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Amino acid sequence and side chain specific synthetic receptors targeting protein phosphorylation
Malmö universitet, Fakulteten för hälsa och samhälle (HS), Institutionen för biomedicinsk vetenskap (BMV).ORCID-id: 0000-0003-1723-9803
2021 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

Antibodies have become a critical component of many diagnostic assays and are used for therapeutic purposes. Nevertheless they often fail to meet the strict performance demands raised in industry and in the clinic (e.g. stability, reproducibility, selectivity, affinity). These issues are especially notable for assays targeting post translational modifications (PTM) of proteins (phosphorylation, glycosylation, sulfation etc.). Antibody-based technologies suffer from problems of a more general nature associated with the analytical use of biological receptors i.e.: i) limited stability requiring cold chain logistics, ii) high production costs, iii) batch to batch variability. The above emphasizes the need for alternative robust, reproducible and low cost “binders” and assays. The aim in this thesis is to design, develop and test molecularly imprinted polymers (MIPs) which were synthesized epitope and stoichiometric imprinting approaches targeting phosphorylation as a PTM. Protein phosphorylationis one of the most common PTM, which is based on covalent attachment of phosphate group to particular amino acids. Misregulation of phosphorylation process is found related with diseases such as cancer, diabetes, and neurodegeneration. MIPs are synthesized through copolymerization of functional monomers and crosslinkers in the presence of N- and C- terminal protected templates. The key recognition element employed in developed synthetic receptors was 1,3-diaryl urea functionalmonomer 1. This monomer is a potent hydrogen bond donor forming strong cyclichydrogen bonds with oxyanions. Amino acid sequence specific and side chain imprinted binders were prepared targeting phosphorylation on tyrosine (pTyr) and on histidine (pHis). pHis MIP-based approach is proposed as a solution to enrich pHis peptides in the presence of other phosphoesters such as phosphoserine (pSer) in complex mixture without pre-treatment like β-elimination. In pTyr, ZAP-70 (zeta associated 70 kDa protein), which is prognosticator for chronic lymphocytic leukemia (CLL), and pTyr-sequence specific motif Src-SH2 domain were chosen as targets to evaluate regio- or stoichiometric selectivity performance of imprinted polymers. The synthesized polymers are used as effective enrichment tools for target phosphorylated peptides from complex mixture prior to mass spectrometry. Overall, the results demonstrate unique proteomics enrichment tools that link with personalized medicine relying on diagnostic coupled cancer treatment strategies based on kinase inhibitors.

Ort, förlag, år, upplaga, sidor
Malmö: Malmö universitet, 2021. , s. 85
Serie
Malmö University Health and Society Dissertations, ISSN 1653-5383 ; 2021:5
Nyckelord [en]
imprinted polymers, synthetic receptors, PTM, phosphorylation, enrichment, diagnosis
Nationell ämneskategori
Biomedicinsk laboratorievetenskap/teknologi
Identifikatorer
URN: urn:nbn:se:mau:diva-46001DOI: 10.24834/isbn.9789178772087ISBN: 9789178772070 (tryckt)ISBN: 9789178772087 (digital)OAI: oai:DiVA.org:mau-46001DiVA, id: diva2:1596621
Disputation
2021-10-07, Aulan, Fakulteten Hälsa & samhälle samt livestreamat, Jan Waldenströms g. 25, Malmö, 10:00 (Engelska)
Opponent
Anmärkning

Paper III and V in dissertation as manuscript

Tillgänglig från: 2021-09-23 Skapad: 2021-09-23 Senast uppdaterad: 2024-03-01Bibliografiskt granskad
Delarbeten
1. Urea-Based Imprinted Polymer Hosts with Switchable Anion Preference
Öppna denna publikation i ny flik eller fönster >>Urea-Based Imprinted Polymer Hosts with Switchable Anion Preference
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2020 (Engelska)Ingår i: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 142, nr 26, s. 11404-11416Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The design of artificial oxyanion receptors with switchable ion preference is a challenging goal in host–guest chemistry. We here report on molecularly imprinted polymers (MIPs) with an external phospho-sulpho switch driven by small molecule modifiers. The polymers were prepared by hydrogen bond-mediated imprinting of the mono- or dianions of phenyl phosphonic acid (PPA), phenyl sulfonic acid (PSA), and benzoic acid (BA) using N-3,5-bis-(trifluoromethyl)-phenyl-Ń-4-vinylphenyl urea (1) as the functional host monomer. The interaction mode between the functional monomer and the monoanions was elucidated by 1H NMR titrations and 1H–1H NMR NOESY supported by molecular dynamic simulation, which confirmed the presence of high-order complexes. PPA imprinted polymers bound PPA with an equilibrium constant Keq = 1.8 × 105 M–1 in acetonitrile (0.1% 1,2,2,6,6-pentamethylpiperidine) and inorganic HPO42– and SO42– with Keq = 2.9 × 103 M–1 and 4.5 × 103 M–1, respectively, in aqueous buffer. Moreover, the chromatographic retentivity of phosphonate versus sulfonate was shown to be completely switched on this polymer when changing from a basic to an acidic modifier. Mechanistic insights into this system were obtained from kinetic investigations and DSC-, MALDI-TOF-MS-, 1H NMR-studies of linear polymers prepared in the presence of template. The results suggest the formation of template induced 1–1 diad repeats in the polymer main chain shedding unique light on the relative contributions of configurational and conformational imprinting.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2020
Nationell ämneskategori
Fysikalisk kemi
Identifikatorer
urn:nbn:se:mau:diva-17605 (URN)10.1021/jacs.0c00707 (DOI)000547329800012 ()32425049 (PubMedID)2-s2.0-85087432794 (Scopus ID)
Tillgänglig från: 2020-06-29 Skapad: 2020-06-29 Senast uppdaterad: 2024-02-05Bibliografiskt granskad
2. Selective Enrichment of Histidine Phosphorylated Peptides Using Molecularly Imprinted Polymers
Öppna denna publikation i ny flik eller fönster >>Selective Enrichment of Histidine Phosphorylated Peptides Using Molecularly Imprinted Polymers
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2021 (Engelska)Ingår i: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 93, nr 8, s. 3857-3866Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Protein histidine phosphorylation (pHis) is involved in molecular signaling networks in bacteria, fungi, plants, and higher eukaryotes including mammals and is implicated in human diseases such as cancer. Detailed investigations of the pHis modification are hampered due to its acid-labile nature and consequent lack of tools to study this post-translational modification (PTM). We here demonstrate three molecularly imprinted polymer (MIP)-based reagents, MIP1-MIP3, for enrichment of pHis peptides and subsequent characterization by chromatography and mass spectrometry (LC-MS). The combination of MIP1 and β-elimination provided some selectivity for improved detection of pHis peptides. MIP2 was amenable to larger pHis peptides, although with poor selectivity. Microsphere-based MIP3 exhibited improved selectivity and was amenable to enrichment and detection by LC-MS of pHis peptides in tryptic digests of protein mixtures. These MIP protocols do not involve any acidic solvents during sample preparation and enrichment, thus preserving the pHis modification. The presented proof-of-concept results will lead to new protocols for highly selective enrichment of labile protein phosphorylations using molecularly imprinted materials.

Ort, förlag, år, upplaga, sidor
American Chemical Society (ACS), 2021
Nationell ämneskategori
Analytisk kemi
Identifikatorer
urn:nbn:se:mau:diva-41178 (URN)10.1021/acs.analchem.0c04474 (DOI)000626269400026 ()33591162 (PubMedID)2-s2.0-85101877466 (Scopus ID)
Tillgänglig från: 2021-03-10 Skapad: 2021-03-10 Senast uppdaterad: 2024-02-05Bibliografiskt granskad
3. MIP-Binders for sequence specific phosphopeptide capture of ZAP-70 regulatory motifs
Öppna denna publikation i ny flik eller fönster >>MIP-Binders for sequence specific phosphopeptide capture of ZAP-70 regulatory motifs
2021 (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Medicinsk bioteknologi
Identifikatorer
urn:nbn:se:mau:diva-45999 (URN)
Tillgänglig från: 2021-09-23 Skapad: 2021-09-23 Senast uppdaterad: 2024-01-16Bibliografiskt granskad
4. High salt compatible oxyanion receptors by dual ion imprinting
Öppna denna publikation i ny flik eller fönster >>High salt compatible oxyanion receptors by dual ion imprinting
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2020 (Engelska)Ingår i: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 11, nr 16, s. 4246-4250Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The design of hosts for either cations or anions is complicated due to the competition for binding by the host or guest counterions. Imprinting relying on self-assembly offers the possibility to stabilize the guest and its counterion in a favorable geometry. We here report on a comprehensive supramolecular approach to anion receptor design relying on concurrent recognition of both anion and cation. This was achieved by high order complex imprinting of the disodium salt of phenyl-phosphonic acid in combination with neutral urea and sodium ion selective 18-crown-6 monomers. The polymers displayed enhanced affinity for the template or inorganic phosphate or sulfate in competitive aqueous buffers, with affinity and selectivity increasing with increasing ionic strength. The presence of engineered sites for both ionic species dramatically increases the salt uptake in strongly competitive media such as brine.

Ort, förlag, år, upplaga, sidor
Royal Society of Chemistry, 2020
Nationell ämneskategori
Organisk kemi
Identifikatorer
urn:nbn:se:mau:diva-17514 (URN)10.1039/c9sc06508c (DOI)000530491400019 ()2-s2.0-85084280307 (Scopus ID)
Tillgänglig från: 2020-06-18 Skapad: 2020-06-18 Senast uppdaterad: 2024-02-05Bibliografiskt granskad
5. Imprinted Src-SH2 domain mimicking: Targeting pYEEI sequence
Öppna denna publikation i ny flik eller fönster >>Imprinted Src-SH2 domain mimicking: Targeting pYEEI sequence
2021 (Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Biomedicinsk laboratorievetenskap/teknologi
Identifikatorer
urn:nbn:se:mau:diva-46000 (URN)
Tillgänglig från: 2021-09-23 Skapad: 2021-09-23 Senast uppdaterad: 2024-01-16Bibliografiskt granskad

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