Abstract
The water ligand observed via a gradient spectroscopy type experiment with 19F detection was applied to selectively detect fluorinated compounds with affinity to the target proteins. The 19F signals of bound and unbound compounds were observed as opposite phases, which was advantageous to distinguish the binding state. The proposed NMR method was optimized based on the 19F{1H} saturation transfer difference pulse sequence, and various inversion pulses for the water resonance were evaluated with the aim of high sensitivity.
Highlights
NMR spectroscopy can be an effective method for screening compounds with affinity to target proteins
Various NMR-based screening methods to observe 1H ligand signals were proposed, such as NOE-pumping [1], saturation transfer difference (STD) [2], water ligand observed via gradient spectroscopy (WaterLOGSY) [3,4], and reverse NOE-pumping [5] experiments
In the 1H→19F STD [9] and 19F{1H} STD experiments [10], on-resonance 1H frequency was set on the protein and off-resonance 1H frequency was set outside the signal region for reference (e.g., −20 ppm)
Summary
NMR spectroscopy can be an effective method for screening compounds with affinity to target proteins. Various NMR-based screening methods to observe 1H ligand signals were proposed, such as NOE-pumping [1], saturation transfer difference (STD) [2], water ligand observed via gradient spectroscopy (WaterLOGSY) [3,4], and reverse NOE-pumping [5] experiments. The 19F signals of the fluorinated compounds bound to proteins were selectively observed in the difference spectra. For the purpose of developing more sensitive NMR-based screening methods for fluorinated compounds, a WaterLOGSY type experiment with 19F detection was optimized using the complex of diflunisal (Figure 1) and human serum albumin (HSA). Diflunisal possesses anti-inflammatory, analgesic, and antipyretic activity and is used in the therapy of chronic arthritis This model system is considered to be suitable for developing the proposed methods
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