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Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids
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: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 18, no 4, 261-269 p.Article in journal (Refereed) Published
Abstract [en]

An efficient method for the heteroconjugation of biomolecules carrying free amino groups was reported previously, where mixed polyfluorophenyl diesters of dicarboxylic acids with varied aliphatic chain length were shown to be efficient reagents for the conjugation of a variety of model biomolecules. The concept was based on the differential reactivity of the esters towards amines. The concept has now been further optimized, and a 2,6-difluorophenyl-pentafluorophenyl diester combination has been demonstrated to be the most efficient, both with respect to selectivity and to reaction rate. A pentafluorophenyl ester reacts faster with an amino group and requires a weaker base than a 2,6-difluorophenyl ester that requires a stronger base and longer reaction time. With the use of this combination of esters, we obtained considerably shortened reaction times compared with those reported previously, yet still retaining the desired selectivity in heteroconjugation. The increased reactivity of the bifunctional reagent allowed the construction of sophisticated peptide heteroconjugates from peptides, carbohydrates and proteins, showing a wide scope of applicability in the field of assembling functional bioconjugates.

Place, publisher, year, edition, pages
2012. Vol. 18, no 4, 261-269 p.
Keyword [en]
conjugation, bioconjugation, linker, heteroconjugation, polyfluorophenyl esters
National Category
Chemical Sciences
Research subject
Chemistry; Chemistry with specialization in Bioorganic Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-161012DOI: 10.1002/psc.2394ISI: 000302017900007OAI: oai:DiVA.org:uu-161012DiVA: diva2:454056
Available from: 2011-11-04 Created: 2011-11-04 Last updated: 2017-12-08Bibliographically 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.Baltzer, Lars

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