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Photochemical Ligation Techniques for Carbohydrate Biosensors and Protein Interaction Studies
KTH, School of Chemical Science and Engineering (CHE), Chemistry, Organic Chemistry.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis concerns the development of surface ligation techniques for the preparation of carbohydrate biosensors. Several methodologies were developed based on efficient photochemical insertion reactions which quickly functionalize polymeric materials, with either carbohydrates or functional groups such as alkynes or alkenes. The alkyne/alkene surfaces were then treated with carbohydrate azides or thiols and reacted under chemoselective Cu-catalyzed azide-alkyne cycloaddition (CuAAC) or photo-radical thiol-ene/yne click chemistry, thus creating a range of carbohydrate biosensor surfaces under ambient conditions. The methodologies were evaluated by quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) flow through instrumentations with recurring injections of a range of lectins, allowing for real-time analysis of the surface interactions. The developed methods were proven robust and versatile, and the generated carbohydrate biosensors showed high specificities and good capacities for lectin binding.  The methods were then used to investigate how varying the glycan linker length and/or a sulfur-linkage affect the subsequent protein binding. The survey was further explored by investigating the impact of sulfur in glycosidic linkages on protein binding, through competition assays with various O/S-linked disaccharides in solution interactions with lectins.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2012. , xi, 89 p.
Series
Trita-CHE-Report, ISSN 1654-1081 ; 2012:10
Keyword [en]
Carbohydrates, Biosensors, CuAAC, Photochemistry, Thiol-ene/yne, Perfluorophenylazide (PFPA), Lectins, Molecular recognition, Quartz crystal microbalance (QCM), Surface plasmon resonance (SPR)
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:kth:diva-90956ISBN: 978-91-7501-271-1 (print)OAI: oai:DiVA.org:kth-90956DiVA: diva2:508185
Public defence
2012-03-30, F3, Lindstedtsvägen 26, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20120309Available from: 2012-03-09 Created: 2012-03-05 Last updated: 2012-03-09Bibliographically approved
List of papers
1. Photo-Click Immobilization of Carbohydrates on Polymeric Surfaces - A Quick Method to Functionalize Surfaces for Biomolecular Recognition Studies
Open this publication in new window or tab >>Photo-Click Immobilization of Carbohydrates on Polymeric Surfaces - A Quick Method to Functionalize Surfaces for Biomolecular Recognition Studies
2009 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 20, no 12, 2364-2370 p.Article in journal (Refereed) Published
Abstract [en]

Methods to rapidly functionalize specific polymeric surfaces with alkynes, which can subsequently be linked to azide-containing carbohydrates, are presented. The methods comprise two main concepts: azide photoligation and Cu-catalyzed azide-alkyne cycloaddition. 2-Azidoethyl-functionalized CL-D-mannopyranoside was synthesized and covalently attached to alkyne-functionalized polymeric surfaces using the techniques. The protein recognition properties of the carbollydrate-presenting surfaces were evaluated using quartz crystal microbalance biosensor instrumentation.

National Category
Polymer Chemistry
Identifiers
urn:nbn:se:kth:diva-19043 (URN)10.1021/bc9003519 (DOI)000272690100021 ()19888719 (PubMedID)2-s2.0-72449192287 (Scopus ID)
Note

QC 20100525

Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
2. Photo-Click Immobilization on Quartz Crystal Microbalance Sensors for Selective Carbohydrate-Protein Interaction Analyses
Open this publication in new window or tab >>Photo-Click Immobilization on Quartz Crystal Microbalance Sensors for Selective Carbohydrate-Protein Interaction Analyses
Show others...
2011 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 83, no 3, 1000-1007 p.Article in journal (Refereed) Published
Abstract [en]

A photoclick method based on azide photoligation and Cu-catalyzed azide-alkyne cycloaddition has been evaluated for the immobilization of carbohydrates to polymeric materials. The biomolecular recognition properties of the materials have been investigated with regard to applicable polymeric substrates and selectivity of protein binding. The method was used to functionalize a range of polymeric surfaces (polystyrene, polyacrylamide, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and polypropene) with various carbohydrate structures (based on alpha-D-mannose, beta-D-galactose, and N-acetyl-beta-D-glucosamine). The functionalized surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance flow through system with a series of different carbohydrate-binding proteins (lectins). The method proved to be robust and versatile, resulting in a range of efficient sensors showing high and predictable protein selectivities.

National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-30971 (URN)10.1021/ac102781u (DOI)000286689600053 ()2-s2.0-79952126793 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20110318Available from: 2011-03-18 Created: 2011-03-07 Last updated: 2017-12-11Bibliographically approved
3. Stereoselective synthesis of light-activatable perfluorophenylazide-conjugated carbohydrates for glycoarray fabrication and evaluation of structural effects on protein binding by SPR imaging
Open this publication in new window or tab >>Stereoselective synthesis of light-activatable perfluorophenylazide-conjugated carbohydrates for glycoarray fabrication and evaluation of structural effects on protein binding by SPR imaging
Show others...
2011 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, no 9, 3188-3198 p.Article in journal (Refereed) Published
Abstract [en]

A series of light-activatable perfluorophenylazide (PFPA)-conjugated carbohydrate structures have been synthesized and applied to glycoarray fabrication. The glycoconjugates were structurally varied with respect to anomeric attachment, S-, and O-linked carbohydrates, respectively, as well as linker structure and length. Efficient stereoselective synthetic routes were developed, leading to the formation of the PFPA-conjugated structures in good yields over few steps. The use of glycosyl thiols as donors proved especially efficient and provided the final compounds in up to 70% total yield with high anomeric purities. PFPA-based photochemistry was subsequently used to generate carbohydrate arrays on a polymeric surface, and surface plasmon resonance imaging (SPRi) was applied for evaluation of carbohydrate-protein interactions using the plant lectin Concanavalin A (Con A) as a probe. The results indicate better performance and equal efficiency of S-and O-linked structures with intermediate linker length.

National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-33214 (URN)10.1039/c1ob05040k (DOI)000289488000018 ()2-s2.0-79954444517 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20110523Available from: 2011-05-23 Created: 2011-05-02 Last updated: 2017-12-11Bibliographically approved
4. Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry in aqueous solution
Open this publication in new window or tab >>Photogenerated lectin sensors produced by thiol-ene/yne photo-click chemistry in aqueous solution
Show others...
2012 (English)In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 34, no 1, 51-56 p.Article in journal (Refereed) Published
Abstract [en]

The photoinitiated radical reactions between thiols and alkenes/alkynes (thiol-ene and thiol-yne chemistry) have been applied to a functionalization methodology to produce carbohydrate-presenting surfaces for analyses of biomolecular interactions. Polymer-coated quartz surfaces were functionalized with alkenes or alkynes in a straightforward photochemical procedure utilizing perfluorophenylazide (PFPA) chemistry. The alkene/alkyne surfaces were subsequently allowed to react with carbohydrate thiols in water under UV-irradiation. The reaction can be carried out in a drop of water directly on the surface without photoinitiator, and any disulfide side products were easily washed away after the functionalization process. The resulting carbohydrate-presenting surfaces were evaluated in real-time studies of protein-carbohydrate interactions using a quartz crystal microbalance (QCM) flow-through system with recurring injections of selected lectins, with intermediate regeneration steps using low pH buffer. The resulting methodology proved fast, efficient and scalable to high-throughput analysis formats, and the produced surfaces showed significant protein binding with expected selectivities of the lectins used in the study.

Keyword
Photochemistry, Click chemistry, Thiol-ene/-yne, Carbohydrates, Lectins, Quartz crystal microbalance
National Category
Analytical Chemistry
Identifiers
urn:nbn:se:kth:diva-90846 (URN)10.1016/j.bios.2012.01.001 (DOI)000302886500007 ()2-s2.0-84858216975 (Scopus ID)
Funder
Swedish Research Council, 621-2007-4709
Note
QC 20120525Available from: 2012-03-05 Created: 2012-03-01 Last updated: 2017-12-07Bibliographically approved
5. Stereocontrolled 1-S-glycosylation and comparative binding studies of photoprobe-thiosaccharide conjugates with their O-linked analogs
Open this publication in new window or tab >>Stereocontrolled 1-S-glycosylation and comparative binding studies of photoprobe-thiosaccharide conjugates with their O-linked analogs
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2013 (English)In: Pure and Applied Chemistry, ISSN 0033-4545, E-ISSN 1365-3075, Vol. 85, no 9, 1789-1801 p.Article in journal (Refereed) Published
Abstract [en]

The use of thioglycosides and other glycan derivatives with anomeric sulfur linkages is gaining increasing interest, both in synthesis and in various biological contexts. Herein, we demonstrate the occurrence and circumvention of anomerization during 1-S-glycosylation reactions, and present highly efficient and stereocontrolled syntheses of a series of photoprobe-thiosaccharide conjugates. Mutarotation of glycosyl thiols proved to be the origin of the anomeric mixtures formed, and kinetic effects could be used to circumvent anomerization. The synthesized carbohydrate conjugates were then evaluated by both solution- and solid-phase-based techniques. Both binding results showed that the S-linked glycosides interact with their cognate lectins comparably to the corresponding O-analogs in the present cases, thus demonstrating the reliability of the solid-support platform built upon our photo-initiated carbohydrate immobilization method for probing protein bindings, and showing the potential of combining these two means for studying carbohydrate-protein inter-actions.

Keyword
carbohydrates, photochemistry, protein interactions, organic materials, organic synthesis, stereocontrol
National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-41972 (URN)10.1351/PAC-CON-12-08-13 (DOI)000323700200004 ()2-s2.0-84883377211 (Scopus ID)
Funder
Swedish Research Council, 622-2003-2343
Note

QC 20130930. Updated from submitted to published.

Available from: 2011-10-04 Created: 2011-10-04 Last updated: 2017-12-08Bibliographically approved
6. Synthesis and Binding Affinity Analysis of α1-2- and α1-6-O/S-linked Dimannosides for the Elucidation of Sulfur in Glycosidic Bonds using Quartz Crystal Microbalance Sensors
Open this publication in new window or tab >>Synthesis and Binding Affinity Analysis of α1-2- and α1-6-O/S-linked Dimannosides for the Elucidation of Sulfur in Glycosidic Bonds using Quartz Crystal Microbalance Sensors
Show others...
(English)Article in journal (Other academic) Submitted
Abstract [en]

Synthetic routes towards α1-2- and α1-6-linked dimannosides with S- or O-glycosidic bonds are presented. A glycosylation method was developed in which a sulfhydryl glycosyl acceptor was coupled to a 1-O-acetyl-glycosyl donor under Lewis acid catalysis. Final debenzylation of the S-linked dimannosides were accomplished through Birch reduction in high yields. The protein recognition properties of the synthesized dimannosides were then evaluated in a competition binding assay with the model lectin Con A, to investigate the effect of sulfur in the glycosidic bonds. Mannose-presenting surfaces were produced according to a previously reported Photo-Click immobilization method and the subsequent binding study was performed in an automated QCM flow through instrumentation. The recorded EC50-values correlated well to previously reported binding affinities for the O-linked dimannosides. Results were in agreement with known binding affinities, where the S-analogs displayed slightly weaker binding and a positive apparent cooperativity.

National Category
Organic Chemistry
Identifiers
urn:nbn:se:kth:diva-90848 (URN)
Note
QS 2012 QS 20120328Available from: 2012-03-05 Created: 2012-03-01 Last updated: 2012-03-28Bibliographically approved

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