Design, Synthesis and Characterization of Oriented Glyco-Affinity Macroligands for Glyco-Capturing, Glycomics and Glycoproteomics Applications

Cell surface carbohydrates existing as parts of glycoproteins, glycolipids, and other conjugates present the first information about cell to the outside world and are intimately involved in various biological events such as intercellular communication, and molecular and cellular targeting. However, mechanisms of most processes at the molecular level are still unclear. Therefore, it is very important to develop carbohydrate-specific binding molecules for rapid, efficient, sensitive purification and accurate analysis of complex carbohydrate structures as well as their functions. Furthermore, carbohydrate-specific binding molecules can be expected to be used in medical diagnostic applications for carbohydrate biomarkers. In this thesis study, oriented and multivalent carbohydrate-binding macromolecules were designed and developed based on a chain-end functionalized boronic acid-containing polymer (boropolymer) for glyco-capturing, glycomics and glycoproteomics applications. Namely, a biotin chain end and O-cyanate chain-end functionalized boropolymers were synthesized via aryalamine initiated cyanoxyl-mediated free radical polymerization in a one-pot fashion. The resultant boropolymers were characterized by 1H-NMR and 13C NMR spectroscopy. In our first study we demonstrated the efficient glyco-capturing followed by direct MALDI mass spectrometry identification of the captured carbohydrate by using magnetic beads functionalized with the biotin boropolymer via streptavidin/biotin interaction. In our second study we demonstrated oriented and covalent immobilization of O-Cyanate chain-end functionalized boropolymer on to amine-modified solid surfaces and its specific glyco-capturing capacity by QCM and AFM techniques. We further studied the multivalent interactions of the immobilized O-Cyanate chain end functionalized boropolymer with five different carbohydrate conjugated AuNPs. Our studies showed that different carbohydrates have different binding constants. Furthermore, the multivalent binding between carbohydrate

Design, Synthesis and Characterization of Oriented Glyco-Affinity Macroligands for Glyco-Capturing, Glycomics and Glycoproteomics Applications

Cell surface carbohydrates existing as parts of glycoproteins, glycolipids, and other conjugates present the first information about cell to the outside world and are intimately involved in various biological events such as intercellular communication, and molecular and cellular targeting. However, mechanisms of most processes at the molecular level are still unclear. Therefore, it is very important to develop carbohydrate-specific binding molecules for rapid, efficient, sensitive purification and accurate analysis of complex carbohydrate structures as well as their functions. Furthermore, carbohydrate-specific binding molecules can be expected to be used in medical diagnostic applications for carbohydrate biomarkers. In this thesis study, oriented and multivalent carbohydrate-binding macromolecules were designed and developed based on a chain-end functionalized boronic acid-containing polymer (boropolymer) for glyco-capturing, glycomics and glycoproteomics applications. Namely, a biotin chain end and O-cyanate chain-end functionalized boropolymers were synthesized via aryalamine initiated cyanoxyl-mediated free radical polymerization in a one-pot fashion. The resultant boropolymers were characterized by 1H-NMR and 13C NMR spectroscopy. In our first study we demonstrated the efficient glyco-capturing followed by direct MALDI mass spectrometry identification of the captured carbohydrate by using magnetic beads functionalized with the biotin boropolymer via streptavidin/biotin interaction. In our second study we demonstrated oriented and covalent immobilization of O-Cyanate chain-end functionalized boropolymer on to amine-modified solid surfaces and its specific glyco-capturing capacity by QCM and AFM techniques. We further studied the multivalent interactions of the immobilized O-Cyanate chain end functionalized boropolymer with five different carbohydrate conjugated AuNPs. Our studies showed that different carbohydrates have different binding constants. Furthermore, the multivalent binding between carbohydrate

Design, Synthesis and Characterization of Oriented Glyco-Affinity Macroligands for Glyco-Capturing, Glycomics and Glycoproteomics Applications

Cell surface carbohydrates existing as parts of glycoproteins, glycolipids, and other conjugates present the first information about cell to the outside world and are intimately involved in various biological events such as intercellular communication, and molecular and cellular targeting. However, mechanisms of most processes at the molecular level are still unclear. Therefore, it is very important to develop carbohydrate-specific binding molecules for rapid, efficient, sensitive purification and accurate analysis of complex carbohydrate structures as well as their functions. Furthermore, carbohydrate-specific binding molecules can be expected to be used in medical diagnostic applications for carbohydrate biomarkers. In this thesis study, oriented and multivalent carbohydrate-binding macromolecules were designed and developed based on a chain-end functionalized boronic acid-containing polymer (boropolymer) for glyco-capturing, glycomics and glycoproteomics applications. Namely, a biotin chain end and O-cyanate chain-end functionalized boropolymers were synthesized via aryalamine initiated cyanoxyl-mediated free radical polymerization in a one-pot fashion. The resultant boropolymers were characterized by 1H-NMR and 13C NMR spectroscopy. In our first study we demonstrated the efficient glyco-capturing followed by direct MALDI mass spectrometry identification of the captured carbohydrate by using magnetic beads functionalized with the biotin boropolymer via streptavidin/biotin interaction. In our second study we demonstrated oriented and covalent immobilization of O-Cyanate chain-end functionalized boropolymer on to amine-modified solid surfaces and its specific glyco-capturing capacity by QCM and AFM techniques. We further studied the multivalent interactions of the immobilized O-Cyanate chain end functionalized boropolymer with five different carbohydrate conjugated AuNPs. Our studies showed that different carbohydrates have different binding constants. Furthermore, the multivalent binding between carbohydrate

Synthesis and Characterization of Biotin Chain-End Functionalized Boronic Acid-Containing Polymer (Boropolymer) as Functional Glyco-Affinity Macroligand

A biotin chain-end functionalized boronic acid-containing polymer (biotin boropolymer) as an oriented glyco-affinity macroligand for efficient carbohydrate and glycoconjugate purification and identification application was described. Briefly, the biotin boropolymer was synthesized via a biotin derivated arylamine initiated cyanoxyl-mediated free-radical polymerization in one-pot fashion. The specific streptavidin binding capacity of biotin boropolymer was confirmed by streptavidin–HABA assay, while the specific carbohydrate binding capacity of biotin boropolymer was evaluated by Alizarin Red S binding assay. Efficient glyco-capturing followed by direct MALDI mass spectrometry identification of the captured carbohydrate was demonstrated by using magnetic bead functionalized with the biotin boropolymer via streptavidin/biotin interaction

Multivalent Interaction-based Carbohydrate Biosensors for Signal Amplification

Multivalent interaction between boronic acids immobilized on quartz crystal microbalance (QCM) sensor surface and the carbohydrates modified Au-nanoparticle (AuNP) has been demonstrated for the development of a sensitive carbohydrate biosensor. Briefly, a boronic acid-containing polymer (boropolymer) as multivalent carbohydrate receptor was oriented immobilized on the cysteamine coated electrode through isourea bond formation. Carbohydrates were conjugated to AuNPs to generate a multivalent carbohydrates moiety to amplify the response signal. Thus, the binding of the carbohydrate conjugated AuNPs to the boropolymer surface are multivalent which could simultaneously increase the binding affinity and specificity. We systematically studied the binding between five carbohydrates conjugated AuNPs and the boropolymer. Our studies show that the associate constant (Ka) was in the order of fucose \u3c glucose \u3c mannose \u3c galactose \u3c maltose. A linear response in the range from 23 μM to 3.83 mM was observed for mannose conjugated AuNPs and the boropolymer recognition elements, with the lower detection limit of 1.5 μM for the carbohydrate analytes. Furthermore, the multivalent binding between carbohydrates and boronic acids are reversible and allow the regeneration of boropolymer surface by using 1 M acetic acid so as to sequentially capture and release the carbohydrate analytes