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Biserka Žinić - One of the best experts on this subject based on the ideXlab platform.
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Mass Spectrometry and Theoretical Studies on N–C Bond Cleavages in the N-Sulfonylamidino Thymine Derivatives
Journal of The American Society for Mass Spectrometry, 2015Co-Authors: Renata Kobetić, Snježana Kazazić, Borislav Kovačević, Zoran Glasovac, Luka Krstulović, Miroslav Bajić, Biserka ŽinićAbstract:The reactivity of new biologically active thymine derivatives substituted with 2-(arylsulfonamidino)ethyl group at N1 and N3 position was investigated in the gas phase using CID experiments (ESI-MS/MS) and by density functional theory (DFT) calculations. Both derivatives show similar chemistry in the negative mode with a Retro-Michael addition (Path A^–) being the most abundant Reaction channel, which correlate well with the fluoride induced Retro-Michael addition observed in solution. The difference in the fragmentation of N -3 substituted thymine 5 and N -1 substituted thymine 1 in the positive mode relates to the preferred cleavage of the sulfonyl group ( m/z 155, Path B) in N -3 isomer and the formation of the acryl sulfonamidine 3 ( m/z 309) via Path A in N -1 isomer. Mechanistic studies of the cleavage Reaction conducted by DFT calculations give the trend of the calculated activation energies that agree well with the experimental observations. A mechanism of the Retro-Michael Reaction was interpreted as a McLafferty type of fragmentation, which includes H_β proton shift to one of the neighboring oxygen atoms in a 1,5-fashion inducing N1(N3)–C_α bond scission. This mechanism was found to be kinetically favorable over other tested mechanisms. Significant difference in the observed fragmentation pattern of N -1 and N -3 isomers proves the ESI-MS/MS technique as an excellent method for tracking the fate of similar sulfonamidine drugs. Also, the observed N -1 and/or N -3 thymine alkylation with in situ formed reactive acryl sulfonamidine 3 as a Michael acceptor may open interesting possibilities for the preparation of other N -3 substituted pyrimidines. Graphical Abstract ᅟ
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Mass spectrometry and theoretical studies on N-C bond cleavages in the N-sulfonylamidino thymine derivatives.
Journal of the American Society for Mass Spectrometry, 2015Co-Authors: Renata Kobetić, Snježana Kazazić, Borislav Kovačević, Zoran Glasovac, Luka Krstulović, Miroslav Bajić, Biserka ŽinićAbstract:The reactivity of new biologically active thymine derivatives substituted with 2-(arylsulfonamidino)ethyl group at N1 and N3 position was investigated in the gas phase using CID experiments (ESI-MS/MS) and by density functional theory (DFT) calculations. Both derivatives show similar chemistry in the negative mode with a Retro-Michael addition (Path A–) being the most abundant Reaction channel, which correlate well with the fluoride induced Retro-Michael addition observed in solution. The difference in the fragmentation of N-3 substituted thymine 5 and N-1 substituted thymine 1 in the positive mode relates to the preferred cleavage of the sulfonyl group (m/z 155, Path B) in N-3 isomer and the formation of the acryl sulfonamidine 3 (m/z 309) via Path A in N-1 isomer. Mechanistic studies of the cleavage Reaction conducted by DFT calculations give the trend of the calculated activation energies that agree well with the experimental observations. A mechanism of the Retro-Michael Reaction was interpreted as a McLafferty type of fragmentation, which includes Hβ proton shift to one of the neighboring oxygen atoms in a 1,5-fashion inducing N1(N3)–Cα bond scission. This mechanism was found to be kinetically favorable over other tested mechanisms. Significant difference in the observed fragmentation pattern of N-1 and N-3 isomers proves the ESI-MS/MS technique as an excellent method for tracking the fate of similar sulfonamidine drugs. Also, the observed N-1 and/or N-3 thymine alkylation with in situ formed reactive acryl sulfonamidine 3 as a Michael acceptor may open interesting possibilities for the preparation of other N-3 substituted pyrimidines.
Yasuhisa Shiraishi - One of the best experts on this subject based on the ideXlab platform.
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One-Step Conjugation Method for Site-Specific Antibody–Drug Conjugates through Reactive Cysteine-Engineered Antibodies
Bioconjugate Chemistry, 2016Co-Authors: Daisuke Shinmi, Eri Taguchi, Junko Iwano, Junichi Enokizono, Kazuhiro Masuda, Tsuyoshi Yamaguchi, Yasuhisa ShiraishiAbstract: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...
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one step conjugation method for site specific antibody drug conjugates through reactive cysteine engineered antibodies
Bioconjugate Chemistry, 2016Co-Authors: Daisuke Shinmi, Eri Taguchi, Junko Iwano, Junichi Enokizono, Kazuhiro Masuda, Tsuyoshi Yamaguchi, Yasuhisa ShiraishiAbstract: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...
Renata Kobetić - One of the best experts on this subject based on the ideXlab platform.
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Mass Spectrometry and Theoretical Studies on N–C Bond Cleavages in the N-Sulfonylamidino Thymine Derivatives
Journal of The American Society for Mass Spectrometry, 2015Co-Authors: Renata Kobetić, Snježana Kazazić, Borislav Kovačević, Zoran Glasovac, Luka Krstulović, Miroslav Bajić, Biserka ŽinićAbstract:The reactivity of new biologically active thymine derivatives substituted with 2-(arylsulfonamidino)ethyl group at N1 and N3 position was investigated in the gas phase using CID experiments (ESI-MS/MS) and by density functional theory (DFT) calculations. Both derivatives show similar chemistry in the negative mode with a Retro-Michael addition (Path A^–) being the most abundant Reaction channel, which correlate well with the fluoride induced Retro-Michael addition observed in solution. The difference in the fragmentation of N -3 substituted thymine 5 and N -1 substituted thymine 1 in the positive mode relates to the preferred cleavage of the sulfonyl group ( m/z 155, Path B) in N -3 isomer and the formation of the acryl sulfonamidine 3 ( m/z 309) via Path A in N -1 isomer. Mechanistic studies of the cleavage Reaction conducted by DFT calculations give the trend of the calculated activation energies that agree well with the experimental observations. A mechanism of the Retro-Michael Reaction was interpreted as a McLafferty type of fragmentation, which includes H_β proton shift to one of the neighboring oxygen atoms in a 1,5-fashion inducing N1(N3)–C_α bond scission. This mechanism was found to be kinetically favorable over other tested mechanisms. Significant difference in the observed fragmentation pattern of N -1 and N -3 isomers proves the ESI-MS/MS technique as an excellent method for tracking the fate of similar sulfonamidine drugs. Also, the observed N -1 and/or N -3 thymine alkylation with in situ formed reactive acryl sulfonamidine 3 as a Michael acceptor may open interesting possibilities for the preparation of other N -3 substituted pyrimidines. Graphical Abstract ᅟ
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Mass spectrometry and theoretical studies on N-C bond cleavages in the N-sulfonylamidino thymine derivatives.
Journal of the American Society for Mass Spectrometry, 2015Co-Authors: Renata Kobetić, Snježana Kazazić, Borislav Kovačević, Zoran Glasovac, Luka Krstulović, Miroslav Bajić, Biserka ŽinićAbstract:The reactivity of new biologically active thymine derivatives substituted with 2-(arylsulfonamidino)ethyl group at N1 and N3 position was investigated in the gas phase using CID experiments (ESI-MS/MS) and by density functional theory (DFT) calculations. Both derivatives show similar chemistry in the negative mode with a Retro-Michael addition (Path A–) being the most abundant Reaction channel, which correlate well with the fluoride induced Retro-Michael addition observed in solution. The difference in the fragmentation of N-3 substituted thymine 5 and N-1 substituted thymine 1 in the positive mode relates to the preferred cleavage of the sulfonyl group (m/z 155, Path B) in N-3 isomer and the formation of the acryl sulfonamidine 3 (m/z 309) via Path A in N-1 isomer. Mechanistic studies of the cleavage Reaction conducted by DFT calculations give the trend of the calculated activation energies that agree well with the experimental observations. A mechanism of the Retro-Michael Reaction was interpreted as a McLafferty type of fragmentation, which includes Hβ proton shift to one of the neighboring oxygen atoms in a 1,5-fashion inducing N1(N3)–Cα bond scission. This mechanism was found to be kinetically favorable over other tested mechanisms. Significant difference in the observed fragmentation pattern of N-1 and N-3 isomers proves the ESI-MS/MS technique as an excellent method for tracking the fate of similar sulfonamidine drugs. Also, the observed N-1 and/or N-3 thymine alkylation with in situ formed reactive acryl sulfonamidine 3 as a Michael acceptor may open interesting possibilities for the preparation of other N-3 substituted pyrimidines.
Daisuke Shinmi - One of the best experts on this subject based on the ideXlab platform.
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One-Step Conjugation Method for Site-Specific Antibody–Drug Conjugates through Reactive Cysteine-Engineered Antibodies
Bioconjugate Chemistry, 2016Co-Authors: Daisuke Shinmi, Eri Taguchi, Junko Iwano, Junichi Enokizono, Kazuhiro Masuda, Tsuyoshi Yamaguchi, Yasuhisa ShiraishiAbstract: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...
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one step conjugation method for site specific antibody drug conjugates through reactive cysteine engineered antibodies
Bioconjugate Chemistry, 2016Co-Authors: Daisuke Shinmi, Eri Taguchi, Junko Iwano, Junichi Enokizono, Kazuhiro Masuda, Tsuyoshi Yamaguchi, Yasuhisa ShiraishiAbstract: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...
Wei Wang - One of the best experts on this subject based on the ideXlab platform.
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Maleimide–thiol adducts stabilized through stretching
Nature Chemistry, 2019Co-Authors: Wenmao Huang, Yifei Yu, Xin Wu, Yulan Chen, Wei WangAbstract:Maleimide–thiol Reactions are widely used to produce protein–polymer conjugates for therapeutics. However, maleimide–thiol adducts are unstable in vivo or in the presence of thiol-containing compounds because of the elimination of the thiosuccinimide linkage through a Retro-Michael Reaction or thiol exchange. Here, using single-molecule force spectroscopy, we show that applying an appropriate stretching force to the thiosuccinimide linkage can considerably stabilize the maleimide–thiol adducts, in effect using conventional mechanochemistry of force-accelerated bond dissociation to unconventionally stabilize an adjacent bond. Single-molecule kinetic analysis and bulk structural characterizations suggest that hydrolysis of the succinimide ring is dominant over the Retro-Michael Reaction through a force-dependent kinetic control mechanism, and this leads to a product that is resistant to elimination. This unconventional mechanochemical approach enabled us to produce stable polymer–protein conjugates by simply applying a mechanical force to the maleimide–thiol adducts through mild ultrasonication. Our results demonstrate the great potential of mechanical force for stimulating important productive chemical transformations. Single-molecule force spectroscopy reveals that maleimide–thiol adducts can be stabilized by stretching through a force-dependent kinetic control mechanism. This unconventional use of mechanochemistry enabled us to produce stable polymer–protein conjugates by simply applying a mechanical force to the maleimide–thiol adducts through mild ultrasonication.
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Maleimide-thiol adducts stabilized through stretching.
Nature chemistry, 2019Co-Authors: Wenmao Huang, Yulan Chen, Xiang Gao, Hai Lei, Zhenshu Zhu, Yi Shi, Meng Qin, Wei WangAbstract:Maleimide-thiol Reactions are widely used to produce protein-polymer conjugates for therapeutics. However, maleimide-thiol adducts are unstable in vivo or in the presence of thiol-containing compounds because of the elimination of the thiosuccinimide linkage through a Retro-Michael Reaction or thiol exchange. Here, using single-molecule force spectroscopy, we show that applying an appropriate stretching force to the thiosuccinimide linkage can considerably stabilize the maleimide-thiol adducts, in effect using conventional mechanochemistry of force-accelerated bond dissociation to unconventionally stabilize an adjacent bond. Single-molecule kinetic analysis and bulk structural characterizations suggest that hydrolysis of the succinimide ring is dominant over the Retro-Michael Reaction through a force-dependent kinetic control mechanism, and this leads to a product that is resistant to elimination. This unconventional mechanochemical approach enabled us to produce stable polymer-protein conjugates by simply applying a mechanical force to the maleimide-thiol adducts through mild ultrasonication. Our results demonstrate the great potential of mechanical force for stimulating important productive chemical transformations.