The Experts below are selected from a list of 303 Experts worldwide ranked by ideXlab platform

Roger Stromberg - One of the best experts on this subject based on the ideXlab platform.

  • copper catalyzed huisgen 1 3 dipolar cycloaddition tailored for phosphorothioate oligonucleotides
    Current protocols in human genetics, 2020
    Co-Authors: Malgorzata Honcharenko, Dmytro Honcharenko, Roger Stromberg
    Abstract:

    An efficient Method for attachment of a variety of reporter groups to oligonucleotides (ONs) is copper (I) [Cu(I)]-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition ("click reaction"). However, in the case of ONs with phosphorothioate modifications as internucleosidic linkages (PS-ONs), this Conjugation Method has to be adjusted to be compatible with the sulfur-containing groups. The Method described here is adapted for PS-ONs, utilizes solid-supported ONs, and implements the Cu(I) bromide dimethyl sulfide complex (CuBr × Me2 S) as a mediator for the click reaction. The solid-supported ONs can be readily transformed into "clickable ONs" by on-line addition of an alkyne-containing linker that subsequently can react with an azido-containing moiety (e.g., a peptide) in the presence of CuBr × Me2 S. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Conjugation on solid support Support Protocol: Removal of 4,4'-dimethoxytrityl group from amino linker Basic Protocol 2: Removal of protecting groups and cleavage from solid support Basic Protocol 3: HPLC purification.

Malgorzata Honcharenko - One of the best experts on this subject based on the ideXlab platform.

  • copper catalyzed huisgen 1 3 dipolar cycloaddition tailored for phosphorothioate oligonucleotides
    Current protocols in human genetics, 2020
    Co-Authors: Malgorzata Honcharenko, Dmytro Honcharenko, Roger Stromberg
    Abstract:

    An efficient Method for attachment of a variety of reporter groups to oligonucleotides (ONs) is copper (I) [Cu(I)]-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition ("click reaction"). However, in the case of ONs with phosphorothioate modifications as internucleosidic linkages (PS-ONs), this Conjugation Method has to be adjusted to be compatible with the sulfur-containing groups. The Method described here is adapted for PS-ONs, utilizes solid-supported ONs, and implements the Cu(I) bromide dimethyl sulfide complex (CuBr × Me2 S) as a mediator for the click reaction. The solid-supported ONs can be readily transformed into "clickable ONs" by on-line addition of an alkyne-containing linker that subsequently can react with an azido-containing moiety (e.g., a peptide) in the presence of CuBr × Me2 S. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Conjugation on solid support Support Protocol: Removal of 4,4'-dimethoxytrityl group from amino linker Basic Protocol 2: Removal of protecting groups and cleavage from solid support Basic Protocol 3: HPLC purification.

  • Efficient Conjugation to Phosphorothioate Oligonucleotides by Cu-Catalyzed Huisgen 1,3-Dipolar Cycloaddition
    2019
    Co-Authors: Malgorzata Honcharenko, Dmytro Honcharenko, Roger Strömberg
    Abstract:

    Improving oligonucleotide delivery is critical for the further development of oligonucleotide-based therapeutics. Covalent attachment of reporter molecules is one of the most promising approaches toward efficient oligonucleotide-based therapies. An efficient Methods for the attachment of a variety of reporter groups is Cu­(I)-catalyzed Huisgen azide–alkyne 1,3-dipolar cycloaddition. However, the majority of potential oligonucleotide (ON) therapeutics in clinical trials are carrying phosphorothioate (PS) linkages, and this robust Conjugation Method is not yet established for these ONs due to a general concern of Cu–S interaction. Here, we developed a Method allowing for efficient Conjugation of peptides to PS oligonucleotides. The Method utilizes solid supported oligonucleotides that can be readily transformed into “clickable ONs” by simple linker Conjugation and further reacted with an azido containing moiety (e.g., a peptide) using the CuBr × Me2S complex as a superior catalyst in that reaction. This study opens the way for further development of PS oligonucleotide-conjugates by means of efficient Cu­(I)-catalyzed Huisgen azide–alkyne 1,3-dipolar cycloaddition

Christopher Barnerkowollik - One of the best experts on this subject based on the ideXlab platform.

  • sunlight induced crosslinking of 1 2 polybutadienes access to fluorescent polymer networks
    Polymer Chemistry, 2014
    Co-Authors: Jan O Mueller, Nathalie K Guimard, Klaus Kim Oehlenschlaeger, F. Schmidt, Christopher Barnerkowollik
    Abstract:

    The efficient sunlight-induced crosslinking of 1,2-polybutadienes to generate fluorescent patterns with spatial resolution is reported. The photochemical Conjugation Method employed is based on a nitrile imine-mediated tetrazole–ene cycloaddition (NITEC) reaction, which proceeds under UV-light irradiation (λmax = 312 nm) at ambient temperature in the absence of any catalyst. The NITEC reaction between 1-pentene and a newly designed di-linker, consisting of two photosensitive diaryl-substituted tetrazoles joined by a tetraethylene glycol spacer, was investigated in an initial study. Detailed characterization of a small molecule model study was performed by size exclusion chromatography (SEC), UV-vis and fluorescence spectroscopy as well as electrospray-ionization mass spectrometry (ESI-MS), which was also employed for monitoring the progress of the reaction (100% conversion in 20 min). Finally, two 1,2-polybutadienes of disparate molar masses were each photocrosslinked with the di-linker. The crosslinking reaction parameters, such as concentration, di-linker fraction and reaction time were optimized via SEC analysis and gravimetric determination of gel fractions. The applicability of the novel crosslinking technology for generating spatially controlled highly fluorescent gel patterns is demonstrated in a solvent-free reaction for 2 h under sunlight. In summary, the current study introduces an efficient light-triggered technology platform for crosslinking polymers carrying non-activated double bonds.

  • sunlight induced crosslinking of 1 2 polybutadienes access to fluorescent polymer networks
    Institute for Future Environments; Science & Engineering Faculty, 2014
    Co-Authors: Jan O Mueller, Nathalie K Guimard, Klaus Kim Oehlenschlaeger, F. Schmidt, Christopher Barnerkowollik
    Abstract:

    The efficient sunlight-induced crosslinking of 1,2-polybutadienes to generate fluorescent patterns with spatial resolution is reported. The photochemical Conjugation Method employed is based on a nitrile imine-mediated tetrazole-ene cycloaddition (NITEC) reaction, which proceeds under UV-light irradiation (λ max = 312 nm) at ambient temperature in the absence of any catalyst. The NITEC reaction between 1-pentene and a newly designed di-linker, consisting of two photosensitive diaryl-substituted tetrazoles joined by a tetraethylene glycol spacer, was investigated in an initial study. Detailed characterization of a small molecule model study was performed by size exclusion chromatography (SEC), UV-vis and fluorescence spectroscopy as well as electrospray-ionization mass spectrometry (ESI-MS), which was also employed for monitoring the progress of the reaction (100% conversion in 20 min). Finally, two 1,2-polybutadienes of disparate molar masses were each photocrosslinked with the di-linker. The crosslinking reaction parameters, such as concentration, di-linker fraction and reaction time were optimized via SEC analysis and gravimetric determination of gel fractions. The applicability of the novel crosslinking technology for generating spatially controlled highly fluorescent gel patterns is demonstrated in a solvent-free reaction for 2 h under sunlight. In summary, the current study introduces an efficient light-triggered technology platform for crosslinking polymers carrying non-activated double bonds. © The Royal Society of Chemistry.

Yasuhisa Shiraishi - One of the best experts on this subject based on the ideXlab platform.

  • an expanded genetic code facilitates antibody chemical Conjugation involving the lambda light chain
    Biochemical and Biophysical Research Communications, 2021
    Co-Authors: Akifumi Kato, Kazumasa Ohtake, Yoshitaka Tanaka, Shigeyuki Yokoyama, Kensaku Sakamoto, Yasuhisa Shiraishi
    Abstract:

    Abstract Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful Conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Ne-(o-azidobenzyloxycarbonyl)- l -lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical Conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical Conjugation Method to the whole spectrum of humanized antibodies, including the λ isotype.

  • one step Conjugation Method for site specific antibody drug conjugates through reactive cysteine engineered antibodies
    Bioconjugate Chemistry, 2016
    Co-Authors: Daisuke Shinmi, Eri Taguchi, Junko Iwano, Junichi Enokizono, Kazuhiro Masuda, Tsuyoshi Yamaguchi, Yasuhisa Shiraishi
    Abstract:

    Engineered cysteine residues are particularly convenient for site-specific Conjugation of antibody–drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to Conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the Conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange proced...

Dmytro Honcharenko - One of the best experts on this subject based on the ideXlab platform.

  • copper catalyzed huisgen 1 3 dipolar cycloaddition tailored for phosphorothioate oligonucleotides
    Current protocols in human genetics, 2020
    Co-Authors: Malgorzata Honcharenko, Dmytro Honcharenko, Roger Stromberg
    Abstract:

    An efficient Method for attachment of a variety of reporter groups to oligonucleotides (ONs) is copper (I) [Cu(I)]-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition ("click reaction"). However, in the case of ONs with phosphorothioate modifications as internucleosidic linkages (PS-ONs), this Conjugation Method has to be adjusted to be compatible with the sulfur-containing groups. The Method described here is adapted for PS-ONs, utilizes solid-supported ONs, and implements the Cu(I) bromide dimethyl sulfide complex (CuBr × Me2 S) as a mediator for the click reaction. The solid-supported ONs can be readily transformed into "clickable ONs" by on-line addition of an alkyne-containing linker that subsequently can react with an azido-containing moiety (e.g., a peptide) in the presence of CuBr × Me2 S. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Conjugation on solid support Support Protocol: Removal of 4,4'-dimethoxytrityl group from amino linker Basic Protocol 2: Removal of protecting groups and cleavage from solid support Basic Protocol 3: HPLC purification.

  • Efficient Conjugation to Phosphorothioate Oligonucleotides by Cu-Catalyzed Huisgen 1,3-Dipolar Cycloaddition
    2019
    Co-Authors: Malgorzata Honcharenko, Dmytro Honcharenko, Roger Strömberg
    Abstract:

    Improving oligonucleotide delivery is critical for the further development of oligonucleotide-based therapeutics. Covalent attachment of reporter molecules is one of the most promising approaches toward efficient oligonucleotide-based therapies. An efficient Methods for the attachment of a variety of reporter groups is Cu­(I)-catalyzed Huisgen azide–alkyne 1,3-dipolar cycloaddition. However, the majority of potential oligonucleotide (ON) therapeutics in clinical trials are carrying phosphorothioate (PS) linkages, and this robust Conjugation Method is not yet established for these ONs due to a general concern of Cu–S interaction. Here, we developed a Method allowing for efficient Conjugation of peptides to PS oligonucleotides. The Method utilizes solid supported oligonucleotides that can be readily transformed into “clickable ONs” by simple linker Conjugation and further reacted with an azido containing moiety (e.g., a peptide) using the CuBr × Me2S complex as a superior catalyst in that reaction. This study opens the way for further development of PS oligonucleotide-conjugates by means of efficient Cu­(I)-catalyzed Huisgen azide–alkyne 1,3-dipolar cycloaddition