Structural Elucidation of Relevant Gibberellic Acid Impurities and <i>In Silico</i> Investigation of Their Interaction with Soluble Gibberellin Receptor GID1

Gibberellin derivatives are a family of tetracyclic diterpenoid plant hormones used in agriculture as plant growth regulators included in the European Directive 91/414. In the pesticide peer review process and to assess their toxicological relevance and product chemical equivalence, the European Food Safety Authority (EFSA) highlighted data gaps such as the identification of hydrolysis products and unknown impurities. The aspect of impurity characterization and quantitation is challenging and requires the use of hyphenated analytical techniques. In this regard, we used an LC-QTOF/MS and NMR analysis for the characterization of gibberellic acid impurities found in technical products. Gibberellic acid impurities such as gibberellin A1 (GA1), 3-isolactone gibberellic acid (iso-GA3), gibberellenic acid, 1α,2α-epoxygibberellin A3 (2-epoxy- GA3), and (1α,2β,3α,4bβ,10β)-2,3,7-trihydroxy-1-methyl-8-methylenegibb-4-ene-1,10-dicarboxylic acid were identified and successfully characterized. Moreover, an in silico investigation on selected gibberellic acid impurities and derivatives and their interactions with a gibberellin insensitive dwarf1 (GID1) receptor has been carried out by means of induced fit docking (IFD), generalized-Born surface area (MM-GBSA), and metadynamics (MTD) experiments. A direct HPLC method with DAD and MS for the detection of gibberellic acid and its impurities in a technical sample has been developed. Moreover, by means of the in silico characterization of the GID1 receptor-binding pocket, we investigated the receptor affinity of the selected gibberellins, identifying compounds (2) and (4) as the most promising hit to lead compounds