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Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit
In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at sur...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8350135/ https://www.ncbi.nlm.nih.gov/pubmed/34381760 http://dx.doi.org/10.3389/fchem.2021.707797 |
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author | Cooper, Chris E. Bird, Matthew Sheng, XiaoBo Choi, Ji-Won Silkstone, Gary G.A. Simons, Michelle Syrett, Natalie Piano, Riccardo Ronda, Luca Bettati, Stefano Paredi, Gianluca Mozzarelli, Andrea Reeder, Brandon J. |
author_facet | Cooper, Chris E. Bird, Matthew Sheng, XiaoBo Choi, Ji-Won Silkstone, Gary G.A. Simons, Michelle Syrett, Natalie Piano, Riccardo Ronda, Luca Bettati, Stefano Paredi, Gianluca Mozzarelli, Andrea Reeder, Brandon J. |
author_sort | Cooper, Chris E. |
collection | PubMed |
description | In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate. |
format | Online Article Text |
id | pubmed-8350135 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-83501352021-08-10 Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit Cooper, Chris E. Bird, Matthew Sheng, XiaoBo Choi, Ji-Won Silkstone, Gary G.A. Simons, Michelle Syrett, Natalie Piano, Riccardo Ronda, Luca Bettati, Stefano Paredi, Gianluca Mozzarelli, Andrea Reeder, Brandon J. Front Chem Chemistry In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate. Frontiers Media S.A. 2021-07-26 /pmc/articles/PMC8350135/ /pubmed/34381760 http://dx.doi.org/10.3389/fchem.2021.707797 Text en Copyright © 2021 Cooper, Bird, Sheng, Choi, Silkstone, Simons, Syrett, Piano, Ronda, Bettati, Paredi, Mozzarelli and Reeder. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Chemistry Cooper, Chris E. Bird, Matthew Sheng, XiaoBo Choi, Ji-Won Silkstone, Gary G.A. Simons, Michelle Syrett, Natalie Piano, Riccardo Ronda, Luca Bettati, Stefano Paredi, Gianluca Mozzarelli, Andrea Reeder, Brandon J. Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit |
title | Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit |
title_full | Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit |
title_fullStr | Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit |
title_full_unstemmed | Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit |
title_short | Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit |
title_sort | stability of maleimide-peg and mono-sulfone-peg conjugation to a novel engineered cysteine in the human hemoglobin alpha subunit |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8350135/ https://www.ncbi.nlm.nih.gov/pubmed/34381760 http://dx.doi.org/10.3389/fchem.2021.707797 |
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