Sweet 2D-IR: Two-dimensional infrared spectroscopy of carbohydrates with vibrational probes

Carbohydrates are a fundamental class of biomolecules, comparable in significance to proteins and lipids. Despite primarily consisting of carbon and water, carbohydrates exhibit remarkable diversity in structure and biochemical function, for instance due to size / degree of polymerization, stereochemistry, conformation, ring forms, chemical modification, glycosidic linkage, and branching patterns. Even "simple" glucose can exist as one open form and four cyclic stereoisomers. It also shares its molecular formula with 24 other hexoses. This diversity, coupled with their closely related atomic composition, makes investigating them with biophysical tools challenging. For infrared (IR) spectroscopy this leads to highly congested spectra with signals that are difficult to assign to specific functional groups. This challenge has been addressed in other complex biomolecules, such as proteins, using site-specific vibrational probes (VP). These probes are functional groups that usually do not occur naturally and exhibit absorption in the free spectral range between 1.800 cm−1 and 2300 cm−1. Using VP allows to investigate the immediate environment of the oscillator with a high degree of spatial resolution. This thesis applies the concept of VP for biophysical IR studies of carbohydrates. The aim is to enable two-dimensional infrared (2D-IR) spectroscopy of carbohydrates with time resolutions ranging from femtoseconds to picoseconds. This will allow for the precise study of the structure and dynamics of biomolecules, providing a detailed understanding of the relationship between structure, dynamics, and function, which is not yet possible in glycoscience. The field of glycoscience has gained strong momentum in recent years. However, biophysical and molecular detail approaches are lacking. This thesis aims to close this scientific gap by establishing 2D-IR spectroscopy of vibrationally labeled carbohydrates for study. Although this technique has been used to study proteins, its application to carbohydrates remains unexplored. The first step in achieving this goal was to synthesize several thiocyanate (SCN) carbohydrates (using a new synthesis scheme) and characterize them using IR methods. SCN is a VP with special properties that make it suitable for biomolecular investigation. It has an outstandingly long vibrational lifetime and is referred to as a Stark label due to its sensitivity to local electric fields, with possible applications in aqueous environments. 2D-IR spectroscopy revealed unexpected structural dynamics that were not observed in MeSCN. The solvatochromism of thiocyanate arabinose (SCN-ara) showed intrinsic structural dynamics of subpopulations related to the SCN vibration. Analysis of the 2D-IR data confirms the presence of two subpopulations within the ensemble caused by the intrinsic structural dynamics of the label and the carbohydrate. Several samples with Azide (N3) as the VP were tested to expand the portfolio of carbohydrates for 2D-IR studies. Unlike SCN, this VP has a much stronger extinction coefficient, which improves the signal-to-noise ratio and allows for experiments at lower concentrations. However, it has a short lifetime and a more complex lineshape. Due to advances in click chemistry, a wide range of small carbohydrates with azides are now readily available. The high extinction coefficient of N3 (up to 921 L mol−1 cm−1) makes it a favorable VP for a proof-of-concept study on protein-carbohydrate interactions. The first proof-of-concept study on protein-carbohydrate interactions targeted the hexokinase (Hk) enzyme in complex with two N3-glucoses (labeled on C6 and C2 position). Monitoring of the active site of Hk showed increased inhomogeneity and an increased amount of structural dynamics inside the protein binding pocket. The two glucose forms probed a similar environment, despite the VP being on opposite sides of the molecule. Collaboration partners further investigated the binding properties using MD simulations and ITC. It was found that 6-N3-Glc binds reversely, leading to increased inhomogeneity through a looser binding. This also explains the comparable absorption properties, as both labels probe close spots in the active site of Hk. The final research chapter of this thesis builds upon the initial enzymatic study, by monitoring the kinetics of the first reaction of the metabolic pathway of glucose (glucose + ATP glucose-6-phosphate + ADP) using 2D-IR spectroscopy. The data analysis focused on the phosphate signals of ATP-ADP rather than the phosphorylated glucose. This work presents the first 2D-IR spectra of enzymatic activity over the course of hours using a pulse shaper setup in the phosphate region. Additionally, it serves as a study on expanding the use of currently employed VPs by application of phosphates. The studies show that 2D-IR spectroscopy of VP is an effective tool for investigating the structure and dynamics of biomolecules, expanding its application to carbohydrates and yielding advances in glycobiology and glycoscience using biophysical methods.