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Insights into dynamic covalent chemistry for bioconjugation applications
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Polymer Chemistry.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Dynamic covalent chemistry (DCC) is currently exploited in several areas of biomedical applications such as in drug discovery, sensing, molecular separation, catalysis etc. Hydrazone and oxime chemistry have several advantages, such as mild reaction conditions, selectivity, efficiency, and biocompatibility and therefore, have the potential to be for bioconjugation applications. However, these reactions suffer from major drawbacks of slow reaction rate and poor bond stability under physiological conditions. In this regard, the work presented in this thesis focuses on designing novel bioconjugation reactions amenable under physiological conditions with tunable reaction kinetics and conjugation stability.

The first part of the thesis presents different strategies of dynamic covalent reactions utilized for biomedical applications. In the next part, a detailed study related to the mechanism and catalysis of oxime chemistry was investigated in the presence of various catalysts. Aniline, carboxylate and saline were selective as target catalysts and their reaction kinetics were compared under physiological conditions (Paper I and II). Then we attempted to explore the potential of those chemistries in fabricating 3D hydrogel scaffolds for regenerative medicine application. A novel mild and regioselective method was devised to introduce an aldehyde moiety onto glycosaminoglycans structure. This involved the introduction of amino glycerol to glycosaminoglycans, followed by regioselective oxidation of tailed flexible diol without affecting the C2-C3 diol groups on the disaccharide repeating unit. The oxidation rate of the tailed flexible diol was 4-times faster than that of C2-C3 diol groups of native glycosaminoglycan. This strategy preserves the structural integrity of the glycosaminoglycans and provides a functional aldehyde moiety (Paper III). Further, different types of hydrazones were designed and their hydrolytic stability under acidic condition was carefully evaluated. The hydrazone linkage with the highest hydrolytic stability was utilized in the preparation of extracellular matrix hydrogel for delivery of bone morphogenetic proteins 2 in bone regeneration (Paper IV) and studied for controlled release of the growth factor (Paper III).

In summary, this thesis presents a selection of strategies for designing bioconjugation chemistries that possess tunable stability and reaction kinetics under physiological conditions. These chemistries are powerful tools for conjugation of biomolecules for the biomedical applications.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. , p. 59
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1554
Keywords [en]
dynamic covalent chemistry, reaction mechanism, hydrogel, bioconjugation, catalysis
National Category
Polymer Chemistry Materials Chemistry Organic Chemistry
Research subject
Chemistry with specialization in Polymer Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-329022ISBN: 978-91-513-0065-8 (print)OAI: oai:DiVA.org:uu-329022DiVA, id: diva2:1138753
Public defence
2017-10-26, Room 10132, Ånströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-10-03 Created: 2017-09-06 Last updated: 2017-10-18
List of papers
1. Insights into the Mechanism and Catalysis of Oxime Coupling Chemistry at Physiological pH
Open this publication in new window or tab >>Insights into the Mechanism and Catalysis of Oxime Coupling Chemistry at Physiological pH
2015 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 21, no 15, p. 5980-5985Article in journal (Refereed) Published
Abstract [en]

The dynamic covalent-coupling reaction involving alpha-effect nucleophiles has revolutionized bioconjugation approaches, due to its ease and high efficiency. Key to its success is the discovery of aniline as a nucleophilic catalyst, which made this reaction feasible under physiological conditions. Aniline however, is not so effective for keto substrates. Here, we investigate the mechanism of aniline activation in the oxime reaction with aldehyde and keto substrates. We also present carboxylates as activating agents that can promote the oxime reaction with both aldehyde and keto substrates at physiological pH. This rate enhancement circumvents the influence of alpha-effect by forming H-bonds with the rate-limiting intermediate, which drives the reaction to completion. The combination of aniline and carboxylates had a synergistic effect, resulting in a similar to 14-31-fold increase in reaction rate at pD 7.4 with keto substrates. The biocompatibility and efficiency of carboxylate as an activating agent is demonstrated by performing cell-surface oxime labeling at physiological pH using acetate, which showed promising results that were comparable with aniline.

Keywords
ketones, kinetics, coupling reaction, oxime, reaction mechanisms
National Category
Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-252690 (URN)10.1002/chem.201406458 (DOI)000352506500042 ()25737419 (PubMedID)
Available from: 2015-05-25 Created: 2015-05-11 Last updated: 2017-12-04Bibliographically approved
2. Saline catalyse oxime reaction at physiological pH: overcoming a major limitation of bioorthogonal reaction
Open this publication in new window or tab >>Saline catalyse oxime reaction at physiological pH: overcoming a major limitation of bioorthogonal reaction
Show others...
(English)In: Article in journal (Refereed) Submitted
Abstract [en]

We have discovered a simple and versatile reaction condition for oxime mediated bioconjugation reaction that could be adapted for both aldehyde and keto substrates. We found that saline accelerated the oxime kinetics in a concentration dependent manner under physiological conditions. The reaction mechanism is validated by computational studies, and the versatility of the reaction is demonstrated by cell-surface labeling experiments. Saline offers an efficient and non-toxic catalytic option for performing the bioorthogonal-coupling reaction of biomolecules at the physiological pH. This saline mediated bioconjugation reaction represents the most bio-friendly, mild and versatile approach for conjugating sensitive biomolecules and does not require any extensive purification step.

Keywords
Oxime reaction, catalysis, kinetics, labeling
National Category
Polymer Chemistry
Identifiers
urn:nbn:se:uu:diva-328905 (URN)
Available from: 2017-09-04 Created: 2017-09-04 Last updated: 2017-09-07Bibliographically approved
3. Mild and Efficient Strategy for Site-Selective Aldehyde Modification of Glycosaminoglycans: Tailoring Hydrogels with Tunable Release of Growth Factor
Open this publication in new window or tab >>Mild and Efficient Strategy for Site-Selective Aldehyde Modification of Glycosaminoglycans: Tailoring Hydrogels with Tunable Release of Growth Factor
2013 (English)In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, no 7, p. 2427-2432Article in journal (Refereed) Published
Abstract [en]

Aldehydes have been used as an important bioorthogonal chemical reporter for conjugation of large polymers and bioactive substances. However, generating aldehyde functionality on carbohydrate-based biopolymers without changing its native chemical structure has always persisted as a challenging task. The common methods employed to achieve this require harsh reaction conditions, which often compromise the structural integrity and biological function of these sensitive molecules. Here we report a mild and simple method to graft aldehydes groups on glycosaminoglycans (GAGs) in a site-selective manner without compromising the structural integrity of the biopolymer. This regio-selective modification was achieved by conjugating the amino-glycerol moiety on the carboxylate residue of the polymer, which allowed selective cleavage of pendent diol groups without interfering with the C2C3 diol groups of the native glucopyranose residue. Kinetic evaluation of this reaction demonstrated significant differences in second-order reaction rate for periodate oxidation (by four-orders of magnitude) between the two types of vicinal diols. We employed this chemistry to develop aldehyde modifications of sulfated and nonsulfated GAGs such as hyaluronic acid (HA), heparin (HP), and chondroitin sulfate (CS). We further utilized these aldehyde grafted GAGs to tailor extracellular matrix mimetic injectable hydrogels and evaluated its rheological properties. The composition of the hydrogels was also found to modulate release of therapeutic protein such as FGF-2, demonstrating controlled release (60%) for over 14 days. In short, our result clearly demonstrates a versatile strategy to graft aldehyde groups on sensitive biopolymers under mild conditions that could be applied for various bioconjugation and biomedical applications such as drug delivery and regenerative medicine.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-204978 (URN)10.1021/bm400612h (DOI)000321793700035 ()
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2013-08-16 Created: 2013-08-13 Last updated: 2017-12-06Bibliographically approved
4. Smart Design of Stable Extracellular Matrix Mimetic Hydrogel: Synthesis, Characterization, and In Vitro and In Vivo Evaluation for Tissue Engineering
Open this publication in new window or tab >>Smart Design of Stable Extracellular Matrix Mimetic Hydrogel: Synthesis, Characterization, and In Vitro and In Vivo Evaluation for Tissue Engineering
Show others...
2013 (English)In: Advanced Functional Materials, ISSN 1616-301X, E-ISSN 1616-3028, Vol. 23, no 10, p. 1273-1280Article in journal (Refereed) Published
Abstract [en]

The simplicity and versatility of hydrazone crosslinking has made it a strategy of choice for the conjugation of bioactive molecules. However, the labile nature of hydrazone linkages and reversibility of this coupling reaction restricts its full potential. Based on the fundamental understanding of hydrazone stability, this problem is circumvented by resonance-stabilization of a developing N2 positive charge in a hydrazone bond. A novel chemistry is presented to develop a resilient hydrazone bond that is stable and non- reversible under physiological conditions. A carbodihydrazide (CDH) type hydrazide derivative of the biomolecule forms intrinsically stabilized hydrazone-linkages that are nearly 15-fold more stable at pH 5 than conventional hydrazone. This chemoselective coupling reaction is catalyst-free, instantaneous, and virtually non-cleavable under physiological conditions, therefore can serve as a catalyst-free alternative to click chemistry. This novel crosslinking reaction is used to tailor a hyaluronan hydrogel, which delivered exceptional hydrolytic stability, mechanical properties, low swelling, and controlled enzymatic degradation. These desired characteristics are achieved without increasing the chemical crosslinking. The in vivo evaluation of this hydrogel revealed neo-bone with highly ordered collagen matrix mimicking natural bone regeneration. The proximity ligation assay or PLA is used to detect blood vessels, which highlighted the quality of engineered tissue.

Keywords
biomimetics, biomedical applications, hydrogels, tissue engineering, hyaluronic acid
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-198387 (URN)10.1002/adfm.201201698 (DOI)000316196100007 ()
Available from: 2013-04-15 Created: 2013-04-15 Last updated: 2017-12-06Bibliographically approved

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