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Development of advanced NMR spectroscopy methods for qualitative and quantitative determination of peptide secondary structure to calibrate solid-state FTIR models


Prof. Jozef Lewandowski, Dept. of Chemistry, University of Warwick

Dr Steph Brookes, Associate Principal Scientist, vibrational spectroscopy (Pharmaceutical Sciences)

Dr Kevin Embrey, Associate Principal Scientist, solution-state NMR spectroscopy (Pharmaceutical technology & development)


Dr Les Hughes, Associate Principal Scientist, solid-state NMR spectroscopy (Pharmaceutical technology & development)

Project outline:

AstraZeneca is a science focussed multinational biopharmaceutical company that delivers life-changing medicines. The next generation of therapeutics includes proteins and peptides that are designed to replace a deficient or abnormal protein, augment an existing pathway, or provide a novel function or activity. Knowing the location of alpha helices, and beta strands within such systems, i.e., secondary structure, is important for building property-function relationships and thus facilitates the development of a medicine that is specific and potent.

Fourier-transform Infra-red spectroscopy (FTIR) is a well-established technique for the quantification of protein/peptide secondary structure in the solution state. Modern FTIR spectrometers utilise solution-based calibration libraries based on transmission spectra of large numbers of well characterised proteins to quantify new materials. However, no such calibration libraries exist for the solid state and there is a need to quantify secondary structure both solution and solid formulations.

Attenuated total reflectance (ATR) is the most commonly used sampling technique for FTIR analysis of samples in the solid state. In the ATR technique, the depth of penetration of the infrared beam varies as a function of the wavelength of light. As a result, the bands at lower frequencies are much stronger than those at higher frequencies. In addition, the ATR effect also causes a significant shift of intense infrared absorption bands. This shift depends on the refractive indices of both the ATR crystal and the sample and is further affected by the angle of incidence of the infrared beam within the crystal.

Owing to the significant differences between ATR and transmission FTIR spectra it is necessary to develop ATR specific calibration models in order to quantify peptide secondary structure in the solid state. This project will utilise solid state NMR as a reference technique to calibrate these FTIR models. Many peptides are amorphous in the solid-state which makes the measurement of their secondary structure challenging. Solid-state NMR methods will be developed to enable the measurement of secondary structure of amorphous systems. This will require the development of advanced solid-state NMR methods and will be supported by computational methods including simulations based on density functional theory calculations.

Training that the project would provide to the student:

This project will allow the student to develop skills in solid-state and solution-state NMR spectroscopy and simulation of peptides to quantify secondary structure. Specifically, 1H 1D and 2D 1H-13C correlation solution-state spectra will be compared with state of the art very high speed 1H MAS solid-state NMR spectra (and possibly solid-state correlation spectra). 

In addition, the student will develop skills in both solid state FTIR spectroscopy, using the ATR technique, and solution state FTIR spectroscopy. Specifically, transmission analysis using Confocheck FTIR technology and novel microfluidic modulation spectroscopy (MMS). The student will engage with a world leading biopharmaceutical company through regular Teams meetings to discuss scientific progress.