- Cell-free protein synthesis and: In situ immobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion
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In the present work, microgels were utilized as a cell-free reaction environment to produce a functional malonyl-CoA synthetase (deGFP-MatB) under geometry-controlled transcription and translation. Our approach combines the straight-forward optimization of overall protein yield of an E. coli-based cell-free protein synthesis (CFPS) system based on concentration screening of magnesium and potassium glutamate, DNA as well as polyethylene glycol (PEG), and its innovative usage in microgel-based production of a key enzyme of the polyketide synthesis pathway. After partial modification of the carboxyl groups of hyaluronic acid (HA) with 5′-methylfuran groups via 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM)-activation, these were further functionalized with dibenzocyclooctyne (DBCO) and nitrilotriacetic acid (NTA) groups by bio-orthogonal [4+2] Diels-Alder cycloaddition to yield a bifunctional macromer. After coupling the DBCO groups with azide-functionalized DNA, containing the genetic information for deGFP-MatB, via strain-promoted azide-alkyne cycloaddition (SPAAC), the DNA-/NTA-functionalized HA macromer was utilized as base material together with maleimide-functionalized PEG (PEG-mal2) as the crosslinker to form bifunctional microgels utilizing water-in-oil (W/O) microemulsions. As-formed microgels were incubated with nickel sulfate to activate the NTA groups and provide binding sites for deGFP-MatB, which contained six histidine residues (His-tag) for that purpose. The optimized CFPS mixture was loaded into the microgels to initiate the formation of deGFP-MatB, which was detected by a clear increase in fluorescence exclusively inside the microgel volume. Functionality of both, the bound and the decoupled enzyme was proven by reaction with malonate to yield malonyl CoA, as confirmed by a colorimetric assay.
- Heida, Thomas,Hoefgen, Sandra,K?hler, Tony,Thiele, Julian,Valiante, Vito,Weigel, Niclas
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Read Online
- Synthesis, characterization, and targeted chemotherapy of SCT200-linker-monomethyl auristatin E conjugates
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Antibody-drug conjugates (ADCs) are currently among the most successful and important strategies for treating patients with solid tumors. ADCs are composed of a monoclonal antibody and warhead, which are conjugated via a linker. Currently, monomethyl auristatin E (MMAE) is the most widely applied warhead in the development of ADCs. However, MMAE-based ADCs are generally constructed using the MC-VC-PABC linker, and this design has limited structural diversity and some disadvantages. Accordingly, in this study, we generated three types of novel linker-MMAE (with alterations in the spacer, catabolizing area, and self-immolative compared with MC-VC-PABC-MMAE) in ADCs, termed SCT200-linker-MMAE conjugates, and then evaluated the linker-drug plasma stability and the rate of drug release by cathepsin B. The binding ability, internalization rates, and efficacy of all SCT200-linker-MMAE ADCs were systematically studied, and the expression of apoptosis-associated proteins and the therapeutic efficacies of SCT200-M-2, -C-2, and -C-4 were evaluated. The results showed that the activities of some of these ADCs were increased for epidermal growth factor receptor-positive tumors. Moreover, the novel linkers designed in this study can be linked with other antibodies to treat other types of cancer. Overall, these findings provide important insights into the application of SCT200-based linkers in ADCs.
- Hu, Xinyue,Jiang, Hailun,Bai, Weiqi,Liu, Xiujun,Miao, Qingfang,Wang, Linlin,Jin, Jie,Cui, Along,Liu, Rui,Li, Zhuorong
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supporting information
(2021/03/08)
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- Development of bifunctional anti-PD-L1 antibody MMAE conjugate with cytotoxicity and immunostimulation
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In recent years, tumor immunotherapy, especially the combination of PD1/PD-L1 inhibitors and chemotherapy, has developed rapidly. However, the systemic side effects induced by chemotherapy remain a crucial problem that needs to be addressed. Antibody drug conjugates (ADCs) are exceptional target-specific prodrugs that greatly improve the therapeutic window of chemotherapy drugs. Therefore, designing PD-L1-targeting ADCs is an interesting research project. In this study, we confirmed for the first time that the commercial anti-PD-L1 antibody Atezolizumab has better endocytosis efficiencies than Avelumab, and was more suitable for ADC design. Then, the most popular cytotoxic payload MMAE was conjugated to Atezolizumab via a classical dipeptide (valine-alanine) linker to generate a bifunctional PD-L1 ADC (ADC 3). An in vitro cytotoxicity test indicated the potent tumor cell inhibitory activity of ADC 3, with EC50 values of 9.75 nM to 11.94 nM. In addition, a co-culture of PBMCs in vitro proved that ADC 3 retained the immune activation effect of the Atezolizumab antibody. Moreover, ADC 3 exhibited a higher tumor inhibition rate and tumor regression rate in humanized immune system mice. To the best of our knowledge, this is the most active PD-L1-ADC reported thus far, which may promote the development of immunotherapy and novel ADCs.
- Xiao, Dian,Luo, Longlong,Li, Jiaguo,Wang, Zhihong,Liu, Lianqi,Xie, Fei,Feng, Jiannan,Zhou, Xinbo
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- Biocompatible organic coatings based on bisphosphonic acid RGD‐derivatives for PEO‐modified titanium implants
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Currently, significant attention is attracted to the problem of the development of the specific architecture and composition of the surface layer in order to control the biocompatibility of implants made of titanium and its alloys. The titanium surface properties can be tuned both by creating an inorganic sublayer with the desired morphology and by organic top coating contributing to bioactivity. In this work, we developed a composite biologically active coatings based on hybrid molecules obtained by chemical crosslinking of amino acid bisphosphonates with a linear tripeptide RGD, in combination with inorganic porous sublayer created on titanium by plasma electrolytic oxidation (PEO). After the addition of organic molecules, the PEO coated surface gets nobler, but corrosion currents increase. In vitro studies on proliferation and viability of fibroblasts, mesenchymal stem cells and osteoblastlike cells showed the significant dependence of the molecule bioactivity on the structure of bisphosphonate anchor and the linker. Several RGDmodified bisphosphonates of β–alanine, γ–aminobutyric and ε–aminocaproic acids with BMPS or SMCC linkers can be recommended as promising candidates for further in vivo research.
- Danilko, Ksenia V.,Dyakonov, Grigory S.,Farrakhov, Ruzil G.,Galimshina, Zulfia R.,Gil'fanova, Guzel U.,Lukina, Elena S.,Mukaeva, Veta R.,Parfenov, Evgeny V.,Parfenova, Lyudmila V.
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- Method for preparing biotin maleimide
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The invention discloses a method for preparing biotin maleimide. The method comprises the following steps of: 1, reacting 6-maleimidohexanoic acid compound (1) with N-hydroxysuccinimide under the action of a condensing agent to obtain a 6-maleimidohexanoic acid N-hydroxysuccinimide ester compound (2); 2, treating the 6-maleimidohexanoic acid N-hydroxysuccinimide ester compound (2) by using hydrazine hydrate to obtain a 6- maleimidocaproyl hydrazide compound (3); 3, reacting a biotin compound (4) with an acyl halide reagent to obtain a biotinyl acyl halide compound (5); and 4, reacting the 6-maleimidocaproyl hydrazide compound (3) with the biotinyl acyl halide compound (5) to obtain a biotin maleimide compound (6). The method for preparing the biotin maleimide, disclosed by the invention, has the beneficial effects of being suitable for scale-up production, easily available in raw materials, simple in preparation process and friendly to environment.
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Paragraph 0025; 0040-0042; 0046; 0048
(2019/04/06)
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- Improved Methodology for the Synthesis of a Cathepsin B Cleavable Dipeptide Linker, Widely Used in Antibody-Drug Conjugate Research
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Antibody-drug conjugates (ADCs) represent an emerging class of biopharmaceutical agents that deliver highly potent anticancer agents (payloads) selectively to tumors or components associated with the tumor microenvironment. The linker, responsible for the connection between the antibody and payload, is a crucial component of ADCs. In certain examples the linker is composed of a cleavable short peptide which imparts an additional aspect of selectivity. Especially prevalent is the cathepsin B cleavable Mc-Val-Cit-PABOH linker utilized in many pre-clinical ADC candidates, as well as the FDA approved ADC ADCETRIS (brentuximab vedotin). An alternative route for the synthesis of the cathepsin B cleavable Mc-Val-Cit-PABOH linker is reported herein that involved six steps from l-Citrulline and proceeded with a 50% overall yield. In this modified route, the spacer (a para-aminobenzyl alcohol moiety) was incorporated via HATU coupling followed by dipeptide formation. Importantly, this route avoided undesirable epimerization and proceeded with improved overall yield. Utilizing this methodology, a drug-linker construct incorporating a potent small-molecule inhibitor of tubulin polymerization (referred to as KGP05), was synthesized as a representative example.
- Mondal, Deboprosad,Ford, Jacob,Pinney, Kevin G.
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supporting information
p. 3594 - 3599
(2018/09/11)
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- HDAC INHIBITORS-BASED ANTIBODY DRUG CONJUGATES (ADCs) AND USE IN THERAPY
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The present invention relates to novel Histone Deacetylase Inhibitors (HDACi)- based antibody drug conjugates particularly with antibodies directed to ErbB1, ErbB2 and ErbB3 receptors, pharmaceutical compositions comprising said antibodies as well as to their use in the treatment of cancer or tumor and other diseases where a modulation of one or more histone deacetylase isoforms can be effective for therapeutic interventions.
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- Cysteine-transformed antibody-toxin conjugate and its preparation method
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The invention discloses a cysteine-transformed antibody-toxin conjugate and its preparation method. The cysteine-transformed antibody-toxin conjugate with excellent uniformity is formed by inserting aheavy chain and a light chain of a target antibody to the cysteine (C) at the fixed point, and performing fixed-point coupling on a free sulfydryl (-SH) of the cysteine inserted at the fixed point and a connector coupled with micromolecular high-activity cell toxin. The insert site of the cysteine disclosed in the invention is the 205th site and/or the 206th site (Kabat number) of the antibody light chain, and/or the 439th site of the heavy chain (Kabat number).
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Paragraph 0064; 0065; 0066
(2018/03/25)
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- Cysteine transformation-based antibody-toxin conjugate
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Heavy chain 235-site serine (S) and light chain 205-site valine (V) of a target antibody are transformed into cysteine (C), and after transformation, free sulfhydryl (-SH) of the cysteine is coupled to a mc-vc-PAB-OH connexon coupled with small molecular high-activity cytotoxin (Payload) in a fixed-point way to form a cysteine transformation-based antibody-toxin conjugate with good uniformity; thetoxin-antibody ratio (DAR) of the cysteine transformation-based antibody-toxin conjugate is 3.2-4.0, and the antibody-toxin conjugate adopts a general formula of 1C7-HC-S235C-LC-V205C-mc-vc-PAB-payload. The invention further discloses a preparation method and a purification method of a TDC medicine and application to treatment of EGFRwt (wild-type epidermal growth factor receptor) overexpressionrelated tumors.
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Paragraph 0011-0013
(2018/11/22)
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- Cysteine engineered antibody - toxin conjugate (TDC) fixed-point coupling site screening (by machine translation)
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The invention discloses a cysteine engineered antibody - toxin conjugate, characterized in that the antibody is the antibody fixed-point inserted cysteine, cysteine insertion site comprises a site selected from the following group of one or more of: light chain constant region of the light chain kappa / λ 110 position, section 111 position, section 142 bit, IgG antibody heavy chain constant region of the heavy chain of the 254 bit, of the 255 bit, of the 258 position, section 259 bit, section 354 bit, section 355 bit, part 357 bit, paragraph 378 bit, article 379 bit, section 386 bit, article 387 bit or 410 bit. (by machine translation)
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Paragraph 0007; 0008; 0009
(2018/11/22)
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- DRUG-LINKER CONJUGATE PHARMACEUTICAL COMPOSITIONS
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Compositions are disclosed having a cytotoxic and/or vascular disrupting agent (VDA) payload attached to a linker. The linker can be a cathepsin B protease cleavable linker or a non-cleavable linker that may degrade intracellularly. Methods for making and using the compositions are also provided. The compositions can be provided to a patient in need thereof with the composition coming into contact with a cancer cell to activate or release the cytotoxic and/or vascular disrupting agent payload.
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Paragraph 000193-000195
(2017/05/02)
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- Development and properties of valine-alanine based antibody-drug conjugates with monomethyl auristatin E as the potent payload
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Antibody-drug conjugates (ADCs), designed to selectively deliver cytotoxic agents to antigen-bearing cells, are poised to become an important class of cancer therapeutics. Human epithelial growth factor receptor (HER2) is considered an effective target for cancer treatment, and a HER2-targeting ADC has shown promising results. Most ADCs undergoing clinical evaluation contain linkers that have a lysosomal protease-cleavable dipeptide, of which the most common is valine-citrulline (VC). However, valine-alanine (VA), another dipeptide comprising two human essential amino acids, has been used in next generation ADCs loading new toxins, but the druggable properties of ADCs loaded the most popular monomethyl auristatin E (MMAE) remain to be further explored. In this study, we generated VA-based ADCs that connected MMAE to an anti-HER2 antibody. We studied the differences in the preparation process, in vitro stability, cathepsin B activity and in vitro cytotoxicity of VA-based ADC compared to the ADC of VC. VA had comparable performance to VC, which preliminarily displays its practicability. Additional efficacy and safety studies in a xenograft model indicate this novel ADC exerted potent anti-tumor activity and negligible toxicity. The results of this study show the application potential of VA-based ADC with MMAE as the payload.
- Wang, Yanming,Fan, Shiyong,Zhong, Wu,Zhou, Xinbo,Li, Song
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- CYTOTOXIC AND ANTI-MITOTIC COMPOUNDS, AND METHODS OF USING THE SAME
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Compounds having cytotoxic and/or anti-mitotic activity are disclosed. Methods associated with preparation and use of such compounds, as well as pharmaceutical compositions comprising such compounds, are also disclosed. Also disclosed are compositions having the structure: (T)-(L)-(D), wherein (T) is a targeting moiety, (L) is an optional linker, and (D) is a compound having cytotoxic and/or anti-mitotic activity.
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Page/Page column 97; 98
(2016/04/09)
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- SULFONAMIDE-CONTAINING LINKAGE SYSTEMS FOR DRUG CONJUGATES
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Sulfonamide-containing linkage systems for release of payload compounds from an attached targeting moiety in drug conjugates. The conjugates have the formula of [(P)-(L)]m-(T), wherein (P) is a payload compound, (L) is a linker, (T) is a targeting moiety and m is an integer from 1- to 10. Also provided are pharmaceutical compositions comprising such conjugates and there use in treating cancer.
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- Azide-alkyne cycloaddition for universal post-synthetic modifications of nucleic acids and effective synthesis of bioactive nucleic acid conjugates
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The regioselective post-synthetic modifications of nucleic acids are essential to studies of these molecules for science and applications. Here we report a facile universal approach by harnessing versatile phosphoramidation reactions to regioselectively incorporate alkynyl/azido groups into post-synthetic nucleic acids primed with phosphate at the 5′ termini. With and without the presence of copper, the modified nucleic acids were subjected to azide-alkyne cycloaddition to afford various nucleic acid conjugates including a peptide-oligonucleotide conjugate (POC) with high yield. The POC was inoculated with human A549 cells and demonstrated excellent cell-penetrating ability despite cell deformation caused by a small amount of residual copper chelated to the POC. The combination of phosphoramidation and azide-alkyne cycloaddition reactions thus provides a universal regioselective strategy to post-synthetically modify nucleic acids. This study also explicated the toxicity of residual copper in synthesized bioconjugates destined for biological systems. This journal is the Partner Organisations 2014.
- Su, Yu-Chih,Lo, Yu-Lun,Hwang, Chi-Ching,Wang, Li-Fang,Wu, Min Hui,Wang, Eng-Chi,Wang, Yun-Ming,Wang, Tzu-Pin
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p. 6624 - 6633
(2014/08/18)
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- Bismuth A3-corroles: Useful precursors for the development of meso- substituted free-base corroles
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Systematic studies on regioselective functionalization reactions employing oxygen-, nitrogen-, and sulfur-containing nucleophiles with bismuth A3-corroles under the influence of a strong non-nucleophilic base are reported. In the case of the thiols and dithiols a high-yielding reaction procedure was established to obtain mono-, di-, and tri-functionalized corroles at room temperature within short reaction times. The described method offers a possibility to attach bifunctional linker molecules to the para-position of the meso-pentafluorophenyl groups at positions 5, 10, and 15 of the corrole macrocycle. The described reaction strategy may serve as a versatile protocol for the covalent binding of corroles to proteins or antibodies and may be utilized to attach corroles on, for example, gold or titanium surfaces to study surface-supported reactions.
- Faschinger, Felix,Aichhorn, Stefan,Himmelsbach, Markus,Schoefberger, Wolfgang
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p. 3085 - 3096
(2015/02/02)
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- Heat-induced morphological transformation of supramolecular nanostructures by retro-Diels-Alder reaction
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Controlling the morphology of supramolecular nanostructures in response to external stimuli is an important challenge in the development of functional soft materials. Here we show that a morphological transformation from 2D nanosheets to a network of 1D nanofibers is triggered by heating, which induces molecular conversion of a bolaamphiphile to a hydrogelator by means of a retro-Diels-Alder reaction, thereby producing a new heat-set supramolecular hydrogel. We anticipate that our design will be a starting point for more sophisticated supramolecular systems that integrate the thermodynamics of molecular assembly and the kinetics of chemical reactions to create complex supramolecular nanostructures. The heat is on: We show that a morphological transformation from 2D nanosheets to a network of 1D nanofibers is triggered by heating (see figure), which induces molecular conversion of a bolaamphiphile to a hydrogelator by means of a retro-Diels-Alder reaction, thereby producing a new heat-set supramolecular hydrogel. Copyright
- Ikeda, Masato,Ochi, Rika,Kurita, Yu-Shi,Pochan, Darrin J.,Hamachi, Itaru
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supporting information
p. 13091 - 13096
(2013/01/15)
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- Synthesis and antitumor efficacy of a β-glucuronidase-responsive albumin-binding prodrug of doxorubicin
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In this paper we describe the synthesis and biological evaluation of the first β-glucuronidase-responsive albumin-binding prodrug designed for the selective delivery of doxorubicin at the tumor site. This prodrug leads to superior antitumor efficacy in mice compared to HMR 1826, a well-known glucuronide prodrug of doxorubicin that cannot bind covalently to circulating albumin. Furthermore, this compound inhibits tumor growth in a manner similar to that of doxorubicin while avoiding side effects induced by the free drug.
- Legigan, Thibaut,Clarhaut, Jonathan,Renoux, Brigitte,Tranoy-Opalinski, Isabelle,Monvoisin, Arnaud,Berjeaud, Jean-Marc,Guilhot, Fran?ois,Papot, Sébastien
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supporting information; experimental part
p. 4516 - 4520
(2012/08/13)
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- In situ formation of N-trifluoroacetoxy succinimide (TFA-NHS): One-pot formation of succinimidyl esters, N-trifluoroacetyl amino acid succinimidyl esters, and N-maleoyl amino acid succinimidyl esters
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A method for the in situ formation of N-trifluoroacetoxy succinimide (TFA-NHS) and its application in the formation of succinimidyl esters is presented. The developed method provides N-trifluoroacetyl and N-maleoyl amino acid succinimidyl esters from a variety of amino acids using a one-pot, high-yielding protocol. Investigations into the formation of an N-maleoyl amino acid succinimidyl ester supported the proposal of a revised reaction mechanism, and contributed to the optimization of the reaction conditions.
- Leonard, Nicholas M.,Brunckova, Jarmila
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p. 9169 - 9174
(2011/12/16)
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- Practical synthesis of maleimides and coumarin-linked probes for protein and antibody labelling via reduction of native disulfides
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The cellular tracking, detection and sensing of protein or antibody movement are important aspects to advance our understanding of biomolecular interactions and activity. Antibodies modified with fluorescent dyes are also valuable tools, especially in immunology research. We describe here a proof-of-principle study of a new water-soluble coumarin probe with a maleimide thiol-reacting unit to fluorescently tag biomolecules. Highlights include: (1) a convenient water-based preparation of N-substituted maleimides, (2) a one-pot preparation of activated maleimido-esters, and (3) a bio-conjugation protocol for the selenol-promoted reduction of native disulfide bonds and the 'site-specific' labelling of antibodies with no significant loss of activity.
- Song, Hong Y.,Ngai, Mun H.,Song, Zhen Y.,MacAry, Paul A.,Hobley, Jonathan,Lear, Martin J.
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experimental part
p. 3400 - 3406
(2010/01/06)
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- Development of elastin-like polypeptide for thermally targeted delivery of doxorubicin
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The chemotherapeutic drug doxorubicin (Dox) is widely used as an antitumor agent in hematological malignancies and solid tumors. However, one of the limitations of its clinical use is that systemic administration of an effective dose of Dox results in nonselective cardiac toxicity and myelosuppression. In order to minimize this nonspecific toxicity, Elastin-like polypeptide (ELP) was examined for its ability to serve as a macromolecular carrier for thermally targeted delivery of Dox. The ELP-based doxorubicin delivery vehicle (Tat-ELP-GFLG-Dox) consists of: (1) a peptide derived from the HIV-1 Tat protein to facilitate its cellular uptake, (2) ELP to allow thermal targeting, and (3) the lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer and a cysteine residue conjugated to a thiol reactive doxorubicin derivative. Cytotoxicity of Tat-ELP-GFLG-Dox in MES-SA uterine sarcoma cells was enhanced 20-fold when aggregation of ELP was induced with hyperthermia. The ELP delivered doxorubicin displayed a cytoplasmic distribution and induced temperature dependent caspase activation.
- Bidwell III, Gene L.,Fokt, Izabela,Priebe, Waldemar,Raucher, Drazen
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p. 620 - 631
(2008/01/27)
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- THERMALLY-TARGETED DELIVERY OF MEDICAMENTS, INCLUDING DOXORUBICIN
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Elastin-like polypeptide (ELP) serves as a vector for thermally-targeted delivery of therapeutics, including cytotoxic chemotherapeutic drugs such as doxorubicin. Examples of an ELP-based delivery vehicle can comprise: (1) a cell penetrating peptide, such as a Tat protein, (2) ELP, and (3) the lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer and a cysteine residue conjugated to therapeutic such as doxorubicin, or a analog thereof.
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Page/Page column 35; 4/9
(2008/06/13)
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- Synthesis of N-Maleoyl-aminoacids and -peptides
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N-Maleoyl-aminoacid-N'-hydroxysuccinimidesters 3 or N-maleoyl-aminobenzoic acids 5 are synthsized from N-maleyl-aminoacids 1 on different ways.N-maleoyl-aminobenzoic-4-nirophenyl-, -2-nitro-phenyl- or 2,4-dinitro-phenylesters 6, 7 and 8 will be obtained from 1 or 5. o-Mercaptoaniline, thiourea or cysteine react with 5 to benzothiazines 9, thiazolidines 10 and 1,4-thiazines 11.From 5 the peptides 12 are yielded.The pentapeptide 13 are formed from 12 by addition of glutathione.
- Augustin, Manfred,Mueller, Wolfgang
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p. 789 - 798
(2007/10/02)
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