Site-Specific PEGylation of Protein Disulfide Bonds Using a Three-Carbon Bridge
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- 作者：Sibu Balan ; Ji-won Choi ; Antony Godwin ; Ian Teo ; Carlos M. Laborde ; Sibylle Heidelberger ; Mire Zloh ; Sunil Shaunak ; Steve Brocchini
- 刊名：Bioconjugate Chemistry
- 出版年：2007
- 出版时间：January 2007
- 年：2007
- 卷：18
- 期：1
- 页码：61 - 76
- 全文大小：5735K
- 年卷期：v.18,no.1(January 2007)
- ISSN：1520-4812
文摘
The covalent conjugation of a functionalized poly(ethylene glycol) (PEG) to multiple nucleophilic amine residuesresults in a heterogeneous mixture of PEG positional isomers. Their physicochemical, biological, and pharmaceuticalproperties vary with the site of conjugation of PEG. Yields are low because of inefficient conjugation chemistryand production costs high because of complex purification procedures. Our solution to these fundamental problemsin PEGylating proteins has been to exploit the latent conjugation selectivity of the two sulfur atoms that arederived from the ubiquitous disulfide bonds of proteins. This approach to PEGylation involves two steps: (1)disulfide reduction to release the two cysteine thiols and (2) re-forming the disulfide by bis-alkylation via a three-carbon bridge to which PEG was covalently attached. During this process, irreversible denaturation of the proteindid not occur. Mechanistically, the conjugation is conducted by a sequential, interactive bis-alkylation using 
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-unsaturated '-monosulfone functionalized PEG reagents. The combination of (a) maintaining the protein's tertiarystructure after disulfide reduction, (b) the mechanism for bis-thiol selectivity of the PEG reagent, and (c) thesteric shielding of PEG ensure that only one PEG molecule is conjugated at each disulfide bond. PEG was site-specifically conjugated via a three-carbon bridge to 2 equiv of the tripeptide glutathione, the cyclic peptide hormonesomatostatin, the tetrameric protein L-asparaginase, and to the disulfides in interferon -2b (IFN). SDS-PAGE,mass spectral, and NMR analyses were used to confirm conjugation, thiol selectivity, and connectivity. Thebiological activity of the L-asparaginase did not change after the attachment of four PEG molecules. In the caseof IFN, a small reduction in biological activity was seen with the single-bridged IFN (without PEG attached). Asignificantly larger reduction in biological activity was seen with the three-carbon disulfide single-bridged PEG-IFNs and with the double-bridged IFN (without PEG attached). The reduction of the PEG-IFN's in vitro biologicalactivity was a consequence of the steric shielding caused by PEG, and it was comparable to that seen with allother forms of PEG-IFNs reported. However, when a three-carbon bridge was used to attach PEG, our PEG-IFN's biological activity was found to be independent of the length of the PEG. This property has not previouslybeen described for PEG-IFNs. Our studies therefore suggest that peptides, proteins, enzymes, and antibody fragmentscan be site-specifically PEGylated across a native disulfide bond using three-carbon bridges without destroyingtheir tertiary structure or abolishing their biological activity. The stoichiometric efficiency of this approach alsoenables recycling of any unreacted protein. It therefore offers the potential to make PEGylated biopharmaceuticalsas cost-effective medicines for global use.
,-unsaturated '-monosulfone functionalized PEG reagents. The combination of (a) maintaining the protein's tertiarystructure after disulfide reduction, (b) the mechanism for bis-thiol selectivity of the PEG reagent, and (c) thesteric shielding of PEG ensure that only one PEG molecule is conjugated at each disulfide bond. PEG was site-specifically conjugated via a three-carbon bridge to 2 equiv of the tripeptide glutathione, the cyclic peptide hormonesomatostatin, the tetrameric protein L-asparaginase, and to the disulfides in interferon -2b (IFN). SDS-PAGE,mass spectral, and NMR analyses were used to confirm conjugation, thiol selectivity, and connectivity. Thebiological activity of the L-asparaginase did not change after the attachment of four PEG molecules. In the caseof IFN, a small reduction in biological activity was seen with the single-bridged IFN (without PEG attached). Asignificantly larger reduction in biological activity was seen with the three-carbon disulfide single-bridged PEG-IFNs and with the double-bridged IFN (without PEG attached). The reduction of the PEG-IFN's in vitro biologicalactivity was a consequence of the steric shielding caused by PEG, and it was comparable to that seen with allother forms of PEG-IFNs reported. However, when a three-carbon bridge was used to attach PEG, our PEG-IFN's biological activity was found to be independent of the length of the PEG. This property has not previouslybeen described for PEG-IFNs. Our studies therefore suggest that peptides, proteins, enzymes, and antibody fragmentscan be site-specifically PEGylated across a native disulfide bond using three-carbon bridges without destroyingtheir tertiary structure or abolishing their biological activity. The stoichiometric efficiency of this approach alsoenables recycling of any unreacted protein. It therefore offers the potential to make PEGylated biopharmaceuticalsas cost-effective medicines for global use.