The method is exceptional because the nucleophilic reaction with [18 F]fluoride takes place in water at neutral pH and at ambient temperature.

Novel biotin linker with alkyne and amino groups for chemical labelling of a target protein of a bioactive small molecule.

We synthesized a novel linker (1) with biotin, alkyne and amino groups for the identification of target proteins using a small molecule that contains an azide group (azide probe). The alkyne in the linker bound the azide probe via an azide-alkyne Huisgen cycloaddition. A protein cross-linker effectively bound the conjugate of the linker and an azide probe with a target protein. The covalently bound complex was detected by western blotting. Linker 1 was applied to a model system using an abscisic acid receptor, RCAR/PYR/PYL (PYL). Cross-linked complexes of linker 1, the azide probes and the target proteins were successfully visualized by western blotting. This method of target protein identification was more effective than a previously developed method that uses a second linker with biotin, alkyne, and benzophenone (linker 2) that acts to photo-crosslink target proteins. The system developed in this study is a method for identifying the target proteins of small bioactive molecules and is different from photo-affinity labelling.

Design and synthesis of tumor-targeting theranostic drug conjugates for SPECT and PET imaging studies.

Theranostics will play a significant role in the next-generation chemotherapy. Two novel tumor-targeting theranostic drug conjugates, bearing imaging arms, were designed and synthesized. These theranostic conjugates consist of biotin as the tumor-targeting moiety, a second generation taxoid, SB-T-1214, as a potent anticancer drug, and two different imaging arms for capturing 99mTc for SPECT (single photon emission computed tomography) and 64Cu for PET (positron emission tomography). To explore the best reaction conditions for capturing radionuclides and work out the chemistry directly applicable to "hot" nuclides, cold chemistry was investigated to capture 185Re(I) and 63Cu(II) species as surrogates for 99mTc and 64Cu, respectively.

Design, synthesis, and initial evaluation of affinity-based small molecular probe for detection of WDR5.

WDR5, a subunit of the SET/MLL complex, plays critical roles in various biological progresses and are abnormally expressed in many cancers. Here we report the design, synthesis, and biochemical characterization of a new chemical tool to capture WDR5 protein. The probe is a biotinylated version of compound 30 that is a potent WDR5 inhibitor we previously reported. Importantly, the probe displayed high affinity to WDR5 protein in vitro binding potency and showed the ability in specifically and real time monitoring WDR5 protein. Further, the biotinylated tag of the probe enabled selectively "chemoprecipitation" of WDR5 from whole cell lysates of MV4-11. This probe provided a new approach to identify the overexpressed WDR5 protein in different cancer cells and applications to proteomic analysis of WDR5 and WDR5-binding partners.

Biotin-tagged platinum(iv) complexes as targeted cytostatic agents against breast cancer cells.

A biotin-guided platinumIV complex is highly cytotoxic against breast cancer cells but hypotoxic against mammary epithelial cells. The mono-biotinylated PtIV complex is superior to the di-biotinylated one and hence a promising drug candidate for the targeted therapy of breast cancer.

A Boronic Acid Assay for the Detection of Mucin-1 Glycoprotein from Cancer Cells.

Cell surface glycoproteins are commonly aberrant in disease and act as biomarkers that facilitate diagnostics. Mucin-1 (MUC1) is a prominent example, exhibiting truncated glycosylation in cancer. We present herein a boronic acid microplate assay for sensitive and high-throughput detection of such glycoproteins. The immobilization of biotin-boronic acid 1 onto streptavidin plates generated a multivalent surface for glycoprotein recruitment and detection. We first validated the binding properties of 1 in solution through titrations with alizarin dye. Next, the microplate assay was explored through horseradish peroxidase (HRP) analysis as a proof-of-concept glycoprotein with chemiluminescence detection. Finally, this platform was applied for the detection of MUC1 directly from MCF-7 human breast cancer cell lysates by using an HRP-tagged antibody that targets the cancerous form of this glycoprotein. Sensitive, dose-dependent detection of MUC1 was observed, showcasing the efficacy of this platform for detecting disease-associated glycoproteins.

A Photo-Crosslinkable Biotin Derivative of the Phosphoantigen (E)-4-Hydroxy-3-Methylbut-2-Enyl Diphosphate (HMBPP) Activates Vγ9Vδ2 T Cells and Binds to the HMBPP Site of BTN3A1.

Vγ9Vδ2 T cells play an important role in the cross talk of the innate and adaptive immune system. For their activation by phosphoantigens (PAgs), both cell surface receptors, the eponymous Vγ9Vδ2 T cell antigen receptors (Vγ9Vδ2 TCRs) on Vγ9Vδ2 T cells and butyrophilin 3A1 (BTN3A1) on the phosphoantigen-"presenting" cell, are mandatory. To find yet undetected but further contributing proteins, a biotinylated, photo-crosslinkable benzophenone probe BioBP-HMBPP (2) was synthesized from a known allyl alcohol in nine steps and overall 16 % yield. 2 is based on the picomolar PAg (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 1). Laser irradiation of 2 at 308 nm initiated the photo-crosslinking reaction with proteins. When the B30.2 domain of BTN3A1, which contains a positively charged PAg-binding pocket, was exposed to increasing amounts of HMBPP (1), labeling by BioBP-HMBPP (2) was reduced significantly. Because BSA labeling was not impaired, 2 clearly binds to the same site as natural ligand 1. Thus, BioBP-HMBPP (2) is a suitable tool to identify co-ligands or receptors involved in PAg-mediated T cell activation.

ReactELISA: Monitoring a Carbon Nucleophilic Metabolite by ELISA-a Study of Lipid Metabolism.

We here present a conceptually novel reaction-based ELISA principle (ReactELISA) for quantitation of the carbon nucleophilic lipid metabolite acetoacetate. Key to the assay is the utilization of a highly chemoselective Friedländer reaction that captures and simultaneously stabilizes the nucleophilic metabolite directly in the biological matrix. By developing a bifunctional biotinylated capture probe, the Friedländer-acetoacetate adduct can be trapped and purified directly in streptavidin coated wells. Finally, we outline the selection and refinement of a highly selective recombinant antibody for specific adduct quantitation. The setup is very robust and, as we demonstrate via miniaturization for microplate format, amenable for screening of compounds or interventions that alter lipid metabolism in liver cell cultures. The assay-principle should be extendable to quantitation of other nucleophilic or electrophilic and perhaps even more reactive metabolites provided suitable capture probes and antibodies.

An efficient synthesis of 1α,25-dihydroxyvitamin D3 LC-biotin.

In recent years the apparent impact of vitamin D deficiency on human health has gained increased awareness. Consequently, the development of appropriate assays to measure the status of medicinally most relevant vitamin D metabolites in human blood, serum or relevant tissue is continuously being improved. Particularly, assaying of 1α,25-dihydroxyvitamin D3, in turn considered as the most active metabolite, is mainly indicated in disorders leading to calcaemia or those resulting from an impaired 1α-hydroxylation of 25-hydroxyvitamin D3. Thus, in some competitive protein binding and ELISA assays, biotin-linked 1α,25-dihydroxyvitamin D3 (1α,25-dihydroxyvitamin D3 LC-biotin) is employed for measurement of actual calicitriol concentration. A new efficient synthesis of 1α,25-dihydroxyvitamin D3 LC-biotin is described, starting with readily available vitamin D2, and combining a classical approach to access 1α,25-dihydroxyvitamin D3, appropriate OH-protective group transformations, and a C-3-O-alkylation, suitable to connect the biotin-linker in a reliable, selective and high yielding strategy. The developed methodology is applicable to the synthesis of a wide variety of C-3-OH-linked vitamin D3 and D2 derivatives.

A biotinylated piperazine-rhodol derivative: a 'turn-on' probe for nitroreductase triggered hypoxia imaging.

We developed a nitroreductase responsive theranostic probe 1; it comprises biotinylated rhodol in conjunction with p-nitrobenzyl functionality. The probe 1 showed a remarkable fluorescence 'turn-on' signal in the presence of nitroreductase under physiological conditions. The probe is considerably stable within a wide biological pH range (6-8) and also is very sensitive toward a reducing micro-environment e.g. liver microsome. Further, it enables providing cellular and in vivo nematode images in a reducing microenvironment.

Synthesis of asymmetric zinc(II) phthalocyanines with two different functional groups & spectroscopic properties and photodynamic activity for photodynamic therapy.

Zinc(II) phthalocyanine containing [2-(tert-butoxycarbonyl)amino]ethoxy and iodine groups (A and B), as well as their deprotected mono-amino and tri-iodine zinc(II) phthalocyanine (2) were obtained. This structure surrounds by substituents with functional groups. From this perspective it can be used a starting material for many reactions and applications, such as sonogashira coupling, carbodiimide coupling. An example of a first diversification reaction of this compound was obtained with conjugation of a biotin. Asymmetrically biotin conjugated and heavy atom bearing zinc(II) phthalocyanine (3) were synthesized characterized for the first time and photophysical, photochemical and photobiological properties of these phthalocyanines were compared in this study.