Complexity of naturally produced polybrominated diphenyl ethers revealed via mass spectrometry

Abstract

Polybrominated diphenyl ethers (PBDEs) are persistent and bioaccumulative anthropogenic and natural chemicals that are broadly distributed in the marine environment. PBDEs are potentially toxic due to inhibition of various mammalian signaling pathways and enzymatic reactions. PBDE isoforms vary in toxicity in accordance with structural differences, primarily in the number and pattern of hydroxyl moieties afforded upon a conserved core structure. Over four decades of isolation and discovery-based efforts have established an impressive repertoire of natural PBDEs. Based on our recent reports describing the bacterial biosyntheses of PBDEs, we predicted the presence of additional classes of PBDEs to those previously identified from marine sources. Using mass spectrometry and NMR spectroscopy, we now establish the existence of new structural classes of PBDEs in marine sponges. Our findings expand the chemical space explored by naturally produced PBDEs, which may inform future environmental toxicology studies. Furthermore, we provide evidence for iodinated PBDEs and direct attention toward the contribution of promiscuous halogenating enzymes in further expanding the diversity of these polyhalogenated marine natural products.

Figure 1

Diversity of aryl biradical coupling outcomes. (a) Basic structure of PBDEs with nomenclature that is used in this report. Intermediates following radical initiation and rearrangement for (b) bromophenols and (c) bromocatechols. Bromine atoms are omitted in the intermediate structures for clarity. Possible regioisomeric outcomes for (d,e) bromophenol homocoupling, (f–h) bromophenol-bromocatechol heterocoupling, and (i,j) bromocatechol homocoupling. Note that methylation of phenoxyls further increases the structural diversity of marine PBDEs, but has been omitted here for clarity.

Figure 2

Dysidea sp. as sources of PBDEs. UV-absorption profile for HPLC separation of extracts from (a) D. granulosa and (b) L. herbacea monitored at 214 nm. Insets show the morphology of the sponges used in this study. Also shown are chemical structures of the PBDEs 1–3, bromophenol 4, bromocatechol 5 and two possible structures (6–7) for the methoxylated bromocatechol detected in Dysidea sp. sponges.

Figure 3

MS/MS based differentiation between structural classes of PBDEs. (a) EIC for [M–H]¹⁻ 420.79 Da identifies two tribrominated OH-BDEs, of which the isomer denoted by * (b) does not display distinctive MS2 product ions, while the isomer denoted by ● (c) demonstrates a dibromohydroquinone MS2 product ion. These disparate MS/MS signatures led to the identification of structural differences between the two isomeric OH-BDEs. (d) EIC for [M–H]¹⁻ 516.69 Da identified two tetrabrominated di-OH-BDEs, of which the isomer denoted by ■ demonstrated (e) a major MS2 product ion corresponding to loss of one bromine atom. The other isomer demonstrates two major MS2 ions, (f) corresponding to dibromophenol and dibromotrihydroxybenzene moieties, that can be rationalized based on the final deduced structure of 8 (vide infra).

Figure 4

Structure elucidation of 8. (a) HMBC correlations observed for 8 which lead to the assignment of Ring A, with (b) two possible structures of Ring B that were differentiated by comparison to authentic synthetic standards 9 and 10.

Figure 5

MS/MS profile of 11. EIC for [M–H]¹⁻ 546.70 Da identifies one major brominated molecule in the extract of L. herbacea. Only one major dibromotrihydroxybenzene MS2 product ion could be observed, analogous to that for 8 (Figure 3f), that can be rationalized on the basis of the deduced structure for 11 (vide infra).

Figure 6

Off-pathway halogenation of PBDEs as revealed by MS. (a) Proposed sequence of chlorination and bromination steps that lead to the formation of two isomers each for 12, 13, and 15, and single products 14 and 16. The proposed structures of the mixed polyhalogenated diphenyl ethers, with additional halogenations upon 3 (vide infra) localized to Ring A are shown. (b) EICs showing two closely eluting isomeric products corresponding to the molecular formulas for 12, 13, and 15, but only single products for 14 and 16. These observations corroborate the scheme shown in Panel A. All EICs are generated within 10 ppm tolerance. (c) MS/MS profile of 3 demonstrates a single major MS2 product ion corresponding to dihydroxydibromobenzene, which can be rationalized to be derived from Ring A as shown. MS2 product ions observed for 12–16 can similarly be rationalized to be derived from their respective Ring As, but with additional halogen adducts as shown. Note that methoxylation of di-OH-BDEs dramatically alters their MS/MS profiles as compared to Figure 3e. The other MS2 ions observed in every MS/MS spectra correspond to the loss of the methyl group, along with a bromine atom from the respective parent molecule. MS1 profiles for 3 and 12–16 are shown in SI Figure 40.

Figure 7

Postulated chemical structures for iodinated PBDEs 17–20. Based on MS, it cannot be determined if the iodine atom for 17 is positioned at either the 3′ or the 5′ position on Ring A. Also note that the 3′-I and 5′-Br atoms for 18, and 3′-Cl and 5′-I atoms for 20 can be interchanged to give identical MS1 and MS2 spectra.