Lecture from Aubert de Lichy
Structure resolution of glucose units in a heparan sulfate mimetic (RGTA®) fragment by NMR and molecular dynamics simulations
Abstract:
ReGeneraTing Agents (RGTA®) are synthetic polysaccharides designed to mimic the structural and functional properties of heparan sulfates (HS), essential components of the extracellular matrix that are degraded during tissue injuries. Among them, OTR4120, marketed as the medical device CACIPLIQ20®, is synthesized from a dextran backbone (α(1–6) linked glucose units) and modified through carboxymethylation and sulfation. Due to the statistical nature of these chemical modifications under Good Manufacturing Practices (GMP), slight variations in glucose unit composition between production batches are expected, leading to a heterogeneous distribution of chain lengths and positions of substituents.
To characterize the glucose unit composition and confirm its structural features, the heterogeneity of 10 batches of OTR4120 was analyzed using 1H NMR at 600 MHz by optimizing pressure and temperature conditions to enhance signal resolution. Higher-field NMR experiments (1H-950 MHz, 13C-237 MHz) were combined with Lorentzian deconvolution to quantify the different glucose units, while 13C-HSQC and HMBC at 950 MHz enabled the assignment of signals corresponding to carboxymethyl and sulfate substitutions. In parallel, a molecular dynamics approach was used to study the interaction of OTR4120 representative fragments with FGF-2. Finally, a quantum simulation study was conducted to understand the differences in reactivity of the various hydroxyl positions of glucose.
OTR4120 exhibits molecular weight polydispersity, originating from a 40 kDa dextran precursor corresponding to an average of 247 glucose units. Post-substitution, the polymer presents 27 statistically possible glucose units based on their substituent combinations, rendering full-length molecular dynamics simulations impractical. NMR analyses revealed minimal differences across the 10 GMP batches, confirming molecular homogeneity. Among the 27 statistically glucose unit combinations, only 13 glucose units with various substituent combinations have been observed by NMR. Additionally, no glucose units remained unsubstituted at the C2 position. This quantification enabled the selection of representative tetra-saccharides for molecular dynamics simulations. The dialogue between theory and experiment has provided valuable insights into the structure–property relationships of these sugar assemblies, known for their particularly complex nature.
Post from Hong-Nhung Vu
Regioselective Carboxymethylation of Dextran: Mechanistic Insights for heparan sulfate mimetic (RGTA®) Synthesis
Abstract:
ReGeneraTing Agents (RGTA®) are synthetic polysaccharides designed to mimic the structural and functional properties of heparan sulfates (HS), an essential component of the extracellular matrix which is degraded during tissue injury. Among them, OTR4120—marketed as the medical device CACIPLIQ20®—is synthesized from a dextran backbone (α(1–6)-linked glucose units) chemically modified by carboxymethylation followed by sulfation. These chemical modifications introduce variations in the substitution patterns across glucose units, leading to a heterogeneous distribution of functional groups along the polymer chain.
The initial carboxymethylation step, a bimolecular nucleophilic substitution (SN2) reaction targeting hydroxyl groups on glucosidic units, was identified as the key stage influencing the structure of the precursor influencing the subsequent sulfation, which is crucial for the final product’s biological activity. Interestingly, significant regioselectivity was observed during this carboxymethylation, as characterized by 13C HMR spectroscopy (100 MHz) of the carboxymethyl dextran (CMD) precursor. To elucidate its origin, we conducted mechanistic studies using quantum mechanical (QM) simulations.
QM simulations have allowed to estimate the activation energies for the carboxymethylation of the different hydroxyl groups within the aqueous solvent, revealing a clear preference for certain reaction pathways. Furthermore, to fully understand the selectivity within the polymeric context, molecular dynamics simulations were performed on various polysaccharide fragments. These allowed us to evaluate the accessibility of the different reactive sites depending on the substitution state of the polymer. Altogether, these results provide new insights into the complex reaction mechanism and open perspectives for the rational design of chemical modifications on dextran and its derivatives.
