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Mixed pentafluorophenyl and o-fluorophenyl esters of aliphatic dicarboxylic acids: efficient tools for peptide and protein conjugation
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry. (Lars Baltzer)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry. (Lars Baltzer)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Physical Organic Chemistry. (Lars Baltzer)
2012 (English)In: RSC Advances, ISSN 2046-2069, Vol. 2, no 3, 908-914 p.Article in journal (Refereed) Published
Abstract [en]

An efficient methodology for the heteroconjugation of biomolecules with exposed free amino groups has been developed. Mixed pentafluorophenyl and o-fluorophenyl esters of aliphatic dicarboxylic acids with aliphatic chains of varying sizes have been prepared and used to conjugate a 42-residue polypeptides with short model peptides as well as a model dodecapeptide with the antigenic determinant of type B blood, a carbohydrate derivative, to form a glycopeptide. The concept is based on the difference in reactivity towards primary amino groups between phenyl esters with leaving groups of unlike pKa. The reactivities of several pentafluorophenyl and o-fluorophenyl esters towards amino groups were carefully determined under reaction conditions to identify leaving group combinations that would provide optimal differences in reactivity for maximum yields of heteroconjugate formation while keeping the reasonable reaction times. Pentafluorophenyl esters react faster with an amino group and require a weaker base, while an o-fluorophenyl ester requires a stronger base and longer reaction time. The method described is economic, quick and gives complete control over the conjugation reaction. The size of the spacer is conveniently varied by selection of the appropriate aliphatic dicarboxylic acid. While the presented examples describe conjugation reactions of polypeptides with a maximum of 42 residues it is envisioned that the bifunctional linkers reported here will find their most important applications in the heteroconjugation of proteins using lysine side chains, a reaction for which currently few alternatives exist, if access to spacers of variable size is required.

Place, publisher, year, edition, pages
2012. Vol. 2, no 3, 908-914 p.
National Category
Chemical Sciences
Research subject
Chemistry; Chemistry with specialization in Bioorganic Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-161011DOI: 10.1039/C1RA00530HISI: 000299177000025OAI: oai:DiVA.org:uu-161011DiVA: diva2:454055
Available from: 2011-11-04 Created: 2011-11-04 Last updated: 2012-03-20Bibliographically approved
In thesis
1. Peptide Conjugates as Useful Molecular Tools
Open this publication in new window or tab >>Peptide Conjugates as Useful Molecular Tools
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The conjugation of a small organic molecule to synthetic polypeptides from a designed set has been shown to give rise to binders with high affinity and selectivity for the phosphorylated model proteins α-casein and β-casein but not for ovoalbumin. The small organic molecule that was used for this purpose is comprised of two di-(2-picolyl)amine groups assembled on a dimethylphenyl scaffold, and is capable of complexing two Zn2+ ions to form chelates that bind the phosphate ion. The designed polypeptides used for binder construction have no precedence in nature and do not show any prior selectivity favouring any single protein. The polypeptide conjugate binders showed high affinity towards the model protein α-casein, the binder molecule 4C15L8-PP1 bound α-casein with a dissociation constant KD of 17 nM, although the di-(2-picolyl) amine based chelate in the presence of Zn2+ bound phosphate ion with dissociation constants in the low mM range. The observed affinity is due to interactions between the Zn2+ chelate and the phosphate groups of α-casein and also to interactions between the polypeptide scaffold and α-casein. The binder was found to selectively extract α-casein from buffer, bovine milk and human serum spiked with α-casein. The flexible construction of the binder permits for flexible modifications like attachment of fluorophores for titrations and quantifications. The binders were shown to efficiently capture α-casein from human serum when immobilized on solid support in a continuous flow system and to release the captured α-casein upon a simple change of pH using 0.1% acqueous trifluoroacetica acid. The developed technology brings new opportunities in investigating posttranslational phosphorylation events that are involved in signaling cascades and triggering many biologically relevant functions.

A new chemical linker technology has also been developed for the purpose of conjugating biomolecules taking advantage of amino groups for the conjugation. By combining two esters with different reactivities, separated by an aliphatic chain, a molecular tool was developed that allows for controlled conjugation of biomolecules. The two esters react at different rates and can therefore be separated and allowed to react under different conditions in each step, thereby allowing for selective linkage formation between the subunits. The size of the spacer can be varied by selecting the appropriate dicarboxylic acid. The developed technology was shown to provide specificity in heteroconjugate formation in the assembly of a variety of heteroconjugates where polypeptides were combined with other peptides, carbohydrates and proteins.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 71 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 883
Keyword
peptide conjugates, designed peptides, polypeptides, molecular recognition, linker, linking technology, bioconjugation, conjugation, conjugates
National Category
Organic Chemistry
Research subject
Chemistry; Chemistry with specialization in Bioorganic Chemistry
Identifiers
urn:nbn:se:uu:diva-161013 (URN)978-91-554-8227-5 (ISBN)
Public defence
2011-12-15, B41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-11-25 Created: 2011-11-04 Last updated: 2012-01-03Bibliographically approved

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Ślósarczyk, Adam T.Norberg, ThomasBaltzer, Lars

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