Glycosaminoglycans (GAGs) are found in every mammalian tissue and play a key role in many biological functions. Unfortunately, their highly complex, heterogeneous and highly sulfated structure, which is the basis for its multifarious activities, introduces major challenges in structural characterization. We have introduced synthetic, non-saccharide GAG mimetics (NSGMs) as probes of biological functions of GAGs and as putative therapeutics. In contrast to natural GAGs, NSGMs are fully homogeneous and synthetically produced smaller molecules that have now been found to modulate cancer stem cells, thrombopoiesis, inflammation and arterial/venous thrombosis. NSGMs are easier to synthesize than natural GAGs, however, characterizing their level of sulfation (-OSO₃Na) is challenging. A robust high-resolution mass spectrometric method for characterization of NSGMs is desirable. Here, we introduce a novel chemical derivatization-based liquid chromatography-mass spectrometry (LC-MS) approach for detection and analysis of the persulfated NSGMs that should aid chemistry, manufacturing & controls (CMC) and pharmacokinetic/pharmacodynamic (PK/PD) studies.
Our approach relies on chemical derivatization of NSGMs using 2,2,2-trifluorodiazoethane (TFD) to achieve complete sulfo-esterification of the available sulfate groups. TFD esterification significantly increases their hydrophobicity and volatility, enhances their LC-based separation, thereby enabling MS detectability. More importantly, this modification effectively stabilizes sulfate group to various chemical and thermal steps necessary for accurate HR-MS characterization. We optimized the reaction conditions by exploring multiple factors such as mode and order of reagent additions, reaction temperature, type of proton donors, and solid/liquid state preference of the substrate. We also defined the minimal substrate threshold for the detection, especially in biological milieu. Overall, the TFD-based chemical derivatization followed by LC-MS and/or HR-MS detection offers a powerful tool for the in-depth characterization of NSGMs, which is expected to help development of these unusual molecules as drugs.

Daniel Afosah

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