Stereochemistry of enzymatic transformations of (+)ß- and (-)ß-HBCD with LinA2--a HCH-degrading bacterial enzyme of Sphingobium indicum B90A.

LinA2, a bacterial enzyme expressed in various Sphingomonadaceae, catalyzes the elimination of HCl from hexachlorocyclohexanes (HCHs) and, as discussed here, the release of HBr from certain hexabromocyclododecanes (HBCDs). Both classes of compounds are persistent organic pollutants now regulated under the Stockholm Convention. LinA2 selectively catalyzes the transformation of ß-HBCDs; other stereoisomers like α-, γ-, and δ-HBCDs are not converted. The transformation of (-)ß-HBCD is considerably faster than that of its enantiomer. Here, we present the XRD crystal structure of 1E,5S,6S,9R,10S-pentabromocyclododecene (PBCDE) and demonstrate that its enantiomer with the 1E,5R,6R,9S,10R-configuration is the only metabolite formed during LinA2-catalyzed dehydrobromination of (-)ß-HBCD. Formation of this product can be rationalized by HBr elimination at C5 and C6. A reasonable enzyme-substrate complex with the catalytic dyad His-73 and Asp-25 approaching the hydrogen at C6 and a cationic pocket of Lys-20, Try-42 and Arg-129 binding the leaving bromine at C5 was found from in silico docking experiments. A second PBCDE of yet unknown configuration was obtained from (+)ß-HBCD. We predicted its stereochemistry to be 1E,5S,6S,9S,10R-PBCDE from docking experiments. The enzyme-substrate complex obtained from LinA2 and an activated conformation of (+)ß-HBCD allows the HBr elimination at C9 and C10 leading to the predicted product. Both modeled enzyme-substrate complexes are in line with 1,2-diaxial HBr eliminations. In conclusion, LinA2, a bacterial enzyme of the HCH-degrading strain Sphingobium indicum B90A was able to stereoselectively convert ß-HBCDs. Configurations of both PBCDE metabolites were predicted by molecular docking experiments and confirmed in one case by XRD data.

Isobutoxypentabromocyclododecanes (iBPBCDs): a new class of polybrominated compounds.

Isobutoxypentabromocyclododecanes (iBPBCDs) represent a new class of polybrominated compounds found in several flame-proofed polystyrene materials and in a technical mixture of hexabromocyclododecanes (HBCDs). Of the 64 stereoisomers possible, we now have analytical evidence for 16 different stereoisomers. By reversed- and chiral-phase liquid chromatography we distinguished 8 diastereomeric pairs of enantiomers, named alpha-, beta-, gamma-, delta-, epsilon-, xi-, eta-, and theta-iBPBCDs in accordance with their chromatographic retention on a C(18)-RP-column. Crystal structure analysis revealed the stereochemistry of the most prominent theta-iBPBCD stereoisomers, which were determined to be (1R)-1-iso-butoxy-(2R,5R,6S,9S,10R)-2,5,6,9,10-pentabromocyclododecane and its enantiomer. The eight iBPBCD diastereomers were also found in several expanded (EPS) and extruded polystyrene materials (XPS). Stereoisomer pattern varied to a large extent with delta-, eta-, and theta-iBPBCDs dominating in EPS- and alpha-, beta-, epsilon-, and xi-isomers in XPS-materials. The substitution of a bromine atom with an alkoxy group is expected to result in more lipophilic compounds than the parent HBCD compounds. The chromatographic retention on the reversed-phase column supports this assumption. Therefore, we expect that certain iBPBCD stereoisomers may also have the potential to accumulate in biota like e.g. alpha-HBCDs. The presented spectroscopic and chromatographic data allow the identification of 16 different iBPBCD stereoisomers in plastic materials, environmental samples, and biota. With this, the occurrence, fate, and toxicological relevance of this new class of polybrominated compounds can now be studied in more detail.