Enantioselective Preparative HPLC Separation of the HBCD— Stereoisomers from the Technical Product and Their Absolute Structure Elucidation Using X-Ray Crystallography ROBERT KOEPPEN, ROLAND BECKER,* FRANZISKA EMMERLING, CHRISTIAN JUNG, AND IRENE NEHLS Department of Analytical Chemistry, Reference Materials, Federal Institute for Materials Research and Testing (BAM), Berlin, Germany ABSTRACT 1,2,5,6,9,10-Hexabromocyclododecane (HBCD) is a widely used flame retardant, which tends to persist in the environment and accumulates in biota. The six stereoisomers (three racemates named a-, b-, and g-HBCD) of the technical mixture were isolated with high-performance liquid chromatography (HPLC). Direct separations were performed on a chiral stationary phase containing permethylated b-cyclodextrin (NUCLEODEX b-PM column) and the pure enantiomers of a-, b-, and g-HBCD were physically characterized for the first time. The absolute configurations of all six isomers were determined by anomalous dispersion using single crystal X-ray crystallography. Optical rotations aD in tetrahydrofuran were þ4.2/4.0 (a-HBCD), þ26.1/27.5 (b- HBCD), and þ68.0/66.3 (g-HBCD). The sense of rotation could be correlated with the absolute configurations of a-, b-, and g-HBCD enantiomers and their order of elution on a chiral permethylated b-cyclodextrin-bonded stationary phase. The diastereomers a-, b-, and g-HBCD displayed distinctly different melting points as well as 1 H-, 13 C NMR, and IR spectra. Chirality 19:214–222, 2007. V V C 2007 Wiley-Liss, Inc. KEY WORDS: brominated flame retardant; chiral separation; anomalous dispersion; crystal structures; optical rotation; absolute configuration The rapidly growing use of plastic materials and syn- thetic fibers, and the introduction of stricter fire regula- tions in the last decades have led to a drastically increased use of brominated flame retardants (BFRs). Because of the substitution of traditional BFRs such as polybromi- nated diphenyl ethers (PBDEs), the production and con- sumption of the alicyclic flame retardant 1,2,5,6,9,10-hex- abromocyclododecane (HBCD) is increasing. 1 HBCD is widely used in various plastic materials, upholstery tex- tiles, 2 adhesives, styrene-acrylonitrile resins (SAN), ex- truded (XEPS), and expanded (EPS) polystyrene foams 2,3 with a global market demand of about 22,000 metric tons in 2003. 4 Technical HBCD is derived from the addition of bromine to (1Z,5E,9E)-cyclododeca-1,5,9-triene 5 and consists of a mixture of three diastereomeric pairs of enantiomers, termed (6) a-, b-, and g-HBCD (Fig. 1) with the g-isomer as main component 6,7 (a: 1%–12%, b: 10%–13% g: 75%–89%, respectively). 8 It is well known that bromination of a car- bon-carbon double bond proceeds via the bimolecular elec- trophilic addition mechanism. The bromine addition is anti to the double bond plane of (1Z,5E,9E)-cyclododeca-1,5,9- triene (anti-stereochemistry). This leads in case of trans- addition to a Z-double bond to a racemic mixture (RR) and (SS), while an E-double bond gives a product with meso configuration (SR or RS). Accordingly, the three distinct diastereomeric pairs of enantiomers are formed. The structural differences between these diastereomeric pairs of enantiomers lead to concomitant variability in physicochemical properties such as hydrophobicity and water solubility, 9,10 resulting in variable propensities for bi- ological uptake and metabolism. The physicochemical properties of HBCD make it persistent in the environment and potentially bioactive. 2,11 In conjunction with their re- sistance to degradation the HBCD diastereomers are bio- accumulative in both terrestrial and aquatic organisms and were found in humans 10 and consequently challenge sci- entists and regulators. 12 In recent years, increasing levels of HBCD were detected in sewage sludges, 2,13 sediments, 2,14,15 and ma- rine biota. 2,3,13,14,16–19 The bioaccumulation 2,20–23 and bio- degradation 24,25 of HBCD are topics of current interest and there is evidence for the biotransformation of HBCD stereoisomers in fish. 26 This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/0899-0042/suppmat. Crystallographic data for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplemen- tary publication no. CCDC 604543–604548. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [Fax: þ44-1223/336-033; E-mail: deposit@ccdc.cam.ac.uk *Correspondence to: R. Becker, Department of Analytical Chemistry and Reference Materials, Federal Institute for Materials Research and Testing (BAM), Richard-Willstaetter-Strasse 11, D-12489 Berlin, Germany. E-mail: roland.becker@bam.de Received for publication 14 September 2006 ; Accepted 14 November 2006 DOI: 10.1002/chir.20366 Published online 16 January 2007 in Wiley InterScience (www.interscience.wiley.com). CHIRALITY 19:214–222 (2007) V V C 2007 Wiley-Liss, Inc.