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Peptide Conjugates as Useful Molecular Tools
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry. (Lars Baltzer)
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 [en]
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: urn:nbn:se:uu:diva-161013ISBN: 978-91-554-8227-5 (print)OAI: oai:DiVA.org:uu-161013DiVA: diva2:454068
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
List of papers
1. The molecular recognition of phosphorylated proteins by designed polypeptides conjugated to a small molecule that binds phosphate
Open this publication in new window or tab >>The molecular recognition of phosphorylated proteins by designed polypeptides conjugated to a small molecule that binds phosphate
2011 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, no 22, 7697-7704 p.Article in journal (Refereed) Published
Abstract [en]

The conjugation of polypeptides from a designed set to the small molecule ligand 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, which in the presence of Zn2+ ions binds inorganic phosphate, has been shown to provide a polypeptide conjugate that binds α-casein, a multiply phosphorylated protein, with a dissociation constant KD of 17 nM. The measured affinity is more than three orders of magnitude higher than that of the small molecule ligand for phosphate and the binding of 500 nM of α-casein was not inhibited by 10 mM phosphate buffer, providing a 2000-fold excess of phosphate ion over protein. The selectivity for phosphoproteins was demonstrated by extraction of α-casein from solutions of various complexity, including milk and human serum spiked with α-casein. In addition to α-casein, β-casein was also recognized but not ovoalbumin. Conjugation of a polypeptide to the zinc chelating ligand was therefore shown to give rise to dramatically increased affinity and also increased selectivity. A set of polypeptide conjugates is expected to be able to capture a large number of phosphorylated proteins, perhaps all, and in combination with electrophoresis or mass spectrometry become a powerful tool for the monitoring of phosphorylation levels. The presented binder can easily be attached to various types of surfaces; here demonstrated for the case of polystyrene particles. The example of phosphoproteins was selected since posttranslational phosphorylation is of fundamental importance in cell biology due to its role in signaling and therefore of great interest in drug development. The reported concept for binder development is, however, quite general and high-affinity binders can conveniently be developed for a variety of proteins including those with posttranslational modifications for which small molecule recognition elements are available.

Keyword
Molecular recognition, binders, peptide - small molecule conjugate, conjugates
National Category
Chemical Sciences
Research subject
Chemistry with specialization in Bioorganic Chemistry; Chemistry
Identifiers
urn:nbn:se:uu:diva-161010 (URN)10.1039/c1ob06154b (DOI)000296203700017 ()
Available from: 2011-11-04 Created: 2011-11-04 Last updated: 2017-12-08Bibliographically approved
2. Mixed pentafluorophenyl and o-fluorophenyl esters of aliphatic dicarboxylic acids: efficient tools for peptide and protein conjugation
Open this publication in new window or tab >>Mixed pentafluorophenyl and o-fluorophenyl esters of aliphatic dicarboxylic acids: efficient tools for peptide and protein conjugation
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.

National Category
Chemical Sciences
Research subject
Chemistry; Chemistry with specialization in Bioorganic Chemistry
Identifiers
urn:nbn:se:uu:diva-161011 (URN)10.1039/C1RA00530H (DOI)000299177000025 ()
Available from: 2011-11-04 Created: 2011-11-04 Last updated: 2012-03-20Bibliographically approved
3. Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids
Open this publication in new window or tab >>Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids
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.

Keyword
conjugation, bioconjugation, linker, heteroconjugation, polyfluorophenyl esters
National Category
Chemical Sciences
Research subject
Chemistry; Chemistry with specialization in Bioorganic Chemistry
Identifiers
urn:nbn:se:uu:diva-161012 (URN)10.1002/psc.2394 (DOI)000302017900007 ()
Available from: 2011-11-04 Created: 2011-11-04 Last updated: 2017-12-08Bibliographically approved

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