Determining the dissociation and binding dynamics of molecules using 1H NMR chemical shift imaging techniques

Hussain, Haider (2026) Determining the dissociation and binding dynamics of molecules using 1H NMR chemical shift imaging techniques. Doctoral thesis, University of East Anglia.

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Abstract

This PhD thesis introduces and applies new methodologies based on Nuclear Magnetic Resonance (NMR) chemical shift imaging (CSI) to measure acid dissociation constants (pKa) and metal–ligand binding dynamics in situ. The research addresses longstanding challenges in characterizing systems that are difficult to analyse by conventional means (such as polymers with broad NMR signals or systems requiring numerous titrations) by leveraging controlled chemical gradients within a single NMR tube. In one approach, a derivative-based fitting method was developed to determine pKa from 1H NMR titration data. By analysing the derivative of solution pH with respect to the observed chemical shift (δobs) of an analyte, this method circumvents the need to fit limiting chemical shift values and avoids convergence issues associated with traditional nonlinear regression. It enables rapid, robust pKa determination via a simple polynomial fit, yielding values in excellent agreement with literature standards while reducing the data requirements and time cost of titrations.

The subsequent chapter expands NMR pKa measurements to NMR-invisible species (e.g. macromolecules or ions whose proton signals are unobservable or too broad) by coupling CSI with indicators that measure the quantity of protons transferred to them from the analyte. Using a controlled pH gradient in a single “one-shot” CSI experiment, the quantity of protons transferred from an “invisible” analyte to an NMR-visible indicator is measured across space, enabling calculation of the analyte’s pKa and concentration without direct observation of its own signals. This technique was validated on model systems including a high-molecular-weight polymer (polyacrylic acid) and a protein (wheat germ agglutinin), successfully yielding pKa values consistent with their known behaviour. The method proved effective even when analyte NMR peaks were severely broadened or overlapping, demonstrating its versatility for complex mixtures. Furthermore, the thesis extends CSI-based titration to heterogeneous solvent systems: by establishing a solvent (water-Dimethyl sulfoxide) gradient, spatially resolved pH measurements were used to determine solvent dependent pKa values (psKa) of organic compounds across different solvent compositions. These data were extrapolated (via the Yasuda Shedlovsky method) to estimate the true aqueous pKa, providing a novel route to obtain dissociation constants of sparingly water-soluble molecules.

Finally, a single-tube CSI method for metal ligand binding is presented. Here, a concentrated salt (calcium or magnesium acetate) is layered beneath an aqueous solution of a ligand (such as a polymer or small organic acid), generating an upward diffusion gradient of metal ions.1H CSI is then used to map spatial profiles of free metal concentration [M2+]f (monitored via weak binding indicator molecules whose chemical shifts report on M2+ binding) and the total diffusant (acetate) concentration. From the mismatch between the acetate profile and the free M2+ profile, the local amount of metal bound to the ligand (B) is quantified. This approach was tested on strong, multivalent binders like oxalate or alginate which showed pronounced metal uptake, essentially sequestering Ca2+ and Mg2+ until saturation of their binding sites. In contrast, weak binders such as carboxymethyl cellulose (CMC) and gallic acid exhibited negligible binding, yielding free ion profiles almost overlapping with the control (no ligand) case. These findings quantitatively confirm expectations (e.g. alginate’s well-known Ca2+ crosslinking behaviour and CMC’s minimal interaction with divalent cations) and provide new insights such as the precise point of Ca2+ induced cellulose nanocrystal aggregation.

Overall, the thesis demonstrates that CSI can be harnessed to obtain detailed thermodynamic parameters such pKa values and complexation capacities directly within an NMR tube. The developed techniques require only standard high-field NMR hardware and small sample quantities, yet achieve spatially resolved, multi point measurements in a single run. The outcomes not only corroborate traditional measurements but also enable analyses previously impractical or impossible with conventional titrations and assays. This work significantly broadens the applicability of NMR in physical chemistry and chemical biology, offering efficient new tools for characterizing acid–base equilibria and metal–ligand interactions in complex or inhomogeneous systems.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
Depositing User:	Chris White
Date Deposited:	10 Feb 2026 11:51
Last Modified:	10 Feb 2026 11:51
URI:	https://ueaeprints.uea.ac.uk/id/eprint/101887
DOI:

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