Polyhalogenated Alkaloids in Environmental and Food Samples

CHAPTER

3 Polyhalogenated Alkaloids in Environmental and Food Samples Walter  Vetter*

Contents

I. Introduction II. Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles and Related Compounds A. Discovery and Variety of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles B. Synthesis of Polyhalogenated 1,1′-Dimethyl-2,2′Bipyrroles and Related Compounds C. Natural Product Verification of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles D. Environmental and Food Concentrations of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles III. Polyhalogenated 1′-Methyl-1,2′-Bipyrroles A. Discovery and Variety of Polyhalogenated 1′-Methyl-1,2′-Bipyrroles B. Synthesis of Polyhalogenated 1′-Methyl-1,2′Bipyrroles and Related Compounds C. Natural Source Verification of Polyhalogenated 1′-Methyl-1,2′-Bipyrroles D. Distribution and Concentrations of Polyhalogenated 1′-Methyl-1,2′-Bipyrroles in the Environment IV. Tetrabromo-1-Methylpyrrole and Related Polybrominated Pyrroles A. Discovery of Tetrabromo-1-Methylpyrrole and Related Compounds

212 214 214 219 221 224 229 229 233 234 235 240 240

Institute of Food Chemistry, University of Hohenheim, Stuttgart, Germany. *Corresponding author. Email: [email protected] The Alkaloids, Volume 71 ISSN 1099-4831, http://dx.doi.org/10.1016/B978-0-12-398282-7.00003-5

© 2012 Elsevier Inc. All rights reserved.

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B. Synthesis and Properties of Tetrabromo- 1-Methylpyrrole C. Natural Source Verification of Tetrabromo- 1-Methylpyrrole D. Concentrations of Tetrabromo-1-Methylpyrrole in the Marine Environment V. Brominated Indoles and 1-Methylindoles A. Discovery and Variety of Brominated Indoles and 1-Methylindoles B. Natural Product Verification of Brominated Indoles and 1-Methylindoles C. Concentrations of Brominated Indoles and 1-Methylindoles in the Marine Environment VI. Analysis of Polyhalogenated Alkaloids A. Analytical Identification and Quantification of Polyhalogenated Alkaloids B. Identification of Polyhalogenated Alkaloids C. Suitable Novel Nontarget Screening Methods for the Detection of Polyhalogenated Alkaloids D. Enantioselective Determination of Chiral Polyhalogenated Alkaloids VII. Physicochemical Properties of Polyhalogenated Alkaloids VIII. Bioactivity of Polyhalogenated Alkaloids IX. Final Remarks and Conclusions References

241 241 244 245 245 248 249 250 250 259 260 262 266 268 271 272

I. INTRODUCTION Man-made polyhalogenated organohalogen compounds are in the focus of environmental and food chemists since the 1950s when residues of dichlorodiphenyltrichloroethane (better known as DDT) were detected in birds and other animals.1 Immediately, it became apparent that DDT and related compounds were not only present in the fatty tissues of the top predators of the marine food chain but that they were also linked with a bioaccumulation effect: The concentrations in the lipids of marine mammals and birds were orders of magnitude higher than in their prey. Compounds that bioaccumulate in the lipid compartments must have a certain lipophilicity that is usually expressed by the logarithmic octanol–water (log KOW or log P) partitioning coefficient. The higher the (experimentally determined) log KOW, the more the compound can be enriched in higher organisms compared to water. Typically, a log KOW > 5 gives rise to bioaccumulation.2 It followed the observation that the occurrence of high concentrations of polyhalogenated compounds in the body of animals can be associated with

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toxic effects. For example, the egg shell thinning of seabirds from the Baltic Sea observed in the 1970s could be traced back to the effect of o,p′-DDT (more specifically, the (−)-enantiomer3). The dimension of the pollution problem was realized when Jensen discovered residues of polychlorinated biphenyls (PCBs) in fish.4 The discovery of DDT in the Antarctic brought afore another adverse feature of the compounds such as DDT and PCBs, that is, their persistence and ability to long-range transportation to remote areas in which they were never used. Consequently, man-made polyhalogenated compounds are regarded as ubiquitous contaminants, and several members have been classified as persistent organic pollutants (POPs) and persistent bioaccumulative and toxic chemicals (PBTs). PBTs include the anthropogenic chloropesticides (e.g., DDT and its metabolites, hexachlorocyclohexanes, chlordane, toxaphene, mirex, etc.), industrial chemicals (e.g., PCBs and different polybrominated flame retardants). These compounds were produced on a million-ton scale. Frequently, the compounds do not represent a single chemical but complex mixtures obtained from the chlorination or bromination of a given backbone to the desired degree of halogenation. The array of structures also includes the unintended combustion products polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). The structures of the anthropogenic compounds are diverse. They were synthesized by halogenation of more or less all backbones available in sufficient amounts. However, none of the environmentally relevant man-made organohalogen compounds contains nitrogen. In 1999, two articles reported on the detection and structure investigation of two abundant, previously unknown, organohalogen compounds in top predators of the marine food chain, which were shown to be polyhalogenated alkaloids.5, 6 The unique elemental compositions immediately gave rise to the question if these could be bioaccumulated halogenated natural products (HNPs), that is, compounds with similar environmental behavior as the anthropogenic POPs.5 Halogenated natural products have been discovered in a huge and steadily increasing variety, predominantly in marine organisms.7–10 In this armada of halogenated natural products, the polyhalogenated alkaloids represent a comparably small but relevant share of compounds. Obviously, the (usually) aromatic system can be excessively halogenated. With the increase of the number of halogens on an organic backbone, the persistence (stability) and lipophilicity (log KOW) is increasing. Consequently, these compounds could share the environmental properties of the anthropogenic POPs. Polyhalogenated alkaloids were long known from ground-breaking articles of the natural product chemists (see Chapter 1 in this issue10); their links to environmental and food issues, however, is relatively new. In environmental sciences, environmental issues of halogenated natural products can be defined by the detection of these compounds in higher organisms, which are not the natural sources but have accumulated

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the natural products in a similar way as anthropogenic POPs.11 The top predators of the marine food chain have received them via food chain accumulation or uptake from the water phase. By contrast, their natural producers, for example, algae, sponges and bacteria, are not in the focus of environmental and food sciences as long as these are neither toxic to the habitat nor consumed as food. Consequently, the halogenated natural products discussed in this chapter need to be persistent and bioaccumulative. Both parameters require the presence of several halogen substituents. For this reason, the major compounds and compound classes are presented as polyhalogenated alkaloids. Although toxic effects of polyhalogenated alkaloids are only insufficiently investigated, their similarity with anthropogenic POPs at least indicates that such possibility needs to be thoroughly investigated. Articles dealing with environmental aspects of polyhalogenated alkaloids came up in the late 1990s.5,6,12 Since significant amounts of anthropogenic polyhalogenated alkaloids have not been produced, the identification of nitrogen in a compound detected in environmental samples was half the proof that the compounds and compound classes discussed in sections II–V. are naturally produced.

II. POLYHALOGENATED 1,1′-DIMETHYL-2,2′-BIPYRROLES AND RELATED COMPOUNDS A. Discovery and Variety of Polyhalogenated 1,1′-Dimethyl2,2′-Bipyrroles Research on polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles was initiated by the detection of a relatively abundant peak in the gas chromatography with mass spectrometry (GC/MS) chromatogram of purified bird egg extracts from Canada.13 Elliot et al. found this “brominated compound” in sample pools of Lerch’s storm petrel eggs from the Pacific Storm Islands collected in 1987.13 In this sample, the “brominated compound” was the most abundant peak in the full scan chromatogram, and its concentration was estimated at 80 µg/kg wet weight. Initially, the compound was assumed to have five bromine substituents and was thought to be related to polybrominated biphenyls.13 At that time, mixed brominated– chlorinated compounds had been scarcely identified in environmental samples (and if, of circumstantial relevance). Nitrogen-containing anthropogenic compounds were simply not on the map and the occurrence of abundant natural products in top predators of the marine food chain simply beyond any horizon. In any case, the abundant signal in the GC/ MS chromatogram from the bird samples justified the attempts to determine its structure. Subsequently, this compound was isolated from bald

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eagle (Haliaeetus leucocephalus) liver (12g) and studied by different GC/ MS techniques.5 More detailed GC/MS investigations and especially the use of high-resolution mass spectrometry (HRMS) clarified that the novel compound carried four bromine and two chlorine substituents, a pattern rather easily to mix with pentabrominated compounds (Figure 1). The minimal differences in the isotope peak abundances (most visible in form of the slightly lower abundant [M+6]+ peak at m/z 546 in the “Br4Cl2” variant, Figure 1(a)), are within the variability of GC/MS measurements in the low ng or pg range. HRMS analysis at a resolution of 10,000 indicated an exact mass of 539.664063Da for the monoisotopic peak, which matched with the molecular formula C10H6Br4Cl2N2.5 (By contrast, the exact mass of the Br5 variant shown in Figure 1(b) is heavier by ~30mDa due to the higher number of hydrogens (cf. section VI.A.2).) C10H6Br4Cl2N2 was the only molecular formula with both the mass and the presence of four

(a)

544

542

540

(b)

546

548

550

m/z 540 541 542 543 544 545 546 547 548 549 550 551 552

[%] 10.5 1.2 51.2 6.2 100 12.1 97.8 11.8 48 5.7 9.5 1.1 0.1

m/z 540 541 542 543 544 545 546 547 548 549 550 551 552 553

[%] 12 1.4 54.4 6.3 100 11.6 94 10.9 47.2 5.4 11.9 1.3 1.2 0.1

Figure 1  Illustration of the similarity of halogen isotope patterns. (a) The tetrabromo– dichloro pattern of C10H6Br4Cl2N2 with peak relative intensities (%) and (b) the pentabromo isotope pattern of the potential C11H13Br5 compound with relative peak intensities (%).

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Br Br

Br Br

N

Br

N

Cl

N

1

Cl

N

Br

Cl

N

Cl

CH3

5

CH3

Br Br

Br

Br

N

CH3

Cl

4

Br Br

N

Cl

N

CH3

CH3

3 Br

CH3

Cl Cl

Cl

Br

N

CH3

Cl

2

Br Br

Br

Br

N

CH3

CH3

CH3

Br Br

Cl

Br

Cl

Br

N H

Br

N H

Br

6

Figure 2  Structures of the known polybrominated 1,1′-dimethyl-2,2′-bipyrroles with 1,1′-dimethyl-3,3′,4,4′,5,5′-hexabromo-2,2´-bipyrrole, Br6-DBP (1); 5′-chloro-1,1′-dimethyl-3,3′,4,4′, 5-pentabromo-2,2′-bipyrrole, Br5Cl–DBP (2); 5,5′-dichloro-1,1′-dimethyl-3,3′,4,4′-tetrabromo2,2′-bipyrrole, Br4Cl2–DBP (3); 1,1′-dimethyl-3,3′,4-tribromo-4′,5,5′-trichloro-2,2′-bipyrrole, Br3Cl3–DBP (4); 1,1′-dimethyl-3,3′,4,4′,5,5′-hexachloro-2,2′-bipyrrole, Cl6–DBP (5) as well as hexabromo-2,2′-bipyrrole (6).

bromine and two chlorine substituents. The immediate synthesis (cf. section II.B) of 5,5′-dichloro-1,1′-dimethyl-3,3′,4,4′-tetrabromo-2,2′-bipyrrole (Br4Cl2–DBP) (3) by Gribble et  al. confirmed the identity of the isolate from bird eggs14 (Figure 2). Tittlemier et  al. simultaneously detected 1,1′-dimethyl-3,3′,4,4′,5,5′-hexabromo-2,2′-bipyrrole (Br6-DBP) (1), and further congeners in form of two Br3Cl3-1,1′-dimethyl-2,2′-bipyrrole isomers (with one being identified as 1,1′-dimethyl-3,3′,4-tribromo4′,5,5′-trichloro-2,2′-bipyrrole (4)) and 5′-chloro-1,1′-dimethyl-3,3′,4,4′, 5-pentabromo-2,2′-bipyrrole (Br5Cl-DBP) (2) (Figure 2).5 The structures of 2 and 4 were elucidated by means of isotope exchange mass spectrometry and verified by synthesis.15 It is noteworthy that polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles had not been described in marine organisms by natural product chemists. In theory, there are 36 hexahalogenated 1,1′-dimethyl-2,2′-bipyrroles based on the occurrence of Br and/or Cl on the 1,1′-dimethyl-2, 2′-bipyrrole backbone (Table 1). For more than a decade, the five

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Table 1  Structural variety of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles, first reports and currently known congeners

Hexahalogenated 1,1′-dimethyl-2,2′bipyrroles (DBPs) Br6 –DBP Br5Cl–DBPs Br4Cl2–DBPs Br3Cl3 –DBPs Br2Cl4 –DBPs BrCl5 –DBPs Cl6 –DBP SUM

Theoretical number of isomers 1 3 9 10 9 3 1 36

1st report

Total number of Exact isomers reported structure to date known

15,14 15,15 15,14 25,15,42 416 316 116 5

1 216 316 416 4 3 1 18

1 1 1 2 1 6

Pentahalogenated 1,1′-dimethyl-2,2′-bipyrroles (DBPs) 3 Br5 –DBPs 18 219 2 Br4Cl–DBPs 27 118 2 Br3Cl2–DBPs 27 Br2Cl3 –DBPs 18 BrCl4 –DBPs 3 Cl5 –DBPs SUM 96 3 4 Tetrahalogenated 1,1′-dimethyl-2,2′-bipyrroles (DBPs) 9 Br4 –DBPs 27 Br3Cl–DBPs 54 319 3 Br2Cl2–DBPs 27 BrCl3 –DBPs 9 Cl4 –DBPs SUM 126 3 3

­ olyhalogenated 1,1′-dimethyl-2,2′-bipyrroles mentioned in the piop neering article by Tittlemier et al. remained the only structurally known hexahalogenated 1,1′-dimethyl-2,2′-bipyrroles. However, very recently Hauler et  al. detected at least 15 novel hexahalogenated 1,1′-dimethyl2,2′-bipyrroles in a dolphin (Sousa chinensis) sample from Queensland (Northeastern Australia) (Figure 3).16 In contrast to other marine mammal species from Queensland, this sample contained polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles with all combinations of Br and Cl including the (nonbrominated) 1,1′-dimethyl-3,3′,4,4′,5,5′-hexachloro-2,2′-bipyrrole (Cl6-DBP) (5) (Figure 3).16 Thus, approximately 18 of the 36 hexahalogenated 1,1′-dimethyl-2,2′-bipyrroles have been detected to date (Table 1). The identity of the Cl6–DBP (5) was verified by means of an authentic reference standard synthesized by Martin et al.17

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[Int.]

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21.9 22.0 22.1 22.2 22.3 22.4 22.5

min

Br4Cl2-DBPs

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* *

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Br5Cl-DBPs

*(*) *

23.2

(e)

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4000 2000

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23.3 min

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Br3Cl3-DBPs

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*

23.8

23.9 min

24.4

24.5

24.6

min

(g)

20000 10000 2000

25.1

25.2

25.3

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Figure 3  GC/ECNI–MS ion traces of the identification of novel polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole congeners in the blubber of a marine mammal sample from the Great Barrier Reef (Australia). (a) 1,1′-dimethyl-3,3′,4,4′,5,5′-hexachloro-2,2′bipyrrole (Cl6–DBP (5)); (b) three bromo-pentachloro-1,1′-dimethyl-2,2′-bipyrroles (BrCl5–DBPs); (c) four dibromo-tetrachloro-1,1′-dimethyl-2,2′-bipyrroles (Br2Cl4–DBPs); (d) four or possibly six tribromo-trichloro-1,1′-dimethyl-2,2′-bipyrroles (Br3Cl3–DBPs); (e) three dichloro-tetrabromo-1,1′-dimethyl-2,2′-bipyrroles (Br4Cl2–DBPs); (f) two chloropentabromo-1,1′-dimethyl-2,2′-bipyrroles (Br5Cl–DBPs); and (g) 1,1′-dimethyl-3,3′,4,4′,5,5′hexabromo-2,2′-bipyrrole (Br6–DBP (1)). Data from Hauler et al.16

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Tittlemier et  al. reported that the apparent lack of unsubstituted positions on the 1,1′-dimethyl-2,2′-bipyrrole backbone gives rise to little metabolism.15 In fact, penta- and tetrahalogenated 1,1′-dimethyl-2,2′bipyrroles were only scarcely mentioned in the literature. Haraguchi et al. first described one Br3Cl2-1,1′-dimethyl-2,2′-bipyrrole and one Br2Cl2-1,1′dimethyl-2,2′-bipyrrole isomer in false killer whale (Pseudorca crassidens) from coastal waters in the south of Japan.18 Hoh et al. not only verified the presence of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles with unsubstituted ring carbons but expanded the variety to three Br2Cl2-1,1′-dimethyl2,2′-bipyrrole isomers, two Br3Cl2-1,1′-dimethyl-2,2′-bipyrrole isomers and one Br4Cl-1,1′-dimethyl-2,2′-bipyrrole isomer in commercial salmon oil sold as dietary supplements.19 While it remained unknown if the same or different tetra- and pentahalogenated 1,1′-dimethyl-2,2′-bipyrroles were identified in both studies, it appeared that the tetra- and pentahalogenated 1,1′-dimethyl-2,2′-bipyrroles were metabolites of the hexahalogenated ones. The salmon oil analyzed by Hoh et al. was refined for use as dietary supplements, and this treatment might be the origin for their formation.19 Altogether, approximately 26 polyhalogenated 1,1′-dimethyl2,2′-bipyrroles have been detected so far in marine environmental samples (Table 1).

B. Synthesis of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles and Related Compounds Gribble et  al. (1999)14 based their immediate synthesis after the discovery of (in those days potential) polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles on the preparation of the known 1,1′-dimethyl-2,2′-bipyrrole backbone (7) from N-methylpyrrole (8) (Figure 4). Oxidative coupling of the 2-lithiated intermediate 2-pyrrolyllithium (obtained by treatment with n-butyllithium or ethyllithium) with CuCl2 or NiCl2 generated the desired backbone.20,21 Kauffmann and Lexy reported that the 1,1′-dimethyl-2,2′-bipyrrole was very unstable and its purification (by chromatography with short columns) has to be performed in a cooled room and in the absence of sunlight.21 Such purification was required due to the parallel formation of the corresponding N-methylated tripyrroles (9), tetrapyrroles (10) (Figure 4), and further oligomers with up to 16 N-methylpyrrolyl units.21 Gribble et al. obtained a yield of 55% for this backbone.14 They assumed that perbromination of the aromatic system will lead to Br6-DBP (1) while Br4Cl2-DBP (3) was thought to have chlorine substituents in 5- and 5′-positions due to the propensity of ortho positions for electrophilic substitution.14 Both assumptions proved to be correct and resulted in elucidation of the structures of the desired compounds. Br4Cl2–DBP (3) was also generated by the initial chlorination with NCS to give 5,5′-dichloro-1,1′-dimethyl-2,2′-bipyrrole, which in turn was converted with NBS into Br4Cl2–DBP (3). Finally, Gribble et al. also synthesized

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N CH3

N

N

CH3

CH3

7

8

N

N

N

CH3

CH3

CH3

9

N

N

CH3

CH3

N

N

CH3

CH3

10

N H

N

N H

Me

11

N H

N H

N H

12

N

13 Figure 4  Structures of 1,1′-dimethyl-2,2′-bipyrrole (7); 1-methylpyrrole (8); 1,1′,1″-trimethyl2,2′,2″-tripyrrole (9); 1,1′,1″,1‴-tetramethyl-2,2′,2″,2‴-tetrapyrrole (10); 2,2′-bipyrrole (11); 1-methyl-2,2′-bipyrrole (12); and a member of the prodigiosene pigments (13).

Cl6–DBP (5).14 The 1,1′-dimethyl-2,2′-bipyrrole (7) and the (nonmethylated) 2,2′-bipyrrole (11) backbones (Figure 4) were synthesized by Dohi et al.22 New synthetic routes for polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles were developed by Fu and Gribble and Martin et al.17, 23 These novel syntheses included the preparation of one of the pyrrole units from nonnitrogenated substrates. Fu and Gribble started their synthesis from pyrrole in which the α-position was metalated using methylmagnesium iodide and reacted with butyrolactone.23 Oxidation of the primary alcohol and Paal–Knorr condensation with different amines not only provided the 1,1′-dimethyl-2,2′-bipyrrole backbone but also 2,2′-bipyrrole (11) and 1-methyl-2,2′-bipyrrole (12).23 Polyhalogenation of these intermediates

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could provide interesting reference standards as well. Natural producers of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles have not been identified in marine organisms (see the next section). However, the related hexabromo-2,2′-bipyrrole (6) is a known secondary metabolite of marine bacteria.24 Thus, it could be that the (bio)methylation of hexabromo-2,2′bipyrrole (6) to give Br6–DBP (1) is occurring at a different stage and/or location and is probably also performed in a different (natural) process (see also section VI.D). Martin et al. used a silver(I)-catalyzed pyrrole cyclization for the generation of the polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole backbone.17 In this manner, they synthesized neat Br6-DBP (1), Br4Cl2–DBP (3), and Cl6– DBP (5).17 The availability of the Cl6–DBP (5) standard proved to be very useful for the verification of its presence in the dolphin sample (Figure 3).16 Gribble et al. also used both NCS and NBS to generate a random mixture of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles.14 They received different polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles including the five congeners initially detected in the bird samples. It can be supposed that the random halogenation produced more than the four hexahalogenated 1,1′-dimethyl-2,2′-bipyrroles reported in their article.14 The synthetic route of Martin et  al.17 can also be used to produce polyhalogenated 1-methyl-2,2′-bipyrroles. In the course of the reaction, one nitrogen was protected by tosylation. Finally, the tosyl protection group was eliminated by means of potassium tert-butoxide to give 1‐methyl‐2,2′‐bipyrrole (12) as an intermediate. Subsequent methylation of the pyrrole nitrogen atom using methyl iodide afforded the 1,1′-dimethyl-2,2′-bipyrroles (7) backbone. Other simple polyhalogenated bicyclic alkaloids of interest are pentabromopseudilin (14), pentachloropseudilin (15), their N-methylated analogs 16 and 17 (all Figure 5), along with mixed brominated/chlorinated representatives, which have all been synthesized by Martin et al.25 Especially the bioactive pentabromopseudilin (14) had been synthesized on different occasions.26–28 However, none of the compounds shown in Figure 5 have been detected so far in environmental samples.

C. Natural Product Verification of Polyhalogenated 1,1′-Dimethyl2,2′-Bipyrroles Although natural producers of polyhalogenated 1,1′-dimethyl-2,2′bipyrroles have not been identified yet, several convincing arguments were collected to give full support for their biogenic origins. The major Br4Cl2–DBP congener (3) contains both bromine and chlorine, a feature scarcely found for anthropogenic polyhalogenated compounds that are usually either brominated or chlorinated. This adds to the fact that the 1,1′-dimethyl-2,2′-bipyrrole backbone has not been produced industrially

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Br

Br Br

HO

Cl

Cl

Br

Cl

N H

HO

N H

Br

Cl

14 Br

Br Br

Cl

15 HO

Cl

Cl

Br

Cl

N

HO

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Me

Me Br

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16

17 Br

Br

Br Br

Cl

N H

HN

Cl

18 Figure 5  Structures of pentabromopseudilin (14); pentachloropseudilin (15); N-methylpentabromopseudilin (16); N-methyl-pentachloropseudilin (17); and 3,3′,4,4-tetrabromo5,5′-dichloro-2,2′-bipyrrole (18).

exceeding gram amounts. Moreover, the absence of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles in fish samples from the Great Lakes (whose borders are heavily populated by urban industries), which was in contrast to marine birds produced evidence that the source of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles can be found in the marine environment.5 The structural similarity of the polyhalogenated 1,1′-dimethyl-2,2′bipyrroles (1–5) with the structurally known natural product Br6–BP (6) was striking. Initially Br6–BP was detected in an autotoxic chromobacterium isolated from North Pacific seawater samples.24 Later, the bacterium was correctly ascribed to Pseudoalteromonas luteoviolaceus.29 Anderson et al. also indicated that Br6–BP (6) was likely produced by bromination of the (prebiosynthesized) 2,2′-bipyrrole (11) backbone.24 It was suggested that the compound was related to the bacterial prodigiosene (13) pigments (Figure 4).30 Cultures of the bacterium isolated from the seawater sample also contained tetrabromopyrrole and pentabromopseudilin (14).24 However, mixed brominated/chlorinated 1,1′-dimethyl-2,2′-bipyrroles or other pyrrole derivatives were not identified by Anderson et al.24 A more ambiguous analytical approach to verify the natural production was conducted by Reddy et  al.31 This research team isolated about 600 µg of Br4Cl2-DBP (3) from the blubber of marine mammals by means of gel permeation chromatography (GPC), column adsorption

Polyhalogenated Alkaloids in Environmental and Food Samples    

600

223

toxaphenes violacene 6 -hydroxyaplysistan

400

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(‰)

200 0 -200 -400 -600 -800

2-(3‘,5‘-dibromo2‘-methoxyphenoxy)3,5-dibromoanisole

2,3,4-tribromopyrrole

johnstonol 2,4-dibromophenol (S. bromophenolosus) industrial HOCs (PCBs, DDT, PBDEs, chlordane, mirex, and 2,4-dibromophenol)

Br4Cl2-DBP

-1000

Figure 6  Radiocarbon content (Δ14C in ‰) of halogenated natural products (including the polyhalogenated alkaloids Br4Cl2–DBP (3) and 2,3,4-tribromopyrrole (34)) and several anthropogenic POPs (labeled industrial HOCs and toxaphene). Data from Reddy et al.31 reprinted with permission of the American Chemical Society.

chromatography, and two-dimensional heart-cut gas chromatography.31 The isolated amount (which corresponded with ~150 µg carbon) was analyzed for its radiocarbon content (Δ14C value in ‰) by accelerator mass spectrometry.31 The half life of the 14C radioisotope is 5730 years. After approximately 8 cycles or approximately 46,000 years, the 14C content is only 0.4% of the current 14C content of organic materials. This small share is usually below the detection limit of the analytical method. Since anthropogenic polyhalogenated compounds, such as PCBs and hexachlorocyclohexane (HCH), are produced from carbon sources obtained from petrol oil that is free of 14C, Δ14C measurements can be used to identify “new” carbon in a polyhalogenated compound. Accordingly, the detection of 14C in a chemical provides the proof that it was not synthesized from materials of the petrol chemistry. The analysis of the isolated Br4Cl2–DBP (3) by means of accelerator MS confirmed the presence of 14C (Figure 6). However, the Δ14C value of −400‰ suggested an average age of approximately 5000 years for the ten Br4Cl2–DBP (3) carbons. Compared to Br4Cl2–DBP (3), six other marine natural products were analyzed including 2,3,4-tribromopyrrole (34) (isolated and purified from the acorn worm Saccoglossus kowalevski, which was harvested according to Giray et  al. from the top 20cm of salt marsh sediments in Massachusetts, in July 200231,32). This polyhalogenated alkaloid and other natural products were considerably richer in 14C (Δ14C value ~ −200‰) than Br4Cl2–DBP (3), and all natural products were different to man-made POPs (Figure 6).31 Reddy et al. suggested that the partial utilization of aged carbon in the biosynthesis or

224    

Walter Vetter

Br Br

Br

Br

Br Br

Br

Br

- 2 [H] Cl

N H

N H

Cl

+ 2 [CH3 ]

Cl

N

N CH3

CH3

18

Cl

3

Figure 7  Potential formation of 5,5′-dichloro-1,1′-dimethyl-3,3′,4,4′-tetrabromo-2,2′bipyrrole (3) by the methylation of 5,5′-dichloro-3,3′,4,4′-tetrabromo-2,2′-bipyrrole (18). CN

Br

F3C

N

Cl OEt

19 Figure 8  Structure of the man-made insecticide 4-bromo-2-(4-chlorophenyl)-1-ethoxymethyl-5-trifluoromethyl-pyrrole-3-carbonitrile (chlorfenapyr) (19), a polyhalogenated alkaloid with bromine, chlorine, and three fluorine substituents.

any isotope discrimination effects were the most relevant explanations for the detection of aged carbon in Br4Cl2–DBP (3).31 It was also proposed that Br4Cl2–2,2′-bipyrrole (18) could be the real natural product while the final methylation was conducted in a subsequent step to give Br4Cl2–DBP (3) (Figure 7).11 In any case, the detection of significant amounts of 14C in Br4Cl2–DBP (3) supported the natural origin hypothesis, despite the questions that remained unanswered. Other support of the natural source attribution of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles is the presence of nitrogen and the blend of chlorine and bromine substituents. Only a few man-made chemicals such as the insecticide and acarizide chlorfenapyr (19) share these features (Figure 8). This pesticide was introduced in 1994, that is, after the first report of (the later identified) polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles in environmental samples.

D. Environmental and Food Concentrations of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles Food chain enrichment is one of the decisive features adding environmental concerns to a chemical. For this reason, top predators of the marine food chain are frequently in the focus of environmental analyses. The identification of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles in marine birds indicated that they may also be found in human tissues. Indeed, pooled human milk from South Canada contained 0.013–4.48 ng/g lipids

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225

Br4Cl2–DBP (3).33 Likewise, 87% of human milk samples from two sites in Japan contained up to 2.7 ng/g lipids Br4Cl2–DBP (3),34 and Br4Cl2–DBP (3) was identified in human milk from the Faroe Islands.35 These results confirmed that polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles can be bioaccumulated in higher trophic biota.

1. Residues of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles in Marine Mammals A first comprehensive study of polyhalogenated 1,1′-dimethyl-2,2′bipyrrole residues in marine mammals from North America, Europe, and Asia was carried out by Tittlemier et al.36 Concentrations and distribution patterns of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles did not correlate with PCBs, which confirmed different sources for both substance classes.36 Highest concentrations (up to 9.8 mg/kg) were determined in two sea lion species from California (Table 2).36 However, the concentrations Table 2.  A selection of polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole concentrations (ng/g lipids) in marine environmental and food samples

Marine mammals Species

Location

Beluga

Canada & North Alaska Pacific/ Arctic California North Pacific California North Pacific Asia North Pacific Japan North Pacific Japan North Pacific

Steller sea lion California sea lion Dall’s ­porpoise Steller sea lion Melon headed whale Killer whales Japan Bottlenose dolphin Hector’s dolphin Dolphins

Ocean

North Pacific Australia South Pacific South New Pacific ­Zealand Philippines

n sumPDBPs

PCB 153

Reference

17 0.5–99

87–1890

36

5

93–9800

36

5

80–8200

910– 90,800 n.d.

5

1430–4710

960–1550

36

5

28–84

49–173

36

37

15 2900–42,300 620–12,100

39

9

41

4

6500–26,000 18,500– 68,200c 230–8800 250–4150b

40

5

15–840

370–1770

36

5

49–2300

260–680

37 (Continued)

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Table 2  (Continued)

Marine mammals Species

Location

Ocean

Beluga Seala

Svalbard Canadian Arctic New ­England Different locations Different locations Canadian Arctic Canada

Arctic 2 North 10 Atlantic North 8 Atlantic 69

Dolphins Harbor seals Ringed seals Black guillemota Seabird eggs offshore surface feeders Seabird eggsa Sharks Marine fisha Freshwater fisha Canned fisha Arctic coda Shrimpa Sediment

n sumPDBPs

Pacific

Reference

0.6–6.0 ~0.1

650–2480

36 48

900–3800

700– 15,800 65– 282,000 44–1770

38

0.02–530

38 0.1–46 6

PCB 153

9.5

36

48

19 32–140

~70–100

5

North Atlantic America Japan Pacific Canada Canada

40 1.7–4.8

>20

5

52 3–4400 62 <0.001–1.1 39 <0.001–0.2

17–11,000 n.d.

76 46 46

Canada Canada Canada Canada

86 5 33 2

Arctic Arctic

0.03–6.7 1.1 <0.001–0.05 ~ 0.03

46 48 46 48

aWet

weight. from response factor of 2-MeO-BDE 68. cSum of PCBs. bEstimated

varied by two orders of magnitude (the same was observed for PCBs).36 Both the order of magnitude and the variations in the polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole content were confirmed by Stapleton et  al., who determined up to 8.2 mg/kg lipids in blubber of California sea lions (Zalophus californianus).37 Accordingly, data from sites where only a few samples were available for analysis have to be evaluated with care. High concentrations (up to 4.7 mg/kg) were also determined in mammals from the North American Pacific (British Columbia), the Philippines (up to 2.3 mg/kg) and the Bay of Bengal (Figure 9).36

Polyhalogenated Alkaloids in Environmental and Food Samples    

0.1

227

0.006 0.05

2.5 0.5

1.5

42

9.8

2.3 1.9

0.8

4.2

Figure 9  Global distribution of Br4Cl2–DBP (3) in marine mammals. Highest concentrations [mg/kg lipids] are shown.

Concentrations in samples from the Atlantic Ocean were about one order of magnitude lower. For instance, dolphins from New England (USA; North Atlantic) contained 0.5 mg/kg lipids or less Br4Cl2–DBP (3).38 The highest concentrations of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles reported to date were measured in melon-headed whale (Peponocephala electra) blubber from the Asia-Pacific Ocean.39 The polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole concentrations ranged from 2.9 to 42.3 mg/kg lipids (Table 2).39 High concentrations in the Pacific region were confirmed by up to approximately 4 mg/kg lipids of Br4Cl2–DBP (3) in a melon-headed whale and two pygmy sperm whales (Kogia breviceps) from the Great Barrier Reef, Australia (Figure 9), while dolphins and other biota did not contain remarkable amounts of this halogenated natural product.40 Very high concentrations were also detected in six female killer whales (Orcinus orca) from the coast of Japan (6.5–18 mg/kg lipids).41 Even higher concentrations could be determined in calves of two of the females (20.4 and 26 mg/kg lipids).41 However, other marine mammal samples from Japan were lower contaminated with polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles.18,36 Feed, species, geographic, and annual reasons may be responsible for these variations. Frequently, pinnipeds were lower contaminated with polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles than cetaceans.36

2. Residues of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles in Marine Birds Studies of marine birds of prey demonstrated that polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles bioaccumulated in tissue, plasma, and liver.42

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Eggs of Leach’s storm petrel from British Columbia (North Pacific Ocean) accumulated highest amounts of polyhalogenated 1,1′-dimethyl-2,2′bipyrroles with up to 0.14 mg/kg wet weight.5 Lipid content was not reported but based on comparable samples, this indicated ppm levels on lipid basis. These lipid-based levels were thus comparable with those in marine mammals from the same region. As for marine mammals, polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole concentrations were lower in samples from the Atlantic Ocean coast, and they were not detected in birds from the Great Lakes.5 In addition, eggs of surface feeders were higher contaminated with polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles than subsurface feeders.5 Likewise, sea eagle eggs from the Norwegian North Sea/Atlantic coast contained traces of Br4Cl2–DBP (3).43 Polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles were shown to be transported to yolk during egg development.42

3. Residues of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrroles in Fish, Marine Organisms, and Seafood Highest concentrations of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles were determined in sharks from Japan with up to 4.4 mg/kg lipids.44 Yet, polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole concentrations in different shark species varied by more than one order of magnitude, and they appeared to be the highest in adult animals.44 Bluefish tuna and groupers contained up to 0.18 mg/kg polyhalogenated 1,1′-dimethyl-2,2′bipyrroles.45 Furthermore, canned fish (epipelagic high-trophic species) contained up to 6.6 µg/kg wet weight polyhalogenated 1,1′-dimethyl-2,2′bipyrroles (a lipid content of ~5% translates into ~0.13 mg/kg lipids). Similar amounts (0.16 mg/kg lipids) were also reported in salmon oil (sold as dietary supplement).19 Other fish samples contained much lower polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole concentrations.19,46 Deep sea squid samples collected at 1000–3000 m below sea level contained <0.1–3.5 µg/kg dry weight (PCBs up to 0.28 µg/g dry weight).47 These concentrations are comparably low, yet this first inspection demonstrates that halogenated natural products can also enter the deep sea food chain. Notably, the deep sea squid samples originated from the North Atlantic region.47 Given the fact that polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole concentrations in biota were much higher in the Pacific region, high(er) levels can be assumed to occur in Pacific deep sea species.

4. Overall Evaluation of Polyhalogenated 1,1′-Dimethyl-2,2′-Bipyrrole Concentrations in the Environment and Food Concentrations of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles were the highest in marine mammals and especially in whales. Moreover, nitrogen stable isotope mass spectrometry (IRMS) analysis of tissue was used to demonstrate that polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles biomag-

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229

nified from invertebrate over fish to seabirds but not to ringed seals.48 Little abiotic data are available, namely, one sediment sample from Canada contained traces of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles, with Br6–DBP (1) being the most prominent congener.48 An estimate of the human exposure to bioaccumulative polyhalogenated 1,1′-dimethyl2,2′-bipyrroles was presented by Tittlemier.46 A deplorable disadvantage in the analysis of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles is the lack of reference standards (cf. section VI.A.2f) and the scarcely used nontarget approach (cf. section VI.C). It is evident from the literature that many research groups have analyzed species from regions in which polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles were detected. The very recent discovery of highly chlorinated 1,1′-dimethyl-2,2′-bipyrroles including Cl6–DBP (5)16 indicated that more research is required to fully understand their ­origins, distribution and fate in the environment.

III. POLYHALOGENATED 1′-METHYL-1,2′-BIPYRROLES A. Discovery and Variety of Polyhalogenated 1′-Methyl-1,2′Bipyrroles Polyhalogenated 1′-methyl-1,2′-bipyrroles were first described in the form of an abundant peak in the GC/MS chromatogram of an Antarctic seal sample.11 The unknown peak was initially labeled Q1 (20) (acronym for “question 1”), and this acronym is still frequently used in the literature. A first partial characterization of Q1 (20) was reported in 1999.49 Given the high abundance in Antarctic samples, high-resolution mass spectrometric analysis of an Antarctic skua liver extract could be used to unravel the molecular formula of Q1 (20) as C9H3Cl7N2.6 In 2002, the exact structure of Q1 (20) was proven to be 2,3,3′,4,4′,5,5′-heptachloro-1-methyl-1,2′bipyrrole (20) (Figure 10) by synthesis.50 Q1 (20) is remarkable as it has a 64% weight percentage of chlorine.51 Seven chlorine substituent is also an extremely high number for a naturally produced compound. Very recently, three natural decachlorinated natural products (no alkaloids) were identified in basidiomycetes.52 A search of the literature for mass spectra indicated that Q1 (20) had also been detected before53–55 but had remained undiscovered.11 The history of the polyhalogenated 1′-methyl1,2′-bipyrroles is reversed to that of the polyhalogenated 1,1′-dimethyl2,2′-bipyrroles: brominated 1′-methyl-1,2′-bipyrroles were unknown until 2006, when Teuten et al. discovered Br7–MBP (21) as well as one Br6Cl-1′methyl-1,2′-bipyrrole (out of five possible isomers) in marine mammals.38 Shortly after, representatives of all possible mixtures of bromine and chlorine on the heptahalogenated backbone were reported.56, 57 Altogether, 80 heptahalogenated 1′-methyl-1,2′-bipyrrole structures can be drawn56

230    

Walter Vetter

(Table 3). The larger variety of heptahalogenated 1′-methyl-1,2′-bipyrroles compared to fully ring-halogenated 1,1′-dimethyl-2,2′-bipyrroles results from the unsymmetric 1,2′-bipyrrole backbone and one additional ring position. Altogether, approximately 28 heptahalogenated 1′-methyl-1,2′bipyrroles have been described, but only three of them (20, 21, and 22) are currently structurally known (Table 3). In addition to the heptahalogenated 1′-methyl-1,2′-bipyrroles, there were also reports on hexa- to tetrahalogenated 1′-methyl-1,2′-bipyrroles. Wu and Vetter isolated and characterized MBP-77 (25), a by-product of

Cl Cl

Cl

Cl

Cl

Br

Br

N

N

CH3 Cl

Cl

Br

CH3 Br

20 Cl

Cl

Cl

Cl

Cl

Br

N

Cl

CH3 Cl

CH3 Cl

Cl

Cl

Cl

N

N

Cl

CH3 Cl

23

Cl

N

Cl

Cl

Cl

CH3 Cl

Cl

CH3 Cl

Cl

N N

28

Cl

HO

27

26

Cl O

Cl

N N H

Me

29

Br

Br

Cl

Me

N

Cl

N

25

N N

Cl

CH3 Cl

24

Cl

Cl

22 Cl

N N

Cl

N

21

Cl

Cl

Br

Cl

Cl

N

N

Br

Br

Br

Br

N

HN

Cl

O Cl

N

N

Br

Br Br

30

Br

OH

31

Figure 10  Structures of polyhalogenated 1′-methyl-1,2′-bipyrroles and related compounds. 2,3,3′,4,4′,5,5′-heptachloro-1′-methyl-1,2′-bipyrrole (Q1 (20)); 2,3,3′,4,4′,5,5′-heptabromo1′-methyl-1,2′-bipyrrole (Br7-MBP (21)); 2-bromo-3,3′,4,4′,5,5′-hexachloro-1′-methyl-1,2′bipyrrole (Br-MBP-77 (22)); 2,3,3′,4,4′,5-hexachloro-1′-methyl-1,2′-bipyrrole (MBP-74 (23)); 2,3,3′,4,4′,5′-hexachloro-1′-methyl-1,2′-bipyrrole (MBP-75 (24)); 2,3,3′,4,5,5′-hexachloro1′-methyl-1,2′-bipyrrole (MBP-77 (25)); 2,3,3′,4,5-pentachloro-1′-methyl-1,2′-bipyrrole (MBP-62 (26)); 2,3,4,5,5′-pentachloro-1′-methyl-1,2′-bipyrrole (MBP-69 (27)); 1′-methyl-1,2′bipyrrole (28); 1,2′-bipyrrole (29); 2,2′,3,4,4′,5,5′-heptabromo-1′-methyl-1,3′-bipyrrole (30) and the natural product (−)-marinopyrrole B (31).

Polyhalogenated Alkaloids in Environmental and Food Samples    

231

the Q1 (20) synthesis.58 Later three further hexachloro-1′-methyl-1,2′bipyrroles and two pentachloro-1′-methyl-1,2′-bipyrroles were generated by UV irradiation of Q1 (20), and several structures could be assigned to these polyhalogenated 1′-methyl-1,2′-bipyrrole congeners.59 However, the total concentration of the hexa- and pentahalogenated 1′-methyl-1,2′bipyrroles in marine samples was <2.2% of the amount of Q1 (20), and it

Table 3  Structural variety of polyhalogenated 1′-methyl-1,2′-bipyrroles, first reports and currently known congeners

Heptahalogenated Theoretical 1′-methyl-1,2′number of bipyrroles (MBPs) isomers (chiral)

First report (number of isomers)

Total number Exact structure of isomers known reported to date

Cl7–MBP BrCl6 –MBPs

1 (–) 5 (2)

1 4

Br2Cl5 –MBPs

13 (8)

Br3Cl4 –MBPs Br4Cl3 –MBPs

21 (14) 21 (14)

Br5Cl2–MBPs

13 (8)

Br6Cl–MBPs

5 (2)

Br7–MBPs SUM

1 (−) 80 (48)

16 456 357 556 257 556 456 257 456 357 138 357 138

Hexahalogenated 1′-methyl-1,2′bipyrroles (MBPs)

Theoretical number of ­isomers

First report (number of ­isomers)

Total number Exact structure of isomers known reported to date

Cl6 –MBPs

5

1131 459

4

BrCl5 –MBPs Br2Cl4 –MBPs Br3Cl3 –MBPs Br4Cl2–MBPs Br5Cl–MBPs Br6 –MBPs SUM

26 42 42 42 26 5 188

157 957 138 160

1 9 657 457 24

764

Q1 (20) Br-MBP-75 (22)

5 4 4 3 1 29

Br7–MBP (21)

MBP-74 (23); MBP-75 (24): MBP-77 (25)

(Continued)

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Walter Vetter

Table 3  (Continued)

Pentahalogenated 1′-methyl-1,2′bipyrroles (MBPs)

Theoretical number of isomers

1st report (number of isomers)

Br5 –MBPs Br4Cl–MBPs Br3Cl2–MBPs

13 42 52

457

Br2Cl3 –MBPs BrCl4 –MBPs Cl5 –MBPs

13

SUM

120

Tetrahalogenated 1′-methyl-1,2′bipyrroles (MBPs)

Theoretical number of isomers

1st report (number of isomers)

Total number Exact of isomers structure reported to known date

Br4 –MBPs Br3Cl–MBPs Br2Cl2–MBPs BrCl3 –MBPs Cl4 –MBPs SUM

9 27 54 27 9 126

157 157 419

1 1 4

118

Total number Exact of isomers structure reported to known date 119 2

257 259

2

MBP-62 (26); MBP-69 (27)a

5

6

aBoth

identified but peak assignment equivocal: both only detected in UV irradiation experiments and not in samples.59

was suggested that they could be metabolites of Q1 (20).59 Teuten et al. identified a Br5Cl-1′-methyl-1,2′-bipyrrole isomer in D. delphis and other marine mammals from the Atlantic coast of New England.38 This and a Br6-1′-methyl-1,2′-bipyrrole isomer were also detected in archived whale samples from 1921.60 Pangallo et  al. expanded the variety of polyhalogenated 1′-methyl-1,2′-bipyrroles by the GC/MS detection of 28 novel hepta- to tetrahalogenated 1′-methyl-1,2′-bipyrroles.57 It is noteworthy that the reports of both research groups were complementary. While the German researchers mainly identified polyhalogenated 1′-methyl-1,2′bipyrroles with clear dominance of Cl compared to Br, the researcher from the U.S.A. reported on polyhalogenated 1′-methyl-1,2′-bipyrroles

Polyhalogenated Alkaloids in Environmental and Food Samples    

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in which Br dominated over Cl substituents. The differences appear to originate from regiospecific differences in the formation of polyhalogenated 1′-methyl-1,2′-bipyrroles, specifically between Australia and the US–North Atlantic coast. Apparently, North-American samples contained more highly brominated 1′-methyl-1,2′-bipyrroles, while samples from Australia (which were investigated by the German research team) contained more highly chlorinated 1′-methyl-1,2′-bipyrroles. These observations could indicate that the natural producer first biosynthesizes the backbone followed by its halogenation. Altogether, >60 polyhalogenated 1′-methyl-1,2′-bipyrrole congeners have been detected in environmental samples (Table 3).

B. Synthesis of Polyhalogenated 1′-Methyl-1,2′-Bipyrroles and Related Compounds The first synthesis of Q1 (20) was performed independently in two laboratories and provided Q1 (20) in 3.6% yield or even less.50 The Q1 (20) synthesis was based on the reaction of N-methylpyrrole with NCS to give the monoaromatic 1,2′-linked bicyclic intermediate N-(1-methyl-1H-pyrrol2-yl)succinimide, which was subsequently converted into 1′-methyl1,2′-bipyrrole (28) using HSi(OEt)3 and Ti(OiPr)4 in THF at 65°C and/or directly chlorinated employing PCl5 to give Q1 (20).50 The structure of Q1 (20) was unexpected since even the simple (nonhalogenated) 1′-methyl1,2′-bipyrrole (28) and 1,2′-bipyrrole (29) backbones had not been synthesized thus far by organic chemists prior to the synthesis of Q1 (20).50 The synthesis was later improved by starting from 2-nitropyrrole.61 Following the scheme mentioned in the synthesis of the polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles, Fu and Gribble used a tin-mediated reductive-acylation with succindialdehyde, which provided a 35% yield for the 1′-methyl-1,2′-bipyrrole backbone (28).61 The subsequent chlorination provided Q1 (20) with a yield of 23%.62 Fu and Gribble also generated the heptabromo-1′-methyl-1,2′-bipyrrole (21).62 Moreover, the corresponding heptabromo-1′-methyl-1,3′-bipyrrole (30) was synthesized.62 This compound has not been identified as a halogenated natural product, but this “unnatural62“ polyhalogenated alkaloid may represent a useful internal standard for monitoring the correct performance of sample cleanup procedures. However, more complex polyhalogenated 1,3′-bipyrrole derivatives (e.g., (−)-marinopyrrole B (31)) have been isolated from marine bacteria.63 Due to the huge variety and different substitution patterns, the selective synthesis of mixed chlorinated-brominated 1′-methyl-1,2′-bipyrrole isomers is more difficult. Simultaneous bromination and chlorination of the 1′-methyl-1,2′-bipyrrole backbone may be a suitable way to generate such compounds. An uncommon approach was presented by Gaul and

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Vetter.64 With only Q1 (20) at hand and thus lacking the nonhalogenated 1′-methyl-1,2′-bipyrrole backbone as starting material, UV irradiation in the presence of bromine initiated the exchange of Cl by Br.64 At least 15 BrCl6-, Br2Cl5-, and Br3Cl4-1′-methyl-1,2′-bipyrroles could be generated this way.64 Dambacher et al. showed that the limiting factor was the loss of bromine during UV irradiation.65 When Br2 was repeatedly added to the irradiation solution, even Br7–MBP (21) could be produced from Q1 (20). They also determined the structure of the main BrCl6-1′-methyl1,2′-bipyrrole to be Br-MBP-75 (22) by means of 2D-NMR.65 The Cl→Brexchange starting from Q1 (20) to give Br-MBP-75 (22) could be performed in an about 30% yield.65 A curiosity was observed during the initial evaluation of the X-ray diffraction (XRD) analysis of Q1 (20). Initially, two independent laboratories indicated that the XRD structure of Q1 (20) would be octachloro2,2′-bipyrrole, based on indicated C4Cl4N units (unpublished data). This discrepancy compared to GC/MS and NMR data caused some delay in the original publication. Evidence by confirmation of the methyl group on Q1 (20) in the solid state was produced by magic angle spinning (proton decoupled) 13C-NMR.66 The help of a colleague who wished to stay anonymous finally allowed unraveling the correct structure of Q1 (20) by means of XRD analysis.50 Q1 (20) was characterized by the two pyrrole units being twisted by approximately 71.5°.50 Molecular modeling (PM3 method) led to the observation that the methylated nitrogen atom of one of the planar pyrrole units has to pass through a pyramidal configuration to surmount the barrier about the interannular N-pyrrole-C-pyrrole bond.67 Accordingly, this produced strong evidence on the one hand that Q1 (20) cannot be planar and on the other hand that the rotational barrier of Q1 (20) cannot be surmounted below temperatures of at least 100°C. This feature may have an impact on the toxicity (cf. section VIII) and ultimately leads to chirality in the case of nonsymmetrically substituted polyhalogenated 1′-methyl-1,2′-bipyrroles (cf. section VI.D).

C. Natural Source Verification of Polyhalogenated 1′-Methyl-1,2′Bipyrroles Neither Q1 (20) nor other polyhalogenated 1′-methyl-1,2′-bipyrroles have been described by natural product chemists, according to present knowledge. Likewise, similar compounds had never been synthesized in the laboratory. The structure of the only unstable reaction intermediate that shares the molecular formula with Q1 (20)68 had never been produced.11 Although the natural producer of polyhalogenated 1′-methyl-1,2′bipyrroles has not been discovered, their assignment to biogenic origins could be based on other information, similarly to the polyhalogenated

Polyhalogenated Alkaloids in Environmental and Food Samples    

235

1,1′-dimethyl-2,2′-bipyrroles. The fact that the 1,2′-bipyrrole (29) backbone was not available and that the chemical synthesis of Q1 (20) only provided a low yield (initially only 3.6%50) supported the natural product theory. It has also been raised that Q1 (20) was more abundant in the southern than in the Northern Hemisphere, which has not been reported for any anthropogenic polyhalogenated compound. The subsequent discovery of brominated analogs of Q1 (20) added more evidence for the biogenic origin of polyhalogenated 1′-methyl-1,2′-bipyrroles because mixed brominated–chlorinated congeners are rather insignificant in anthropogenic synthesis in the laboratory. Moreover, the distinctly different 1′-methyl1,2′-bipyrrole congener patterns at different sites do not point to anthropogenic synthesis. Also, the high abundance in marine environments and not in terrestrial samples supports the natural production of Q1 (20) and other polyhalogenated 1′-methyl-1,2′-bipyrroles in the marine environment. The stable carbon isotope ratio (δ13C values in ‰) of synthesized Q1 (20) and Q1 (20) in marine mammals differed by approximately 12‰, and this was interpreted as a sign that the chemical product (and similarly synthesized Q1 (20)) was not the source of Q1 (20) in the marine mammals.69 Unfortunately, attempts to verify the natural origin by means of radiocarbon measurements failed because the amount of Q1 (20) isolated from Australian dolphins proved to be too small.70 The halogenated natural product structurally most closely connected with Q1 (20) is the pentachloro-2-pyrrolyl-2-phenol (15), which is better known under the trivial name pentachloropseudilin.71 Surprisingly, pentachloropseudilin (15) was initially isolated from a terrestrial (!) bacterium Actinoplanes sp. ATCC 33002.71 By contrast, the corresponding pentabromopseudilin (14) was isolated from the marine bacterium Pseudoalteromonas luteoviolaceus.72 Presumably, the production of chlorinated versus brominated 2-pyrrolyl2-phenols could be also depending on the location.

D. Distribution and Concentrations of Polyhalogenated 1′-Methyl1,2′-Bipyrroles in the Environment Given the different distribution of polyhalogenated 1′-methyl-1,2′bipyrroles in samples from Australia (Q1 (20) dominates) and the New England/North Atlantic region (highly brominated 1′-methyl-1,2′bipyrroles are more prominent than Q1 (20)), it is difficult to assess the global distribution of polyhalogenated 1′-methyl-1,2′-bipyrroles. The following results refer only to Q1 (20) except where noted differently. Q1 (20) was the first halogenated natural product detected in human milk at 12–230 ng/g lipids (Table 4).73 These Q1 (20) concentrations in milk were orders of magnitude higher than polyhalogenated 1,1′-dimethyl-2,2′bipyrrole residues in human milk from Canada (cf. section II.D). This is

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likely due to the fact that the Faroe milk samples were taken from women who regularly consumed fish and marine mammal meat.73 Q1 (20) was also detected in 90% of human milk samples from Japan with the highest concentration of 0.94 ng/g lipids.34 Despite the wide distribution with positive results in samples from all continents, there were reports from marine samples in which Q1 (20) was undetectable: For instance, Q1 (20) was not detected in ringed seals from both Spitsbergen and the Canadian Arctic, as well as in Baltic seals.51 Q1 (20) was neither detected in fish from Hong Kong (unclear if marine fish or not) nor in seals from Lake Baikal.51 Less surprisingly, Q1 (20) was not detected in a snake sample from Australia.74 Like for the other polyhalogenated alkaloids dealt here, terrestrial samples were scarcely analyzed on polyhalogenated 1′-methyl-1,2′-bipyrroles.

1. Residues of Polyhalogenated 1′-Methyl-1,2′-Bipyrroles in Marine Mammals Cetaceans from Australia contained up to 14.0 mg/kg lipids of Q1 (20) (Table 4).40,66,74 In these samples, the brominated–chlorinated 1′-methyl1,2′-bipyrroles contributed additional 7–32% of the concentration of Q1 (20) to the polyhalogenated 1′-methyl-1,2′-bipyrrole content.56 The maximum concentration in the herbivorous dugongs was almost two orders of magnitude lower (0.17 mg/kg lipids).74 This indicated food chain enrichment of polyhalogenated 1′-methyl-1,2′-bipyrroles. Seals from Africa (Mauritania and Namibia) contained 0.01–0.54 mg/kg (Table 4). Four tissues (blubber, kidney, liver, and lung) of an Antarctic fur seal contained Q1 (20) on the same level and ratio as the major DDT-related compound in marine organisms, p,p′-dichlorodiphenyldichloroethene (p,p′-DDE).75 This suggested a similar body distribution of biogenic Q1 (20), and the anthropogenic POP.75 Q1 (20) was also detected (but not quantified) in melon-headed whales (Peponocephala electra) blubber from the Asia-Pacific Ocean.39 Low abundance of Q1 (20) (~0.1% of trans-nonachlor) was determined in beluga from Canada.51 High concentrations were detected in six female killer whales (Orcinus orca) from the coast of Japan (0.5–1.0 mg/kg lipids).41 As observed for polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles, Q1 (20) was also enriched in the killer whale calves (1.3–1.8 mg/kg lipids).41 Teuten et al. reported the detection of Q1 (20) and estimated the Br6-1′methyl-1,2′-bipyrrole and Br7-MBP (20) content in diverse marine mammals stranded at the coast of New England (both up to 2.7 mg/kg lipids).38 Likewise, polyhalogenated 1′-methyl-1,2′-bipyrroles were detected in different cetacean species (up to 3.0 mg/kg lipids) but not in seals (gray seals and harp seals).76 Concentrations in the liver were about the same level as in blubber.76 Archived whale blubber from 1921 was found to bear 0.02 mg/kg lipids, while a contemporary fin whale (Balaenoptera physalus)

Table 4  Concentrations (selection) of Q1 (20) (ng /g lipids) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) in the marine environment and food

Location

Ocean

n

Q1 (20)

PCB 153

Killer whales South African fur seal Bottlenose & Common dolphins Monk seal Shark Dugongs African fur seal Cetaceans, blubber (liver)

Japan Cape Cross (Namibia) Queensland, Australia

North Pacific Atlantic South Pacific

9 11

520–1880 43–540a 690–14,000a

n.d. 2–273 175–8800

Mauritania Japan Queensland Namibia New England

Atlantic Pacific South Pacific Atlantic North Atlantic

14 52 1 18

Seals blubber (liver)

New England

North Atlantic

7

Melon-headed whale Killer whale Skua eggs (n = 11) Wet weight White-tailed sea eagle

Japan Japan Potter peninsula (Antarctic) Norway

Pacific Pacific

15 9

Atlantic

9–117a 3.5–2300 n.d.–246a 323b ~1000–3000c (~40–>3000) <2 (<2)c 0.5–7.6 500–1900 3–190 3–4 ng ww

References 41 74 51

n.d. 19–170 n.d. ~600–1900 <53–>2400 150 ± 70 (1100 ± 1500) 0.6–12.1 18,500–68,200 1.5–150

74 51 76 76 39 41 78 43 (Continued)

Polyhalogenated Alkaloids in Environmental and Food Samples    

Species

237

Location

Ocean

Shark Fish liver (1987/1996) Green turtle Sting rays Fish Human milk Antarctic air Air

Japan Antarctic Queensland, Australia Brazil USA Faroe Islands Signy Island (Antarctic) Lista, southern Norway

Pacific South Pacific South Pacific Atlantic North Atlantic

aConcentrations

North Sea

corrected relative to estimations in the original articles.11 in other tissues: blubber/kidney/liver/lung: 323/62/11/2.1 µg/kg. cEstimates; highly brominated congeners (no Q1 (20)). dSum of hexachlorobiphenyls. bConcentration

n

1 5 20 4 3

Q1 (20)

PCB 153

3.5–2300 0.4–4.9 10 0.002–0.08 <2–170 12–230 ng/g 1.1–1.4a fg/m3 <4–92 fg/m3

17–11,000 0.4/2.1 70 1600–10,400d 14–480 n.d. n.d. n.d.

References 45 79 74 74 85 73 73 83

Walter Vetter

Species

238    

Table 4  (Continued)

Polyhalogenated Alkaloids in Environmental and Food Samples    

239

blubber sample contained 0.2 mg/kg Q1 (20). Further polyhalogenated 1′-methyl-1,2′-bipyrroles ranged from <0.005 to 0.02 (1921 sample) and 0.005–0.9 (2004 sample) mg/kg lipids.60 Cetaceans from Massachusetts (USA) contained approximately 2.0–3.0 (0.080–3.0) mg/kg lipids polyha­ logenated 1′-methyl-1,2′-bipyrroles (except Q1 (20)) in blubber (liver).76 Although initially detected in Antarctic Weddell seals (Leptonychotes weddelli), Q1 (20) was only quantified in one sample. The Q1 (20) concentration (0.0054 mg/kg) was low but at the same level as the most abundant anthropogenic POPs in the sample.77

2. Residues of Polyhalogenated 1′-Methyl-1,2′-Bipyrroles in Further Marine Samples Comparably high Q1 (20) concentrations were determined in Antarctic skua eggs (0.003–0.19 mg/kg wet weight).78 Transferred to lipid weight, the Q1 (20) amounts were up to 3.0 mg/kg lipids.78 White-tailed sea eagles from Norway contained 3–4 µg/kg wet weight.43 A green turtle from Australia contained 10 µg/kg Q1 (20).74 Fish liver from the Antarctic contained 0.4–4.9 µg/kg lipids.79 The concentrations were lower in the bottom invertebrate feeder than in fishfeeding species. It was suggested that the natural source of Q1 (20) may be in the euphotic zone of the upper water column.79 Despite the rather low levels, Q1 (20) was the third most abundant compound (after hexachlorobenzene (HCB) and p,p′-DDE).79 In the fish feeder, Q1 (20), p,p′-DDE, and 2,2′,3,4,4′,5′-hexachlorobiphenyl (PCB 138) ranked the highest.79 Fish oil capsules from Norway contained up to 3 µg/kg Q1 (20).19 Higher Q1 (20) concentrations were detected in a commercial fish oil capsule (concentration ~0.1 mg/kg oil), and Q1 (20) was also detected in cod livers canned in 1948.80 Pangallo and Reddy determined highly brominated polyhalogenated 1′-methyl-1,2′-bipyrroles in different fish species from the US North Atlantic coast. Up to 0.17 mg/kg lipids were detected in these samples. In addition, Q1 (20) was detected in fish food and in fish fed with the respective food.80 Hackenberg et al. reported highest Q1 (20) concentrations in fish from the South Atlantic and deep-sea fish from the North Atlantic.81 Likewise, Q1 (20) was detected in Mediterranean deep-sea fish.51 A sample of a green turtle contained 0.01 mg/kg lipids.74 Q1 (20) was also determined in two sting ray species from Brazil (0.002–0.08 mg/kg lipids).82 Br5Cl- and Br4Cl-1′-methyl-1,2′-bipyrroles were detected in Atlantic deep sea cephalapods (cf. section II.D.3), while Q1 (20) was not mentioned to occur.47

3. Residues of Polyhalogenated 1′-Methyl-1,2′-bipyrroles in Abiotic Samples Antarctic air from Signy Island (60° 72′ S, 45° 60′ W) contained traces of Q1 (20) (1.1–1.4fg/m3) (Table 4).73 The Q1 (20) concentrations in Antarctic air were low; however, no other compound than Q1 (20) was more abundant

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in Antarctic than in Arctic air.73 Melcher et  al. measured the weekly air concentrations at the southern Norwegian coast and found up to 90fg/m3 Q1 (20).83 Highest concentrations were determined between September and December.83 However, anthropogenic (and more volatile) hexachlorocyclohexane (HCH) isomers were generally in the pg range and thus much higher concentrated.83 Passive water samplers (cf. section IV.D) deployed in the Great Barrier Reef contained Q1 (20) with frequencies of approximately 65% but Q1 (20) was not detected in river samples.84 For the hydrophobic Q1 (20) (log KOW > 5), the uptake is usually water phase controlled and hence related to the thickness of the water boundary layer that is affected by flow/ turbulences.84 Based on sampling rates of 16L/day and average water temperatures of 23–29°C in the Great Barrier Reef, the highest estimated water concentrations were 97pg/L.84 The passive water samplers contained a range of polyhalogenated natural products but no anthropogenic POPs.84

4. Overall Assessment of Polyhalogenated 1′-Methyl-1,2′-bipyrrole Concentrations in the Environment and Food Although detected in marine samples from all six continents, the highest concentrations of polyhalogenated 1′-methyl-1,2′-bipyrroles in the ppm range are found in the Southern Hemisphere and particularly in samples from Australia. The highest concentrations were detected in marine mammals, especially in dolphins. It appears that cetaceans accumulate much higher Q1 (20) concentrations than seals from the same habitat.66, 76 Pangallo and Reddy produced evidence that polyhalogenated 1′-methyl-1,2′bipyrroles are biomagnified in the marine food chain.76 This was further substantiated by determining the trophic level by means of stable nitrogen isotope ratios (δ15N values in ‰).85 They showed that the concentrations of polyhalogenated 1′-methyl-1,2′-bipyrroles increased with the trophic level except for seals.85 They also found age-dependent concentrations.76

IV. TETRABROMO-1-METHYLPYRROLE AND RELATED POLYBROMINATED PYRROLES A. Discovery of Tetrabromo-1-Methylpyrrole and Related Compounds The heptachlorinated bipyrrole Q1 (20) was identified in various species of Australian marine mammals including the strictly herbivorous dugongs (Dugong dugon). The diet spectrum of the dugong is rather narrow and consists almost exclusively of seagrass, with distinct preference for species of the genera Halodule and Halophila.86 GC/MS analysis of the seagrass Halophila ovalis revealed the presence of a few polybrominated

Polyhalogenated Alkaloids in Environmental and Food Samples    

241

compounds one of which was identified by synthesis to be tetrabromo1-methylpyrrole (32) (Figure 11).87 The synthetic product fully matched the GC and MS values of the “seagrass” peak.87 Later the compound was also detected in fish and mussels.87 However, screening for mono-, di-, and tribrominated pyrroles as well as for chlorotribromopyrrole and the corresponding 1-methylpyrroles was unsuccessful to date.

B. Synthesis and Properties of Tetrabromo-1-Methylpyrrole Gaul et al. synthesized tetrabromo-1-methylpyrrole (32) (CAS-no.: 5645429-6) according to existing protocols.87 Gilow and Burton had shown that the commercially available 1-methylpyrrole (8) (dissolved in 20mL tetrahydrofuran) could be tetrabrominated with NBS at −78°C.88 The solid tetrabromo-1-methylpyrrole (32) can be stored under argon at −18°C for a few weeks until it turns slightly yellow.89 Impure product was reported to decompose even more rapidly.89 Solutions in brown glass appear to be more stable, and colored glass equipment is recommended to be used during the chemical analysis of environmental and food samples. The instability of bromopyrroles is also a known fact. Owing to their electrophilic nature, 2/5-substituted pyrroles are easily produced virtually without formation of 3/4-substituted pyrroles.90 In agreement with that, only 2,3,5-tribromopyrrole (35) but no 2,3,4-tribromopyrrole (34), was obtained from the NBS-mediated bromination of pyrrole.91 However, compared to other substituents, 2-halopyrrole proved to be unstable with 2-bromopyrrole being decomposed in air within 60s.90 Likewise, tetrachloropyrrole decomposed on standing,92 and tetrabromopyrrole (29) was extremely sensitive to light.24

C. Natural Source Verification of Tetrabromo-1-Methylpyrrole Commercial syntheses (for use as reaction intermediate) resulted in 5–100 g or mg amounts of tetrabromo-1-methylpyrrole (32).93 The lack of large-scale industrial syntheses makes an anthropogenic source for

Br

Br Br

N CH 3

32

Br

Br Br

Br

N H

33

Br

Br Br

Br

N H

34

Br Br

N H

Br

35

Figure 11  Structures of tetrabromo-1-methylpyrrole (32); tetrabromopyrrole (33); 2,3,4-tribromopyrrole (34); and 2,3,5-tribromopyrrole (35).

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this compound very unlikely.87 However, tetrabromo-1-methylpyrrole (32) was not reported as a natural product either. Concentrations of tetrabromo-1-methylpyrrole (32) in the seagrass were low (~2.2 ng/g dry weight). Despite the wide distribution of this seagrass and known variations in the annual cycle of halogenated natural product production, it seemed unlikely that seagrass is the natural producer of tetrabromo-1methylpyrrole (32). It rather appears that the sample accumulated the compound either from the water phase or from sediment (e.g., by root uptake). However, the structurally related tetrabromopyrrole (33) was identified in cultures of the isolated marine bacterium Pseudoalteromonas luteoviolaceus (initially ascribed to Chromobacterium sp., cf. section II.C).24 P. luteoviolaceus produced also the polyhalogenated 1,1′-dimethyl-2, 2′-bipyrrole-related hexabromo-2,2′-bipyrrole (6) (cf. section II.C). The yield (10 mg) represented 0.2% of the crude benzene triturate from broth cultures of P. luteoviolaceus.24 Low quantities of tetrabromopyrrole (33) were also synthesized in the laboratory. Interestingly, a range of further brominated 1H-pyrroles such as 2,3,4-tribromopyrrole (34) have been isolated from bacteria or have been identified as secondary metabolites of low-trophic marine biota.94,95 In addition, the radiocarbon content of 2,3,4-tribromopyrrole (30) in the range of Δ14C = −175‰ confirmed the natural origin of this isolate from the acorn worm Saccoglossus kowalevski.31 Moreover, the passive water samplers from the Great Barrier Reef contained various abundant halogenated natural products but no appreciable amounts of anthropogenic polyhalogenated compounds.84,87,96 All polyhalogenated alkaloids reported in this article may exist in both the N-H- and the N-methyl forms. In the case of the halopyrroles, only the nonmethylated polyhalogenated pyrroles and bipyrroles were isolated by natural product chemists, while higher marine organisms exclusively contained the N-methylated tetrabromo-1-methylpyrrole (32), polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles, and 1′-methyl-1,2′-bipyrroles. Likewise, the (N-methyl-) pyrroles are the basic building blocks of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles and 1′-methyl-1,2′-bipyrroles as well as the pentahalopseudilins (Figure 12). Similarly, 2,4-dibromo- and 2,4,6-tribromophenols are precursors of brominated phenoxyphenols. The corresponding phenoxyanisoles and the dibenzo-p-dioxins are regularly detected in environmental samples.11,97 It would thus also be possible that 14 is converted into 36 (Figure 12). To date, neither 36 nor its chlorinated analogon 37 has been detected by natural product chemists or environmental analytical chemists. However, 1.1 mg of 2,3,5,7-tetrabromo-1Hbenzofuro[3,2-b]pyrrole (38) was recently isolated from the large-scale fermentation of the marine bacterium Pseudoalteromonas sp. (DMMED 290).98 The bacterium was initially isolated from the surface of a nudibranch collected in the shallow waters at Hawaii.98 Compound 38 is thought to be formed from pentabromopseudilin (14) (Figure 13). Both 14

Polyhalogenated Alkaloids in Environmental and Food Samples    

Hal

Hal

OH

Hal Hal

Hal Hal

N

Hal

R

– Hal2

R

Hal

N R

N

Hal

Hal

Hal

Hal HO

Hal

Hal

Hal

N R

14, 15 – H2

Hal

– Hal2

A

– RHal

Hal

Hal

Hal

N

2x A Hal

+

Hal

Hal Hal

Hal

Hal

2x A

N

R

243

Hal

Hal

14, 15 (R = H) 16, 17 (R = Me)

Hal

N

Hal

O Hal

36, 37 Figure 12  Potential dimerization of tetrahalopyrrole (R = H) or tetrahalo-1-methylpyrrole (R = methyl). Hal refers to Br and/or Cl, with 14, 16 and 36: Hal = Br and 15, 17, 37: Hal = Cl. Subsequent methylation (R = H → R = CH3 and/or OH → OMe) may follow.

and 38 showed antimicrobial activity against methicillin-resistant Staphylococcus aureus with ID50 values in the sub- or low µM range, with 14 being one order of magnitude more active.98 Interestingly, tetrabromo-1-methylpyrrole (32), polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles, and 1′-methyl-1,2′-bipyrroles were previously identified in passive water sampler extracts from the Great Barrier Reef (Queensland, Australia).84,87 Despite this obvious similarity in the basic structure, it should be recalled that the polyhalogenated 1′-methyl1,2′-bipyrroles from the Great Barrier Reef were predominantly polychlorinated, while only the tetrabrominated 1-methylpyrrole (32) has been reported to date. Hence, there is still some mystery on the appearance of the pyrrole alkaloids in the Australian samples. The structurally related tetrabromopyrrole (33) and hexabromo-2,2′bipyrrole (6) were isolated from a marine seawater bacterium (cf. section II.C).24 This indicated that also tetrabromo-1-methylpyrrole (32), and polyhalogenated 1′-methyl-1,2′-bipyrroles and 1,1′-dimethyl-2,2′-bipyrroles – or at least their nonmethylated precursors – are produced by marine bacteria. Anderson et al. mentioned that the natural antibiotic tetrabromopyrrole (33) could accumulate in seawater to concentrations, which could

244    

Walter Vetter

Br HN Br

Br OH Br

14

H N

Br

-HBr O

Br

Br Br

Br

38

Figure 13  Proposed formation of 2,3,5,7-tetrabromo-1Hbenzofuro[3,2-b]pyrrole (38) from pentabromopseudilin (14).98

explain the natural antibiotic seawater activity.24 Such a process would add an environmental dimension to an ecological event.

D. Concentrations of Tetrabromo-1-Methylpyrrole in the Marine Environment Shortly after its first isolation from seagrass, tetrabromo-1-methylpyrrole (32) was also detected in passive water sampler extracts from the Great Barrier Reef (Queensland, Australia).87 Standard passive samplers in the form of semipermeable membrane devices take advantage of 92cm × 2.5cm low-density polyethylene (LDPE) tubings (wall thickness 60–90µm) filled with 1mL of triolein.99 The semipermeable membrane device (SPMD) tubes are mounted on iron cages that are typically deployed for approximately 30 days at about 1m below sea level. Due to its lipid ingredient triolein, the SPMD sampler is also referred to as artificial fish.100 Based on a nominal sampling rate of 16L/day at the investigated sites and a (calculated) log KOW for tetrabromo-1-methylpyrrole (32) of 4.03,93 it was assumed that the concentration of tetrabromo-1-methylpyrrole (32) in the samplers was near equilibrium between the passive samplers and the water at the time of retrieval.84 For the passive water sampler with the highest amount accumulated, the estimated water concentration corresponded with 280pg/L of tetrabromo-1-methylpyrrole (32). However, the stability of tetrabromo-1-methylpyrrole (32) under these conditions remained unstudied (no precaution had been taken during sample cleanup either), so that a certain amount of tetrabromo-1-methylpyrrole (32) may have been degraded by UV light.87 Still it appeared that the highest proportions of tetrabromo-1-methylpyrrole (32) were released during the Australian summer/autumn.87 These analyses of passive samplers indicated that tetrabromo-1-methylpyrrole (32) could be enriched in the water phase. First evidence for the presence of tetrabromo-1-methylpyrrole (32) in living organisms was produced by its identification in a sample of farmed mussels from New Zealand (0.013 µg/kg lipids tetrabromo-1-

Polyhalogenated Alkaloids in Environmental and Food Samples    

245

methylpyrrole (32)) and two samples of farmed salmon (~100 µg/kg and ~0.56 µg/kg lipids tetrabromo-1-methylpyrrole (32)) from Chile. Due to its occurrence in samples from Australia, New Zealand, and Chile, it is likely that tetrabromo-1-methylpyrrole (32) is present throughout the (Southern) Pacific Ocean (coastline), and this verified the environmental dimension of tetrabromo-1-methylpyrrole (32). Noteworthy, the salmon samples also contained Q1 (20) (and a few PCB congeners).87 The salmon samples were from the same producer and site but taken at a different season (best before June and August 2010, respectively).87 Month-dependent variations had already been observed in the case of tetrabromo-1-methylpyrrole (32) in the passive water samplers. This was also found for other halogenated natural products in air83 and water samples.84 Fish farms are frequently located close to the coast, which seems to be in the habitat of the natural producers. It might be speculated if excreta of the fish would increase the nutrient status and in this way could initiate a stronger growth of the natural producer and thus in turn of tetrabromo-1-methylpyrrole (32) and other halogenated natural products.

V. BROMINATED INDOLES AND 1-METHYLINDOLES A. Discovery and Variety of Brominated Indoles and 1-Methylindoles Beside more complex larger halogenated natural products with an indolebuilding block (among them the well-known tyrian purple (39), which is produced by marine mollusks), there are also simple naturally produced bromoindoles (BIs) (40–42,44–49), 3-chloroindole, 6-bromo-3-chloroindole (43), and brominated 1-methylindoles (50–53) (Figure 14). Since the 1970s, different simple brominated indoles and 1-methylindoles were discovered in acorn worms, the red seaweed Laurentia sp. and the brittle star.101–105 The simple bromoindoles add a iodoform odor to these species.101,102 The shared occurrence of bromoindoles and bromo-1-methylindoles in one natural producer103 is different to the polyhalogenated pyrroles and bipyrroles. These simple halogenated natural products are a further class of polyhalogenated alkaloids repeatedly found in food and environmental samples. In theory, there are six monobromoindoles, 15 dibromoindoles, 20 tribromoindoles, 15 tetrabromoindoles, six pentabromoindoles, and one hexabromoindole. Likewise, bromo-1-methylindoles exist in a theoretical variety of 63 congeners. The first connection of bromoindoles to environmental and food issues was provided by Maruya who identified two dibromoindole isomers and one tribromoindole by means of their mass spectra in common oyster (Crassostrea virginica) from an estuary in coastal Georgia, U.S. east coast.106

246    

Walter Vetter

O

H N

N H

Br

39

4 5 6 7

O

N H

3 1 N H

N H

40 Br

Cl

Br

Br

Br

Br

42

N H

43 Br

Br

Br

Br

N H

Br

41

N

Br

44 Br

45 Br

Br

Br

Br

Br N H

Br

N H

Br

46

Br N Me

Br

Br Br

Br N

Br

N

Br

Br

Me

Me

51

49

Br

Br Br

N

Br

N

Br

48 Br

Br

50

Br

47 Br

N H

52

53

Figure 14  Structures of bromoindole-related compounds. Tyrian purple (39); 3-bromoindole (40); 4-bromoindole (41); 6-bromoindole (42); 3-chloro-6-bromoindole (43); 3,4-dibromoindole (44); 3,6-dibromoindole (45); 4,6-dibromoindole (46); 3,4,6-tribromoindole (47); 3,5,7-tribromoindole (48); 2,3,5,6-tetrabromoindole (49); 2,3,5-tribromo-1methylindole (50); 2,3,6-tribromo-1-methylindole (51); 2,3,5,6-tetrabromo-1-methylindole (52); 2,3,4,6-tetrabromo-1-methylindole (53);

Although no reference standards were available, the identification has been supported based on a 99.4% match of a reference spectrum in the National Institute of Standards and Technology (NIST) library.106 The common oyster is exclusively feeding on suspended particulates, which may be the source for the bromoindole uptake.106 Detailed structures of the bromoindoles remained unknown at that time. In 2005, two monobromoindoles, two dibromoindoles, and one tribromoindole were detected in seawater samples from the German Bight (North Sea).107 Weigel et al. studied a full array of anthropogenic compounds and found industrial, agricultural, and household chemicals and also halogenated natural products in seawater. They reported that the halogenated natural products (bromoindoles and phenols) could contribute with abundant peaks to the

Polyhalogenated Alkaloids in Environmental and Food Samples    

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Figure 15  GC/EI–MS chromatogram (TIC) of a purified water sample extract from the North Sea indicating the presence of bromoindoles with DMP = dimethyl-, DEP = diethyl-, and DBP = di-n-butyl-phthalate. Modified by new peak labels from Weigel et al.109 reprinted with permission of Elsevier.

resulting peak mix in the chromatograms (Figure 15).107 They concluded that a substantial part of the halogenated natural products is emitted to the surface waters. Biselli et  al. detected mono- and dibromoindoles in the sediment from the North and Baltic Seas.108 The monobromoindoles could be characterized as 4-bromoindole (41) and 6-bromoindole (42), but the sources of standards and level of verification were not provided at this point.108 In a follow-up study, they used commercially available bromoindole standards (41, 5-bromoindole, 42, 46) along with self-synthesized compounds (44,45, prepared by selective bromination of 41 and 42 in 3-position) for quantification. By means of these standards, they identified 4-bromoindole (41) and 6-bromoindole (42), 3,4-dibromoindole (44), 3,6-dibromoindole (45), 4,6-dibromoindole (46), and tribromoindoles with a clear dominance of 3,6-dibromoindole (45).109 Subsequent evaluation of the previous articles and the GC retention times provided produced evidence that the two major dibromoindoles in the samples are most likely 3,4-dibromoindole (44) (first eluted, medium evidence) and 3,6-dibromoindole (45) (second eluted, strong evidence) (Figure 15). The latter, 45, had been isolated from the ascidian Distaplia regina from Palau (east of Philippines in the South Pacific Ocean).105 There is strong evidence that the two major dibromindoles reported by the German group are the same as those described by Maruya.106,109 However, there was a reversal in the abundance: In Maruya’s samples from the U.S. coast, the first eluting (and potential 3,4-dibromoindole, 44) isomer was dominant.106 The reports from the German group also featured an unknown tribromoindole. Hot

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candidates are 47 and 48 (Figure 14). Bromoindoles (one bromoindole and two dibromoindoles19 and one tribromoindole110) were also detected by means of GCxGC–TOF–MS in fish oil sold as dietary supplements.19 In one sample, the later eluting and probable 3,6-dibromoindole (45) (59 ng/g) was more abundant than p,p′-DDE.19 This salmon oil was reported to originate from Norway. The concentrations in three other fish oils were more than one order of magnitude lower. However, these oils were partly treated for pollutant removal, for example, by molecular distillation.19 At this point, the bromoindoles were not tabulated with the halogenated natural products but with anthropogenic POPs. The bromoindoles were quantified by means of the response of a standard of 4,6-dibromoindole (46), which was not detected in the fish oils.110 These findings support that Hoh et  al. also detected 3,4-dibromoindole (44) and 3,6-dibromoindole (45). In the same samples, Hoh et  al. reported the identification of a dibromo-1-methylindole isomer.110 Using a nontarget approach, Rosenfelder et  al. detected one tribromoindole, one tetrabromoindole along with two tribromo-1-methylindoles and one tetrabromo-1-methylindole in passive water samples from the Great Barrier Reef (Australia).96 Potential candidates are 50–53 (Figure 14). The identification was based on mass spectrometric features, and the positions of the substituents were not identified. Liu and Gribble synthesized the most relevant N-methyl-indoles (2,3,6-tribromo- (51) and 2,3,5,6-tetrabromo-1-methylindole (52)) detected in algae and brittle star.111 These could be among the brominated 1-methylindoles detected in the passive water samples and fish.96, 110 Remarkably, one of the tribromo-1-methylindoles (molecular ion m/z 365) coeluted with Q1 (20) (molecular ion m/z 384) from the DB-5 column.96 Laurencia sp. is a known producer of both tri- and tetrabrominated 1-methylindoles and (simple) bromoindoles.112–114 However, brominated 1-methylindoles have not been described in samples from Australian waters even though Laurencia sp. was reported to be abundant in the Great Barrier Reef.115 The preferential detection of N-methylated alkaloids in Australian samples is in agreement with polyhalogenated 1′-methyl-1,2′-bipyrroles and 1,1′-dimethyl-2,2′-bipyrroles, as well as tetrabromo-1-methylpyrrole (32). Passive water samplers from the Great Barrier Reef also contained one tribromoindole and one tetrabromoindole.96

B. Natural Product Verification of Brominated Indoles and 1-Methylindoles Biogenic sources of bromoindoles are undisputed due to the repeated identification of bromoindoles by natural product chemists (Figure 14).102,105,116 It is noteworthy that the 3,4-dibromoindole (44) detected by Reineke et al. by the retention time from sediment and water extracts from the North

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and Baltic Seas109 has not been characterized by natural product chemists. This could be due to the following reasons: (i) it has not been discovered yet or (ii) it is the metabolite of a known tri- or tetrabromoindole (Figure 14) or a compound with a more complex backbone. Further verification for the biogenic nature of bromoindoles can be derived from the fact that the bromoindole concentrations in oysters varied from year to year.106 Such distinct seasonal or annual variations106 and special variations within one ecosystem109 are uncommon for anthropogenic compounds.

C. Concentrations of Brominated Indoles and 1-Methylindoles in the Marine Environment Given the fact that (i) only a few research teams have reported results, (ii) local/seasonal differences in the bromindole abundances were found, and (iii) authentic reference standards were scarcely used, a thorough evaluation of bromoindoles is currently impossible. However, bromoindoles were already detected in samples from three continents.

1. Biological Samples Maruya estimated that oyster samples collected from coastal Georgia (USA) in November 1999 contained approximately 50 ng/g wet weight (of the potential) 3,4-dibromoindole (44) and tribromoindole and approximately 20 ng/g (of the potential) 3,6-dibromoindole (45).106 At the same site, the concentrations in August 2000 and March 2001 samples ranged from 1 to 10 ng/g wet weight (with the same relative abundances of the three bromoindoles).106 The oysters are exclusively feeding on suspended particulates that most likely contained the bromoindoles.106 The GC/ ECD chromatograms showed no other abundant halogenated residues.106 Oysters are commercially and ecologically important species, and a thorough control by food inspectors would be desirable. Hoh et  al. quantified one bromoindole (0.9–8 ng/g oil), the potential 3,4-dibromoindole (44) (0.03–2.2 ng/g oil), and 3,6-dibromoindole (45) (0.08–59 ng/g oil) in encapsulated fish oils.19 It is noteworthy that the fish oils were sold as dietary supplements, and there was strong evidence that the sample with the lower concentrations of bromoindoles was treated for removal of PCBs and other comparably volatile organohalogen compounds (including the bromoindoles).19 In the untreated sample, the concentration of the assumed 3,6-dibromoindole (45) was at one level with p,p′-DDE.19 The dibromo-1-methylindole reported in these fish oils was not quantified.110 It is noteworthy that neither bromoindoles nor bromo-1-methylindoles were thus far detected in marine mammals. One reason could be the low number of bromine substituents and the expectable lower lipophilicity of the nonmethylated indoles (cf. section VII).

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2. Abiotic Matrices Huge differences were determined in the concentrations of 4-bromoindole (41) and 6-bromoindole (42) in sediments at three different sites and times that spanned over more than three orders of magnitude from 110 ng/g dry weight to nondetectable.108 In one sample, 13 µg/g total organic carbon (TOC) originated from one dibromoindole isomer (most likely 45) in North Sea sediment108 and water samples from the German Bight could contain up to 913 ng/L 3,6-dibromoindole (45).109 However, reports are scarce, and a full picture on the natural occurrence and distribution of these compounds is currently not possible.

VI. ANALYSIS OF POLYHALOGENATED ALKALOIDS A. Analytical Identification and Quantification of Polyhalogenated Alkaloids On most occasions, the polyhalogenated alkaloids discovered by natural product chemists were highly concentrated in algae, sponges, and other organisms. The natural products are usually obtained by extraction with organic solvents, followed by as little as possible treatment of the extracts for their isolation. The techniques applied are designed for the detection of compounds in the percent or permil range of dried sample materials. The range at which polyhalogenated compounds usually occur in environmental and food samples is several orders of magnitude lower, that is, in the µg/g range or lower. Thus, the requirements on methods used by natural product chemists and environmental analysts for isolation/purification (cf. section VI.A1) and identification (cf. section VI.B) are different if not diametrical. Moreover, quantification of residues is the main goal in food and environmental analysis (cf. section VI.A2). For environmental analysis, the following estimations can be made. Typically, concentrations of polyhalogenated compounds in environmental samples are in the ppm–ppb range (i.e., µg/g–ng/g), but analytical methods are designed to detect trace compounds even in the sub-ng/g lipids range. Frequently, standard sample preparation methods are based on the processing of 1g lipids (for instance, this amount is the capacity limit of gel permeation chromatography). The total amount in such a sample aliquot would thus be <1–1000ng per analyte. Before the measurement, the purified sample extract will be concentrated to about 50–1000 µL, from which 1 µL is injected into the GC system. Accordingly, the final method for GC determination must be extremely sensitive. Typically, the limit of detection (based on a signal-to-noise ratio of 3) of polyhalogenated alkaloids should be in the low picogram to subpicogram range (cf. section VI.A2). This in turn means that a few nanograms leftover of

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the sample matrix in the final extract can impede the GC analysis of the target compounds. As a further consequence, the GC/MS determination method should be not only sensitive but also selective for polyhalogenated compounds. Detection systems particularly suited for the analysis of polyhalogenated compounds are GC with electron capture detection (GC/ECD) and GC with electron capture negative ion mass spectrometry (GC/ECNI–MS). These techniques were also those that enabled the first detection of the polyhalogenated alkaloids in environmental samples.

1. Sample Cleanup Methods for the Determination of Polyhalogenated Alkaloids in Environmental and Food Samples Owing to similar lipophilicity and persistence, polyhalogenated alkaloids coexist with anthropogenic POPs in environmental and food samples. Although many different biotic and abiotic sample matrices are analyzed for polyhalogenated compounds, their lipophilicity and bioaccumulative nature give rise to the highest levels in fatty tissues. For this reason, fish, marine birds, and marine mammals are among the sample matrices most frequently analyzed. The processing of these sample matrices is based on methods developed for quantification of anthropogenic POPs. The clear fact that anthropogenic POPs and polyhalogenated alkaloids can be analyzed with the same standard methods also raised the question if they share further properties such as persistence, the bioaccumulative nature, and toxicity. In residue analysis, the primary goal is quantification. Although the (properties of the) target compounds are the same, no standard procedures have yet been set for polyhalogenated compounds.117 Instead, different analytical approaches for both sample preparation and instrumental analysis have been established.117 Usually, the methods were developed and validated for the quantitative determination of one or a few groups of organohalogen compounds. Step by step, these initial methods are usually expanded and validated for the determination of additional groups of pollutants. This can be done by the processing of samples spiked with reference standards. Since reference standards are not readily available, this has been scarcely done, and it is no surprise that the polyhalogenated alkaloids have not been quantified more frequently in marine samples. Most residue analysis methods are based on the following steps. Owing to the lipophilic character of the polyhalogenated compounds, they are bioaccumulated in the sample lipids, from which they can be gathered with organic solvents. Suitable methods include soxhlet extraction, microwave-assisted extraction (MAE), accelerated solvent extraction (ASE) as well as by liquid/liquid partitioning.117 The resulting organic extracts also contain other lipophilic compounds, namely, the lipids which need to be separated due to their high excess. The bulk of the lipids (the triacylglycerides) can be effectively separated by gel permeation chromatography (a.k.a. size-exclusion chromatography).118 A cheaper alternative is

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available in the form of treatment with sulfuric acid, which can be used for the cleanup of acid-stable analytes. This is fulfilled for the polyhalogenated alkaloids (polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles and 1′-methyl1,2′-bipyrroles, tetrabromo-1-methylpyrrole (32) and most likely also the bromoindoles). However, other – probably unknown – organohalogen compounds may be destroyed by this procedure. Hence, the treatment with sulfuric acid is best suited for targeted analyses, in which the method can be validated with spikes of authentic reference standards. Adsorption chromatography with different sorbents (e.g., 1–8 g silica in 10 mm i.d. glass columns) and sulfuric acid can be used for the removal of remaining matrix compounds. However, each sample cleanup step (including changes from one solvent to another or concentration of solutions to low volumes) is accompanied with minor losses of the analyte, and it adds extra effort to the routine work, which at some point makes it inefficient. On the other hand, the elimination of matrix compounds is essential when the selectivity of the method is decreased (see below). In an ideal situation, the resulting fraction now (only) contains anthropogenic POPs and halogenated natural products (including the polyhalogenated alkaloids). With other words, the processed sample has no measurable weight. Adsorption chromatography can also be used to further separate the organohalogen fraction into aromatic polychlorinated compounds (fraction 1) and the more polar organobromine compounds (brominated flame retardants – BFRs) and aliphatic chloropesticides (fraction 2). This step usually benefits from the separation of the PCBs, which can be one or two orders of magnitude more concentrated than the analytes in fraction 2. Since all polyhalogenated alkaloids (except Q1 (20) and Cl6-DBP (5)) are at least in part brominated, such fractionation will simplify the analysis on polyhalogenated alkaloids, which share the GC retention range and the mass range with anthropogenic polyhalogenated pollutants. Last not least, it must be mentioned that quality control issues require the use of internal standards to monitor and correct for analyte losses during the sample cleanup. Internal standards are also used to even-out instrumental variations from run to run. Finally, the quantification is performed against external reference standards.

2. Determination of Polyhalogenated Alkaloids Residue analysis of environmental and food samples on polyhalogenated compounds is predominantly performed by GC/EI–MS or GC/ECNI– MS operated in the selected ion monitoring (SIM) mode. Polyhalogenated compounds simplify this approach notable by the distinct halogen isotope pattern, which can be distinguished visually and which give rise to abundant isotope peaks (e.g., cf. Figure 1 ). Since the ratio of the isotopic peaks is unchanged and determinable with high precision, the required sensitivity in trace analysis can be obtained by the exclusive recording

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of two abundant isotopic peaks from the isotope pattern of the molecular ion or a more abundant high mass fragment ion (Table 5). The classic quantification by GC/EI–MS in the SIM mode takes advantage of the more abundant ion for quantification and the lower abundant isotopic peak for verification (to confirm the correct peak assignment). In the GC/ MS–SIM mode, the analyte is identified by a full match of the retention time with the external standard along with the presence of the quantification and verification ion in the correct ratio within a margin of typically ±10% (Table 5). Using these parameters, the level of identification closely matches that of full scan runs, and coeluting peaks only falsify the result if they also form the ions used for quantification of the analyte. The SIM mode benefits from low background, which improves the signal-to-noise ratio of the detection by approximately 2 orders of magnitude when quadrupole mass filters are used. Due to the mass defect, especially of bromine and chlorine, the resulting exact mass of the isotopic peaks of polyhalogenated compounds are lower than the nominal mass. For instance, the nominal mass of the monoisotopic peak of M+ of Br4Cl2–DBP (3) is m/z 540, while the exact mass obtainable from high-resolution mass spectrometry is m/z 539.6642 (Table 5). Due to this significant mass defect, it can be advantageous to take the first decimal place into account in measurements performed with low-resolution mass spectrometers such as quadrupole systems. Accordingly, Br4Cl2–DBP (3) may be determined with m/z 543.7 (quantification ion) and m/z 545.7 (verification ion) instead of m/z 544 and m/z 546 (Table 5). With increasing number of bromine substituents on the polyhalogenated alkaloids, the decimal places become smaller and smaller (cf. Table 5). For this reason, the exact mass of Br7–MBP (21) is m/z 691.46, which is rounded to m/z 691 (and not to m/z 692). A further beneficial effect of the SIM method is the high selectivity for the analyte (compared to other groups of polyhalogenated compounds). In the ideal case, other polyhalogenated compounds give no response to the quantification and verification ion of the analyte and thus remain undetected. For the same reason, such gain in selectivity and sensitivity has to be paid with drawbacks (cf. section VI.B). The use of targeted GC/ EI-MS-SIM methods for anthropogenic POPs does not give response to (nontargeted) polyhalogenated alkaloids. This may partly explain why polyhalogenated alkaloids are not quantified more frequently and why it took so long before they were detected in environmental samples. Moreover, the classic GC/EI–MS–SIM method of choice is based on isotope dilution analysis. In isotope dilution analysis, a defined amount of isotopically (usually fully 13C) labeled (synthetic) analyte standard is added to the sample (or sample extract). The native and the labeled analytes have the same retention time but due to the different masses, they can be determined without interference. Thus, the recovery rate of the labeled standard can be determined at the end of the analysis. On the one

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Molecular ion Quantification ion Verification ion (%)a [m/z] (=100%) [m/z] [m/z]

Compound

Molecular formula

Cl6 –DBP BrCl5 –DBP Br2Cl4 –DBP Br3Cl3 –DBP Br4Cl2–DBP Br5Cl–DBP Br6 –DBP

Hexahalogenated 1,1′-dimethyl-2,2′-bipyrroles (DBPs) C10H6Cl6N2 364 366 368 (81.0) C10H6BrCl5N2 408 412 410 (98.3) C10H6Br2Cl4N2 452 456 454 (78.8) C10H6Br3Cl3N2 496 500 502 (77.7) C10H6Br4Cl2N2 540 544 546 (94.0) C10H6Br5ClN2 584 590 588 (89.5) C10H6Br6N2 628 634 632 (76.7)

363.8662 407.8157 451.7652 495.7147 539.6642 583.6136 627.5631

Cl7–MBP BrCl6 –MBP Br2Cl5 –MBP Br3Cl4 –MBP Br4Cl3 –MBP Br5Cl2–MBP Br6Cl–MBP Br7–MBP

Heptahalogenated 1′-methyl-1,2′-bipyrroles (MBPs) C9H3Cl7N2 384 386 C9H3BrCl6N2 428 432 C9H3Br2Cl5N2 472 476 C9H3Br3Cl4N2 516 520 C9H3Br4Cl3N2 560 566 C9H3Br5Cl2N2 604 610 C9H3Br6ClN2 648 654 C9H3Br7N2 691 697

383.8116 427.7611 471.7106 515.6601 559.6095 603.5590 647.5085 691.4580

388 (97.5) 430 (83.9) 478 (76.4) 522 (91.0) 564 (93.1) 608 (79.9) 652 (69.2) 699 (97.7)

High-resolved mass of the monoisotopic peak [m/z]

Walter Vetter

Table 5  Mass spectrometric data of polyhalogenated alkaloids

aFor

peak identification, the verification ion must be present at the given %-abundance relative to the quantification ion (=100%).

392.6999 428.6999 350.7894 272.8789 194.9684 442.7155 364.8050 286.8945 208.9840

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Tetrabromo-1-methylpyrrole (32) as well as brominated indoles and 1-methylindoles 395 397 395 (68.3) Tetrabromo-1-methylpyrrole C5H3Br4N (32) 429 433 431 (68.3) Tetrabromoindole C8H3NBr4 351 353 355 (97.7) Tribromoindole C8H4NBr3 273 275 273 (51.2) Dibromoindole C8H5NBr2 195 195 197 (97.8) Bromoindole C8H6NBr 443 447 445 (68.2) Tetrabromo-1-methylindole C9H5NBr4 365 367 369 (97.8) Tribromo-1-methylindole C9H6NBr3 287 289 287 (51.2) Dibromo-1-methylindole C9H7NBr2 209 209 211 (97.9) Bromo-1-methylindole C9H8NBr

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hand, a high recovery rate serves to verify the proper performance of the cleanup. On the other hand, the isotope dilution technique can be used to correct for analyte losses because it is claimed that the native and labeled compound will behave the same and the losses (in %) will be identical. Thus, the analytical result can be corrected by extrapolating the recovery rate of the (known added amount of the) labeled standard to 100%. For instance, if the amount of the analyte is 20 ng/g lipids and the recovery rate of the labeled standard is 80%, the isotope dilution analysis will allow us to extrapolate that the correct amount found in the sample was (20 ng/g × 100/80 =) 25 ng/g. However, polyhalogenated alkaloids are not available in the form of labeled reference standards so that the isotope dilution method cannot be applied (cf. section VI.A.2f). Since the 1980s, GC/ECNI–MS has been used as a valuable tool for the sensitive detection of polyhalogenated compounds in environmental samples.119 In GC/ECNI–MS, a reagent gas is introduced into the ion source. This measure increases the pressure (reduces the vacuum) in the source to about 1–10Torr (~10−3–10−2mbar). As a consequence, the fast electrons emitted from the filament first collide with the reagent gas molecules (which are present in high excess compared to the analyte). The resulting slowed-down, low-energy electrons in turn can be captured by polyhalogenated compounds. Typically, the resulting negatively charged molecular ion (M−) is in excess of energy, and it is cleaved into few stable, negatively charged fragment ions. A common feature of GC/ECNI–MS mass spectra of polybrominated compounds is the formation of bromide ion isotopes (m/z 79 and m/z 81), while polychlorinated compounds feature the chloride ion (m/z 35 and m/z 37) isotopes (albeit to a lesser degree because of the higher stability of the C–Cl bond). Due to the equal natural abundance of the bromine isotopes, response to m/z 79 and m/z 81 at the same abundance allows for a sensitive detection of all organobromine compounds in environmental and food samples.120 Outstanding sensitivities can be reached with this method in the SIM mode. However, GC/ ECNI–MS–SIM chromatograms only indicate the presence of polybrominated compounds along with the retention time. Isotope dilution analysis cannot be performed in the GC/ECNI–MS mode because the 13C-labeled standard is not separated by GC from the native analyte. In addition, coelutions of polyhalogenated alkaloids have been reported.110,121,122 Thus, additional low-mass fragment ions (especially Br2− and HBr2−) can be used to distinguish members of different classes of polybrominated compounds.58 However, none of the polyhalogenated alkaloids produce such ions. Instead, polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles and 1′-methyl-1,2′-bipyrroles as well as tetrabromo-1-methylpyrrole (32) form abundant molecular ions. For instance, the GC/ECNI–MS spectrum of Br4Cl2-DBP (3) was characterized by an equal abundance of M− (m/z 544) and Br−.5 Even without reference standards, the suitable SIM masses of

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other polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles can be determined by adding 44Da per exchange of Cl with Br (or subtracting 44Da per exchange of Br with Cl) (Table 5). With higher numbers of bromine, the abundance of M− relative to Br− is increased. a. Polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles. Tittlemier et  al. could base their investigations on the use of reference standards. Limits of detection (S/N > 3) of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles in the GC/ECNI–MS–SIM mode was in the subpicogram level (10–250 fg).11 The dominant Br4Cl2–DBP (3) eluted in the last third between 2,2′,4,4′tetrabromodiphenyl ether (BDE 47) and 2,2′,4,4′,6-pentabromodiphenyl ether (BDE 100) from DB-5-like GC stationary phases.66,121 More comprehensive retention data for the classic polyhalogenated 1,1′-dimethyl2,2′-bipyrroles on different columns were published by Tittlemier et al.15 Only a few research groups have had access to standards,16,19,38,41,65 and reports on polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles are mostly restricted to these groups. Teuten et  al. reported the use of “customsynthesized” Cl6–DBP (5) for use as IS because it had not been detected in environmental samples.38 b. Polyhalogenated 1′-methyl-1,2′-bipyrroles. GC/ECNI–MS is also the method providing the highest selectivity and sensitivity for Q1 (20) and other polyhalogenated 1′-methyl-1,2′-bipyrroles.66 In the GC/ECNI–MS– SIM, the limit of detection (S/N > 3) for Q1 (20) by means of m/z 386 and m/z 388 was approximately 130fg.73 Other polyhalogenated 1′-methyl1,2′-bipyrroles can be detected by using the SIM masses shown in Table 5. The highly brominated 1′-methyl-1,2′-bipyrroles produced abundant M− and [M-Br]− (base peak) ions but gave little or no response to Br−.38 GC/ EI–MS was about 1–2 orders of magnitude less sensitive for Q1 (20) than GC/ECNI–MS. The [M-Cl]+ (SIM masses m/z 351 and m/z 353) and/or the [M-2Cl]+ (SIM masses m/z 316 and m/z 314) are usually more abundant than M+.75 Likewise, the highly brominated 1′-methyl-1,2′-bipyrroles showed [M]+, and [M- Halx]+ fragment ions, with a higher abundance for Br loss.38 c. Tetrabromo-1-methylpyrrole (32). The highest sensitivity for the determination of tetrabromo-1-methylpyrrole (32) was also reached with GC/ ECNI–MS in the SIM mode, by screening the most abundant isotopic peaks of M− (Table 5).87 The limit of detection (S/N > 3) was 25fg.87 Care has to be taken upon analysis because solutions of tetrabromo-1-methylpyrrole (32) are sensitive to light exposure (half life in clear glass ~1d).87 Likewise, the solid compound is only of limited stability.89 d. Brominated indoles and 1-methylindoles. For dibromo- and tribromoindoles, GC/ECNI–MS also provided the highest sensitivity.106 While the bromide ion isotopes were detected, the base peak in the mass spectrum originated from M− (cf. Table 5).106 However, limits of detection have not been determined due to the lack of reference standards. GC/EI–MS also provided abundant M+ for both di- and tribromoindoles107 as well as

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brominated 1-methylindoles.96 Hoh et  al. reported that the dibromo-1methylindole detected in fish eluted between the potential 3,4-dibromoindole (44) and 3,6-dibromoindole (45) from the DB-5-like column.110 e. LC/MSMS analysis of polyhalogenated alkaloids. While GC/ECNI–MS proved to be the most sensitive method for polyhalogenated alkaloids, alternative methods for Br4Cl2–DBP (3) and Q1 (20) were recently introduced by means of liquid chromatography coupled to atmospheric pressure chemical ionization tandem mass spectrometry (APCI–LC/MS/MS) performed in the negative ion mode.39 The selected reaction monitoring (SRM) mode was used for quantification. Reversed phase HPLC proved to be suitable for separating different halogenated natural products on a 150-mm C18 column using methanol as the solvent. Transitions from [M-Cl+O]− to Br− (for Br4Cl2-DBP (3)) or C4 NCl4− (for Q1 (20)) were used (Figure 16). The sensitivity (limit of detection: 90–570pg) was reported to be similar to GC/EI–MS, allowing the determination in the µg/kg lipid weight range.39 f. Reference standards. Sources for reference standards are scarce. Q1 (20) is commercially available from one source,11 while SciFinder reports two sources for tetrabromo-1-methylpyrrole (32).93 Different bromoindoles are commercially available,110 but the most relevant congeners apparently not. Polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles are currently not commercially available as reference standards. This restricted availability of standards hampers a more thorough worldwide study of their relevance.

Figure 16  (−)-APCI–LC/MS mass spectra (full scan) of (a) Q1 (20) and (b) Br4Cl2–DBP (3) and product ion scan of [M-Cl+O]− ions for (c) Q1 (20) and (d) Br4Cl2–DBP (3). Data from Haraguchi et al.39 reprinted with permission of the American Chemical Society.

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B. Identification of Polyhalogenated Alkaloids The GC/MS–SIM approach described in section VI.A aimed at the selective determination of polyhalogenated alkaloids and other compound classes in samples while those not targeted are usually overlooked. The changes to identify a novel contaminant by means of GC/EI–MS (either performed in the SIM or full scan mode) are small. In fact, none of the polyhalogenated alkaloids reported in this chapter were first described by means of GC/ EI–MS in environmental samples. Instead, their discovery took its starting point in the identification of unusual abundant peaks in GC/ECNI– MS full scan chromatograms (other halogenated natural products such as the pentahalogenated monoterpene MHC-1 and two polybrominated hexahydroxanthene derivatives were first detected by GC/ECD123,124). In GC/ECNI–MS mode, extraction of m/z 79/81 and m/z 35/37 from the full scan run can be used for verification of polyhalogenated compounds. The retention times of the peaks detected can be compared with the organobromine compounds listed in data bases.121 In order to further investigate known or unknown compounds, GC/ECNI–MS full scan spectra can be recorded. However, these are characterized by little fragmentation because breakage of C–C bonds scarcely occurs. Thus, the structural information obtained from GC/ECNI–MS full scan measurements is frequently equivocal. Switching to GC/EI–MS requires about a 100-fold higher amount to be injected. Usually, sample extracts need to be concentrated by the same factor, which may require further purification steps. In such concentrates, remainders of the sample matrix can hamper the identification (see below). When the molecular ion is detected, the bromine and chlorine isotope patterns are very distinct, but caution is to be exercised for certain mixed chlorinated/ brominated compounds, which can look similar to homohalogen patterns. For instance Br4Cl2–DBP (3) was initially thought to be a pentabrominated compound (Figure 1).5 Likewise, the BrCl6 pattern is similar to Cl8.56 Such similarity in the isotope pattern and mass range may be one reason for the late discovery of polyhalogenated alkaloids in environmental and food samples. Excellent mass spectra must be recorded in order to exactly determine the halogen pattern. For this purpose, the more accurate SIM technique can be applied in which four or more isotope peaks are measured with high precision. In addition to the molecular ion, higher abundant fragment ions such as [M-Br]+, [M-2Br]+, and/or [M-Cl]+ might be suitable to distinguish unequivocal isotope patterns of the molecular ions. The identified isotope pattern can be used to determine the mass contribution of the halogens to the analyte, and the remaining mass usually originates from C, H, O, and N. Typically, different elemental compositions can be constructed on the basis of all combinations of these elements, but the correct molecular formula can be unraveled by means of high-resolution mass spectrometry (HRMS) in the EI mode. Different HRMS techniques have been used to determine the

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molecular formula of polyhalogenated alkaloids. For instance, Tittlemier et al. screened the monoisotopic peak of M+ and different fragment ions in the GC/EI–MS spectrum of Br4Cl2–DBP (3)5, while Vetter et al. measured in steps of 4 mDa the mass range of the monoisotopic peak of M+ of Q1 (20) in the SIM mode for highest response.6 HRMS is also frequently used by natural product chemists who, of course, have more material at hand. For instance, Qureshi and Faulkner determined the high resolved mass of 3,6-dibromoindole (45) to be m/z 272.8796, which only differed by 0.7 mDa from the theoretical value (Table 5).105 HRMS measurements are based on the fact that for all backbone elements on organic compounds (C, H, N, O), the mass defect is highest (and positive) for hydrogen. On first instance, the mass of isobaric peaks is the higher the more hydrogen atoms are present. For this purpose, the molecular ion can be scanned or the full isotope pattern can be measured in the SIM mode, which provides higher precision. These measurements can be expanded on fragment ions.5 Finally, the search in databases may provide hints on the identity such as in the case of the previously known bromoindoles (cf. section V). However, polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles and 1′-methyl-1,2′-bipyrroles were not known at the time of their HRMS study, and the details unraveled from their mass spectra were used to initiate their syntheses.

C. Suitable Novel Nontarget Screening Methods for the Detection of Polyhalogenated Alkaloids For a thorough sample analysis, proactive screening approaches are required to detect not only the known organohalogen compounds but also their metabolites, emerging contaminants and, last not least, polyhalogenated alkaloids and further halogenated natural products. For this purpose, new analytical methods are required to cover such a wide range of chemicals.125 These methods should be more informative, more sensitive, more selective, and faster than the current routine methods used in the analysis of polyhalogenated compounds in food and environmental samples.110 A suitable method for this purpose is available in the form of comprehensive two-dimensional gas chromatography (GC×GC) paired with time-of-flight mass spectrometry (TOF-MS). GC×GC delivers a much better separation power than conventional (1D) GC. Moreover, the 2D plot provides a topographic map in which structurally related compounds are grouped in distinct patterns. The combination with a TOF–MS allows one to collect full mass spectra typically with better sensitivity than with quadruples mass analyzers.110 Hoh et  al. combined GCxGC/TOF-MS with direct sample introduction (DSI)110 according to Jing and Amirav.126 DSI is based on the injection of the sample extract into a disposable microvial in a liner, which is then placed into the inlet. After evaporation of the solvent at low temperature, the inlet is heated rapidly to transfer the semivolatile

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compounds onto the GC column.110 Nonvolatile compounds remain in the microvial, which is renewed after each injection. Thus, efforts in sample preparation can be reduced, while high volume injections lead to better sensitivity.110,127 Hoh et al. used this DSI-GCxGC/TOF–MS approach with minimal sample cleanup for the comprehensive analysis of multiple classes of POPs and untargeted organic compounds in fish oil.19,110 Even coeluting compounds can be sorted out by using the molecular ion for identification. Mass spectral matches with those listed in databases led to a large array of compounds identifiable in the samples. The array can be widened by active screening for isomers (with the same backbone) by means of mass spectral data available from isomeric reference standards, and congeners that can be tentatively deduced from the mass spectra of other congeners and isotope ratios of their molecular ions.110 By this means, DSI-GCxGC–TOF–MS enabled the identification of seven novel polyhalogenated 1,1′-dimethyl2,2′-bipyrrole congeners (Figure 17).110 All in all, Hoh et  al. detected up to eleven polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles, seven polyhalogenated 1′-methyl-1,2′-bipyrroles, along with four bromoindoles and one dibromo-N-methylindole in four cod liver oil samples.19,110 Despite the merits of DSI-GCxGC–TOF–MS, there are also drawbacks. The concentrations of different polyhalogenated compounds span over several orders of magnitude, which makes it difficult to determine small peaks. In addition, the data handling of the countless peaks in the 2D plot

Figure 17  GCxGC–TOF–MS chromatogram of the detection of novel polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles (DBPs) in fish oil capsules. Data from Hoh et al.110 reprinted with permission of the American Chemical Society.

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is a challenge. Last not least, DSI-GCxGC–TOF–MS is currently only available in very few laboratories. A more simple approach from the instrumental side was suggested by Rosenfelder et al.96 They overcame the low sensitivity of quadrupole systems along with nonspecificity for polyhalogenated trace compounds of GC/EI–MS in the full scan mode by focusing on the (relatively high) mass range of the molecular ions of polyhalogenated compounds. This measure eliminated background from (nonhalogenated) matrix compounds.96 The observation that the GC retention times of nonpolar polyhalogenated compounds are increasing with the molecular weight could be used to refine the method. The chromatogram was divided into three time segments, and an increasingly higher mass range of 112u was assigned to each segment. The entire mass range of 112u was monitored in the SIM mode by eight GC runs of 15 consecutive SIM ions, respectively. The resulting SIM chromatograms provided high-quality isotope patterns and enabled the detection of 38 polyhalogenated compounds in passive water sampler extracts from Australia. Seven could be identified by means of reference standards and 15 further could be traced back to potential halogenated natural products.96 In addition to polyhalogenated 1′-methyl1,2′-bipyrroles, several bromoindoles and N-methylbromoindoles were identified in this way.96 Polyhalogenated alkaloids with an odd number of nitrogen can be identified by means of the odd mass of the molecular ion. In this article, potential candidates were detected in the form of a Br2Cl compound at m/z 309 (309 − 158 − 35 = 116u; 8 × 12 = 96 − 14 = 110Da) and a Br2 compound with M+ at m/z 325 (325 − 158 = 167u for C, H, N, O and further hetero atoms).96 Such screening methods offer opportunities to identify further polyhalogenated alkaloids. Another option for the selective detection of polyhalogenated alkaloids is available in form of the phosphorus–nitrogen detection (PND).128 Due to the very limited use of anthropogenic halogenated alkaloids, response in GC/PND chromatograms of marine samples can be linked with high probability that the response originated from halogenated natural products.128 With a nitrogen content of 7.3% of Q1 (20), the detection limit in the PND was approximately 20pg, that is, in the range of GC/EI–MS and LC/MSMS measurements. However, this detector does not provide structural information for the identification of the detected polyhalogenated alkaloids.

D. Enantioselective Determination of Chiral Polyhalogenated Alkaloids Already Kauffmann and Lexy reported that methyl groups on the nitrogen atoms of the 1,1′-dimethyl-2,2′-bipyrrole backbone (7) sterically hinder the coplanar conformation of the two aromatic ring moieties.21 Distortion

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from planarity gradually decreases the intramolecular π-conjugation of the two rings.129 As a consequence, the UV spectrum of the (nonplanar) 1,1′-dimethyl-2,2′-bipyrrole backbone featured a blue shift of λmax compared to the planar 2,2′-bifuran and 2,2′-bithiophen molecules.21 Likewise, molecular modeling (PM3 method) demonstrated that Q1 (20) is not planar and that the barrier between the two units is insurmountable at environmental temperatures (i.e., <100°C).67 Much energy must be applied to overcome the steric hindrance (comparable to tri-ortho-substituted PCBs130), including a change from planar (unstressed system) to pyramidal configuration of the N-methyl nitrogen atom during the passage of the barrier.67 Hindered rotation about the interannular ring–ring bond mainly due to bulky substituents in several vicinal positions is one prerequisite for axial chirality. This feature paired with the nonexistence of a center of symmetry along the interannular bond of two aromatic substituents leads to the formation of atropisomers. Polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles and 1′-methyl-1,2′-bipyrroles are chiral provided that the substitution pattern on the individual ring units cannot be aligned by mirroring at the interannular axis. A view from the interannular axis on Q1 (20) (Figure 18(a)) shows that this prerequisite is generally fulfilled in the 1-methylpyrrole unit (see arrow A) while the 1-pyrrole unit is symmetrically substituted (arrow B). For this reason, Q1 (20) is achiral. Different substitution patterns in either the α- (2-/5-position) or the β-positions (3-/4-positions) are required to generate a stereogenic axis in the interannular pyrrole–pyrrole bond of polyhalogenated 1′-methyl-1,2′-bipyrroles (Figure 18(b), arrow D). Accordingly, two of the five hexachlorinated 1′-methyl-1,2′-bipyrroles (i.e., MBP-77 (25) and MBP-75 (24)) are axially chiral. Altogether 48 of the 80 (i.e., 60%) heptahalogenated 1′-methyl-1,2′-bipyrroles are chiral (Table 3). Enantioselective chromatography with chiral stationary phases can be used to separate the racemates. The first gas chromatographic enantiomer separation of the polybrominated alkaloids dealt in this chapter was reported for MBP-77 (25), which had been isolated as a by-product of the synthesis of Q1 (20).131 The successful application of enantioselective gas chromatography using a chiral stationary phase (CSP) in the form of Chirasil-Dex in fact helped to elucidate the structure of this racemic synthetic product.131 It is important to note that the availability of racemic standards is essential for testing the CSP because the ability of a CSP to resolve the enantiomers cannot be predicted.132 Thermodynamically, most enantiomer separations including those of polyhalogenated compounds are enthalpy controlled, and thus, the best enantiomeric resolution is achieved by choosing a low isothermal temperature.132 Due to the high molecular weight of the polyhalogenated alkaloids, the separation of enantiomers of polybrominated compounds is rather difficult. Dambacher et al. also separated the enantiomers of a synthesized Br-MBP-75 (22) standard by both enantioselective GC and HPLC.65 However, enantioselective studies of chiral polyhalogenated

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Cl

Cl

Cl

A Cl

B

CH3

Cl

Cl

Br

D

N CH3

(b)

N Cl

Br

Br

E Br

Cl

Cl

Br

Br

Cl

Cl

C

(a)

N

N

Cl

Cl

(c)

F

N

N

CH3

CH3

Cl

Figure 18  Illustration of the chirality of three polyhalogenated bipyrroles with artificially elongated interannular bonds. (a) 2,3,3′,4,4′,5,5′-heptachloro-1′-methyl-1,2′-bipyrrole (Q1, 20); 2-bromo-3,3′,4,4′,5,5′-hexachloro-1′-methyl-1,2′-bipyrrole (22); 5′-chloro-3,3′,4,4′,5pentabromo-1,1′-dimethyl-2,2′-bipyrrole (2); letters refer to remarks noted in the text.

1′-methyl-1,2′-bipyrroles in real samples have not been carried out so far. The key problem is that Q1 (20), which is dominant in many samples, is achiral (cf. section III). On the other hand, MBP-77 (25) and Br-MBP-75 (22) are not only of lower abundance but they are also accompanied by further isomers, which makes it difficult to exclude coelutions during the enantioselective analysis. Hexahalogenated 1,1′-dimethyl-2,2′-bipyrroles are generally chiral, irrespective of the substitution pattern (Figure 18(c), arrows E,F). Rosenfelder et al. succeeded in the GC separation of the enantiomers of a synthetic Br4Cl2–DBP (3) standard (Figure 19(a)).133 Likewise, enantioselective HPLC was used to fractionate the racemate, and the resulting pure atropisomers could also be studied by polarity and XRD measurements.133 Moreover, enantioselective GC/MS was used to study the atropisomer composition of the natural Br4Cl2–DBP (3) in two marine mammal samples (Figure 19(b),(c)). In both cases, the (−)-Br4Cl2-DBP atropisomer was dominant, but the dextrorotary enantiomer was detected as well.133 The occurrence of two abundant enantiomers was unexpected. For instance, the natural 1,3′-bipyrrole derivative (−)-marinopyrrole B (31) was found

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Figure 19  GC/EI–MS ion chromatograms (m/z 543.7) of the enantiomer separation of the atropisomeric Br4Cl2–DBP (3) on a β-PMCD column: (a) synthesized racemic Br4Cl2– DBP (3), as well as Br4Cl2–DBP (3) in (b) melon-headed whale extract and (c) pygmy sperm whale extract. Data from Rosenfelder et al.133 reprinted with permission of Elsevier.

to be enantiopure. Chirality of 31 emerges from the four bulky substituents adjacent to the pyrrole–pyrrole bond (Figure 10). Since the generation of the second enantiomer from pure (−)-Br4Cl2-DBP in the environment is not plausible, it rather appears that the synthesis of Br4Cl2-DBP (3) in the environment is not enantioselective. As mentioned before, Br4Cl2– DBP (3) might originate from the corresponding Br4Cl2–2,2′-bipyrrole (18). This molecule only possesses two bulky substituents adjacent to the pyrrole-pyrrole bond and rotation about the interannual C–C bond is most likely possible under environmental conditions. The crucial step in the

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formation of Br4Cl2–DBP (3) leading to chirality is thus the addition of the methyl groups, which could be the terminal step of the biosynthesis (Figure 7). If so, this reaction is likely performed with poor (if any) enantioselectivity. It could be possible that Br4Cl2–DBP (3) is formed from (18) as the racemate in nature (Figure 7) and that the observed nonracemic proportions of Br4Cl2–DBP (3) in the marine mammals were the result of enantioselective metabolism, which has been frequently observed in top predators with anthropogenic organohalogen compounds including atropisomeric PCBs.132 It is noteworthy as well that a different natural organism could be involved in the methylation reaction shown in Figure 7. In this case, the carbon sources used by the two organisms do not need to be necessarily identical. There are many questions related to these issues, and it is deplorable that only few enantioselective studies have been carried out so far with chiral polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles and 1′-methyl-1,2′-bipyrroles.

VII. PHYSICOCHEMICAL PROPERTIES OF POLYHALOGENATED ALKALOIDS The bioaccumulation/bioconcentration (BCF/BAF) criterion is an important issue in the environmental assessment of polyhalogenated compounds.2 Evidence for exposure to a chemical due to bioaccumulation is produced if the bioaccumulation/bioconcentration factor (BCF/BAF) in aquatic species for the substance is >5000. If experimental data are missing, the bioaccumulative character of a polyhalogenated compound can be estimated from log KOW values of >5.2 While the vapor pressure decreases with increasing molecular weight of polyhalogenated compounds (this was also observed for polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles), the substitution of Cl with Br and vice versa has only a small effect on the water solubility and log KOW of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles (Table 6).134 The experimentally determined log KOW values (~6.5 to 6.7) of the polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole congeners were comparable with penta- and hexachlorinated biphenyls.134 These log KOW values are the only measured ones available for polyhalogenated alkaloids. Tittlemier et al. noted that according to Schwarzenbach et al.,135 the small effect of Br/Cl exchange on log KOW was expected because the value is primarily dependent on the water solubility,134 which is barely affected by the halogen pattern (Table 6). A narrow range of log KOW (~6.2 to 6.5) and a quite good agreement with the experimental data were also found for estimated values of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles (Table 6). Only Cl6-DBP (5) had a lower predicted log KOW than the bromine-containing 1,1′-dimethyl-2,2′-bipyrrole congeners (Table 6). In either case, the log KOW values >5 clearly demonstrate that polyhalogenated

Table 6  Physicochemical parameters of selected polyhalogenated alkaloids

Vapor pressure (estimated)

m.w.

log KOW (log P) (estimated)a

Cl6 –DBP (5)b Br3Cl3 –DBP (4) Br4Cl2–DBP (3) Br5Cl–DBP (2) Br6 –DBP (1) Br6 –2,2′-bipyrrole Q1 (20) Br7–MBP (21)c 2,3,6-Tribromo-1methylindole (46) 2,3,6-Tribromoindole Tetrabromo-1-meth­ ylpyrrole (32)

1.20 × 10−6 Torr 6.90 × 10−8 Torr 2.03 × 10−8 Torra 1.64 × 10−8 Torr 1.32 × 10−8 Torr 1.98 × 10−11 Torr 3.08 × 10−9 Torr 2.64 × 10−13 Torr

366.89 500.24 544.69 589.14 633.59 605.54 387.30 698.46 367.86

5.697 ± 1.514 6.435 ± 1.532 6.506 ± 1.539 6.337 ± 1.543 6.169 ± 1.547 4.484 ± 1.468 6.928 ± 1.612 7.521 ± 1.110 3.779 ± 1.108

353.84 396.70

4.624 ± 0.527 4.031 ± 1.439

aCalculated

7.13 × 10−5 Torr

log KOW (measured Water solubility H25 [Pa m3/mol] or calculated) SW,25 [g/L] 6.5 ± 0.3134 6.5 ± 0.3134 6.6 ± 0.3134 6.7 ± 0.3134

2.2 10−5 134 9 10−6 134 2.7 10−5 134 1.4 10−5 134

0.030 ± 0.004134 0.036 ± 0.004134 0.0068 ± 0.0005134 0.0020 ± 0.0004134

5.9 – 6.451, 81

4.6 10−6 67

n. d.

using Advanced Chemistry Development (ACD/Labs) Software V11.02 (© 1994–2012 ACD/Labs) as implemented in the SciFinder data base.93 refers to 1,1′-dimethyl-2,2′-bipyrrole(s). cMBP(s) refers to 1′-methyl-1,2′-bipyrrole(s).

bDBP(s)

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1,1′-dimethyl-2,2′-bipyrroles are candidates for food chain enrichment (cf. section II.D.4). In contrast, a lower log KOW of 4.5 was calculated for (the nonmethylated) hexabromo-2,2′-bipyrrole (6). This value questions if this compound will be effectively enriched in marine mammals. The predicted log KOW of Q1 (20) (Table 6) was higher than of the polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles, but the other sources reported lower (and probably) more realistic values. Hackenberg et al. presented a novel GC technique for the estimation of log KOW and water solubility, which was introduced with Q1 (20) as the model compound.81 The reported log KOW of 6.481 was slightly higher than the value predicted from other calculations (Table 6). The predicted log KOW value of Br7-MBP (21) was higher by approximately 0.6 units than Q1 (20) (Table 6). In either case, the resulting lipophilicity of the polyhalogenated 1′-methyl-1,2′-bipyrroles is high and food chain enrichment as reported in section III.D.4 is in full agreement with these measurements. The predicted log KOW values of tetrabromo-1-methylpyrrole (32), 2,3,6-tribromoindole and 2,3,6-tribromo-1-methylindole (51) ranged from 3.8 to 4.6 (Table 6). Log KOW values in this range do not suggest bioaccumulation of these alkaloids in marine mammals. Remarkably, the predicted log KOW of the tribromoindole was higher than that of its analogous tribromo-1-methylindole, which is rather unrealistic since methylation should decrease the water solubility and thus the log KOW value (see above for Br6-DMP/Br6-DP). For example, the water solubility of pyrrole (0.26mol/L) is higher than that of 1-methylpyrrole (8) (0.095mol/L) and the water solubility of indole (0.014mol/L) exceeds the one of N-methylindole (0.011mol/L).93 In another study, the log KOW of dibromoindoles was estimated at 4.45,109, that is, in the medium range where bioaccumulation is not warranted. In accordance, the log KOW value of 5-bromoindole was only 2.97.136 This illustrates that the potential for bioaccumulation increases with the number of halogen substituents.

VIII. BIOACTIVITY OF POLYHALOGENATED ALKALOIDS Environmental exposure of organisms to polyhalogenated compounds can induce different biochemical changes. Several studies with polyhalogenated alkaloids have been conducted in order to find indications for effects similar to those reported for anthropogenic compounds. Effects of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles on reproduction were tested by Tittlemier et al. by feeding 64 adult captive birds (Falco sparverius) whose diets were spiked with low, medium, and high doses of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles.137 Individual samples were taken stepwise from before pairing until after hatching.137 Polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles were both detected in the bodies

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of the adults, in the eggs, and in the breed.137 However, only about 1–3% of the administered amount of polyhalogenated 1,1′-dimethyl-2, 2′-bipyrroles remained in the bodies (dietary absorption efficiencies <0.03). In a similar experiment, >85% of PCBs were accumulated in ring doves (Streptopelia risoria).138 This indicated that polyhalogenated 1,1′-dimethyl2,2′-bipyrroles were mostly eliminated and/or metabolized.137 However, previous environmental studies indicated no pronounced metabolism of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles by birds.48 Effects were virtually absent even at the highest dose and this suggested that polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles do not pose an acute threat to avian reproduction.137 In another study, Tittlemier et  al. tested three individual polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles and two polyhalogenated 1,1′-dimethyl-2,2′-bipyrrole mixtures for dioxin-like toxicity by means of the ethoxyresorufin-O-deethylase (EROD) enzyme activity assay.42 The EROD assay measures the induction of the cytochrome P4501A gene which is mediated by binding of an inducer to the aryl hydrogen (AH) receptor. Typically, the strength of the binding is measured competitively by means of the reference 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is among the compounds inducing the strongest known effect. Typically, the effects of the polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles were in the range of mono-ortho substituted PCBs, which is remarkably high.42 The polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles were most likely the first polyhalogenated natural products positively tested as AH receptor ligands.42 The relative high response of Br4Cl2–DBP (3) to the AH receptor was unexpected due to the nonplanarity of the molecule (cf. section VI.D). However, in recent years, other nonplanar compounds were also found to bind to the AH receptor. By contrast, Q1 (20) was tested negative in the EROD assay (~107 less potent than TCDD) and also in the sulforhodamine B assay.67 Comprehensive standard pesticide tests on the insecticidal, herbicidal, and fungicidal activity of Q1 (20) were also negative even at the highest doses.67 It appeared that the compound was completely biologically inactive. These documented differences between Br4Cl2-DBP (3) and Q1 (20) can be attributed to various reasons. The 1,2′-bipyrrole backbone (compared to 2,2′-bipyrroles) could play a role as could the absence of bromine substituents in the case of Q1 (20). In addition, the slightly better water solubility of polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles compared to polyhalogenated 1′-methyl-1,2′-bipyrroles (Table 6) could also have an effect. Additional effects may arise from the N-methyl substituents. Clearly, the introduction of N-methyl groups will lower the polarity and will result in the formation of stable atropisomers. For instance, the more polar, structurally related pentabromopseudilin (14) proved to be antibiotic and cytotoxic.72,139 Moreover, pentachloropseudilin (15)

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was shown to act as a reversible and allosteric myosin ATPase inhibitor in mammalian cells.140 Martin et  al. investigated the allosteric myosin ATPase inhibition of different pentabromopseudilin (14) derivatives.25 They found that the O-methyl-pentabromopseudilin and the N-methylpentabromopseudilin (16) were much weaker inhibitors than pentabromopseudilin (14) (Figure 20).25 Moreover, the bioactivity decreased in the order pentabromo > dichlorotribromo > pentachloropseudilin (see section I,VI,IV, Figure 20).25 It would be interesting to test whether such different bioactivity is also found in other tests. Indications for antipredatory chemical defense properties were observed with tetrabromo-1-methylpyrrole (32).91,95 Actually two fish species (Leiostomus xanthurus and Fundulus heteroclithus) rejected the consumption of squid when tetrabromo-1-methylpyrrole (32) was present in the tissues.95 However, crabs (Callinectes similis) spiked with 32 were not rejected.95 Comparable results were also reported for polybrominated pyrroles. The acorn worm Saccoglossus otagoensis, which contains high concentrations of tribromopyrrole, is not readily predated by fish.32,141 Pellets spiked with 2,3,4-tribromopyrrole (34) administered to predatory fish had a deterrent effect, and only six of 14 fish actually consumed the pellets.91 The unpalatability of S. otagoensis to flounder was attributed to the “iodoform-like” flavor of tribromopyrrole.141 Attempts to detect the bromopyrroles in fish tissue were not undertaken.

100

Myosin 2 ATPase activity [%]*

16

80

60

15

40

20

0

14

I

II

III

IV

V

VI

VII

VIII

Figure 20  Inhibition of skeleton muscle myosin-2 ATPase activity relative to control (no inhibitor added = 100%). Data from Martin et al.25

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Reineke et  al. conducted a bacterial bioluminescence inhibition assay with the marine bacterium Vibrio fischeri and tested individual bromoindoles and selected water extracts.109 Bromoindoles proved to be strong inhibitors of bacterial luminescence.109 The lowest EC50 value (and thus the highest toxicity) was found for the unnatural 5-bromoindole (0.08 mg/L).109 The 4-bromoindole (41) and 4,6-dibromoindole (46) showed the lowest EC50 (1.0 ± 0.2 mg/L) of the congeners detected in water and sediments.109 Sample extracts containing highest concentrations of organobromine compounds were tested as well, but the effects could not be equivocally assigned to the polyhalogenated alkaloids.109 A broader spectrum of substances was expected to be responsible for the observed effects.109 Likewise, the zebrafish (Danio rerio) embryo test was performed with bromoindoles according to Kammann et al.142 Sediment extracts in which the brominated compounds (bromophenols and bromoindoles) were detected showed teratogenic activity in the zebrafish test. Reported EC50 values in the two samples with the highest content of bromoindole and bromophenol content showed 98% mortality versus 0% mortality plus 98% malformations (mostly in the form of yolk sac edema).109 Overall, the toxicity of the brominated indoles was considered low.109 In addition, 2,3,6-tribromo-1-methylindole (51), 2,3,5-tribromo-1-methylindole (50), 2,3,5,6-tetrabromo-1-methylindole (52), and 2,3,5,6-tetrabromoindole (49) were tested for antibacterial activity against Bacillus subtilis and Saccharomyces cerevisiae.103 However, only the (nonmethylated) 2,3,5,6-tetrabromoindole (49) was active in this test. This again highlights the impact of the N-methyl substitution on the properties of the polyhalogenated alkaloids.

IX. FINAL REMARKS AND CONCLUSIONS For polyhalogenated 1,1′-dimethyl-2,2′-bipyrroles and 1′-methyl-1, 2′-bipyrroles, as well as tetrabromo-1-methylpyrrole (32), the natural producers are still unknown. However, their natural origin cannot be disputed anymore. Especially for the polyhalogenated 1,1′-dimethyl-2,2′bipyrroles and 1′-methyl-1,2′-bipyrroles log KOW values and analysis of different trophic level biota suggests persistence and bioaccumulation in the marine food web (an exception may form pinnipeds, cf. section II.D.1). In the 1970s, when the marine environment was most heavily polluted with anthropogenic POPs such as DDT and PCBs, residues of 50–70 mg/kg lipids PCBs were made responsible for reproductive failure in seals.143 The melon-headed whale (Peponocephala electra) sample from Japan with the highest concentration of polyhalogenated alkaloids has reached this target level merely by the two key compounds Br4Cl2-DBP

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(3) (42.3 mg/kg) and Q1 (20) (7.62 mg/kg).39 By contrast, tetrabromo-1methylpyrrole (32) as well as bromoindoles appear to be less bioaccumulative. The backbone of the former might be too small while the latter might be not bioaccumulative due the low log KOW values. This issue again raises the question as to the particular role of methyl groups on the nitrogen(s). Still, the concentrated release of bromoindoles into the marine environment might lead regionally to toxic effects. Consequently, halogenated natural products including the polyhalogenated alkaloids have to be considered as common residues in marine organisms. This new view on environmental contaminants was addressed by Ballschmiter’s classification of polyhalogenated compounds into144: (i) biogenic polyhalogenated compounds (including the polyhalogenated alkaloids), (ii) natural/geogenic (probably specific halogenated dibenzo-p-dioxins in clay), (iii) nonhalogenated precursors (e.g., phenols that are halogenated in the environment), (iv) halogenated precursors (halophenols that are converted into haloanisoles), and (v) anthropogenic polyhalogenated compounds (chloropesticides, PCBs). Although residues of polyhalogenated alkaloids and other halogenated natural products are frequently detected in marine biota (cf. sections II–V), the knowledge of their environmental role lags behind that of anthropogenic POPs. For instance, the modeling of the distribution of polyhalogenated alkaloids has not been thoroughly carried out because the halogenated natural products do not fit with the current environmental models. Their global natural production is difficult to assess due to the uneven distribution with hotspots and seasonal and annual variations in their occurrence. On the one hand, it was suggested that the biogenic compounds may have initiated the evolutionary development of halorespiring bacteria, which in turn enabled the partial transformation of anthropogenic POPs.145 On the other hand, like the anthropogenic pollutants, polyhalogenated alkaloids and other halogenated natural products may also pose pronounced hazards to the marine environment and ultimately to humans.146 Whether this hazard translates to risk depends on factors such as exposures and persistence in the marine environment.146 Moore et al. pointed out that we live in a rather ‘data-rich’ environment with regard to anthropogenic organohalogen compounds as opposed to a ‘data-poor’ environment when dealing with our understanding of naturally produced compounds.146

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