*2.1. Brominated Indole Derivatives*

The hypobranchial gland of muricid mollusks is the source of the ancient dye Tyrian purple, for which the main pigment is well established to be a brominated derivative of indole, 6,6 dibromoindigo (**6**, Figure 1) [1–3]. Original observations of the hypobranchial glands confirmed that the dye pigment itself is not present in the live mollusk, but rather is generated after a series of enzymatic, oxidative and photolytic reactions. In 1685, Cole [53] first described the changes in the hypobranchial glands of muricid mollusks, from a white fluid to yellow, through various shades of green and blue, before obtaining the final purple color after exposure to sunlight. This series of color reactions was also noted by Baker [1,8,9] in the hypobranchial glands from the Australian species *D. orbita*; illustrated in Figure 1. The indole precursors span a range of chemical properties (Table 1a) from the water soluble salt of tyrindoxyl sulfate (M.W. 337, 339, log *p* < −0.3) to the highly insoluble tyriverdin (M.W. 514, 516, 518, log *p* > 4.6).

Baker and Sutherland [8] first isolated a salt of tyrindoxyl sulphate (**1**, Figure 1) from an ethanol extract of the hypobranchial gland of *D. orbita* and identified this as the ultimate precursor to the dye Tyrian purple. They also isolated an enzyme with sulfatase activity capable of hydrolyzing tyrindoxyl sulfate and initiating the production of Tyrian purple by exposure to sunlight [8]. Baker and Duke [6,7,10,11] subsequently isolated and identified the intermediate precursors tyrindoxyl (**2**) and tyrindoleninone (6-bromo-2-methylthio-3*H*-indol-3-one) (**3**), as well as tyrindolinone (**4**), a methanethiol adduct of tyrindoleninone (Figure 1a). Using various organic solvents, Baker and Sutherland were also able to isolate a yellow light insensitive compound identified as 6-bromoisatin, and the immediate precursor to Tyrian purple, a green light sensitive compound tyriverdin [8]. The structure of tyriverdin (**5**, Figure 1) was subsequently corrected by Christophersen *et al.* [54] as an indole dimer that forms spontaneously from the reaction between tyrindoxyl and tyrindoleninone (Figure 1a). 6-Bromoisatin (**8**, Figure 1) is considered to be an oxidation artifact in this sequence of reactions [2,8] and is a precursor of the red Tyrian purple isomer 6,6 -dibromoindirubin (**7**) [55]. These oxidation products do occur naturally in small amounts of the extracts from males, but were not detected in female *D. orbita* hyprobranchial gland and gonad extracts (Figure 1b), suggesting sex specific differences in the chemical environment of these glands [13].


**Table 1.** Molecular properties of (**A**) brominated indoles and (**B**) choline esters isolated from *Dicathais orbita* using Molinspiration Cheminformatics (2012). Molecular weight for Br79 isotopes.

(**B**) <sup>a</sup> Log *p* is based on octanol-water partition coefficient; <sup>b</sup> H bond acceptors include O & N atoms; <sup>c</sup> H bond donors include OH and NH groups; <sup>d</sup> Rule of 5 violations are based on the molecular descriptors used by Lipinski *et al.* [56] for "drug-like" molecules (log *p* ≤ 5, molecular weight ≤500, number of hydrogen bond acceptors ≤10, and number of hydrogen bond donors ≤5).

An interesting point of difference in *D. orbita* indole chemistry, relative to other Muricidae, is the production of a single brominated ultimate precursor molecule [2,8,57]. Four prochromogens including brominated and nonbrominated indoxyl sulfates have been suggested for *Murex brandaris* [58], which then generate a mixture of purple 6,6 dibromoindigo, as well as blue indigo and monobromoindigo [2]. Baker [1] also demonstrated the complexity of purple precursors obtained from the hypobranchial glands of some other Muricidae species. These Tyrian purple precursors are also transferred to the egg masses of *D. orbita* (Figure 1b) and other Muricidae mollusks [12,59]. Similar to the hypobranchial glands, the egg masses of other Muricidae were found to contain a more complex mixture of brominated and non brominated indole, as well as other brominated compounds including imidazoles, quinolones and quinoxalines [17,60,61]. Consequently, the Australian species *D. orbita* appears to be a particularly pure source of 6,6 dibromoindigo and the simplicity of the single precursor make it a good model for biosynthetic studies of brominated indoles. On the other hand, the diversity of indoles and brominated compounds in the Muricidae family more broadly provides a good opportunity for phylogenetic investigations into the evolution of secondary metabolism.

#### *2.2. Choline Esters*

In 1976, Baker and Duke made an important breakthrough when they isolated choline from the hypobranchial glands of *D. orbita* and demonstrated that tyrindoxyl sulfate is stored as a choline ester salt [7]. This salt is hydrolysed by an arylsulphatase enzyme, which is also stored within the hypobranchial gland [8], to generate the intermediate precursors of Tyrian purple (Figure 1a). Both choline, and to a lesser extent murexine (β-imidazolyl-4(5)acrylcholine) (Table 1b) were found to be associated with tyrindoxyl sulfate [7]. *N*-Methylmurexine was also suggested to be present in the hypobranchial gland extracts [7], but this was subsequently questioned by Duke *et al*. [62,63].

In 1996, Roseghini *et al*. [64] reported a survey of choline esters and biogenic amines from the hypobranchial glands of 55 species of gastropods. *Dicathais* (*Neothais*) *orbita* was found to contain significant quantities of murexine and senecioylcholine (Table 1b). Dihydromurexine was the dominant choline ester found in some other Muricidae species, but was not detected in *D. orbita* [64]. Shiomi *et al.* [65] have also identified tigloylcholine (Table 1b) in other muricids from the genus *Thais*. These authors pointed out that senecioylcholine is a structural isomer of tigloylcholine and since senecioylcholine was only previously identified by thin layer chromatography and is indistinguishable from tigloylcholine using this method, it may have been misidentified in the earlier studies [65].

### *2.3. Mycosporine-Like Amino Acids, Fatty Acids and Sterols in the Egg Masses*

In addition to reports on the indole derivatives in *D. orbita* egg masses [60,66], the composition of mycosporine-like amino acids (MAAs) and fatty acids has been documented for this species. MAAs are small sunscreening compounds with an absorption maxima of 310–360 nm [67]. They are produced via the shikimate pathway in algae, fungi and bacteria, but animals, including marine invertebrates, are thought to acquire these secondary metabolites through diet or symbiosis [67,68]. Przeslawski *et al*. [69] revealed that mycosporine-glycine and shinorine were the dominant MAAs in *D. orbita*, along with porphyra-334 and mycosporine-2-glycine and trace amounts of palythine. Mycosporine-taurine, palythene, asterina-330 and palythinol were not detected in this species, although an additional unknown peak with an absorption maxima of λ 307 nm was reported in *D. orbita*, along with two other Muricidae [69]. The composition of MAAs was found to be strongly influenced by phylogeny in molluskan egg masses, but not by the adult diet or levels of UV exposure in the spawning habitat [69]. This suggests that predatory marine mollusks, such as *D. orbita*, are able to bioaccumulate MAAs from their prey and transfer these into the egg masses to protect their developing embryos. Higher MAA concentrations were found in *D. orbita* egg masses with viable embryos in comparison to inviable egg masses [69]. The inviable eggs of *D. orbita* typically appear pink or purple in color, as opposed to the usual yellow color [59], thus indicating further chemical changes, likely due to the photolytic degradation of Tyrian purple precursors. By absorbing UV radiation in normally developing Muricidae

egg masses, MAAs may play an essential role in maintaining the bioactive indole precursors prior to larval hatching. Alternatively, by absorbing in the UV spectra [13,27], the brominated indoles may provide further protection against harmful UV rays.

In a comparative study of lipophylic extracts of the egg masses from a range of molluskan species, Benkendorff *et al*. [70] revealed that *D. orbita* egg capsules predominately contain palmitic and stearic acid. Unlike many other gastropod egg masses, no unsaturated fatty acids were found in the leathery egg capsules of *D. orbita* and related neogastropods [70]. The extracts from *D. orbita* egg masses contained a large amount of sterol, predominately cholesterol, but with smaller amounts of cholestadienol, cholestanol, methyl cholestadienol and methylcholestenol [70]. No cholestadiene or stigmatenone were found, although some unknown sterols were detected. It is unclear why Neogastropoda with leathery egg capsules, such as *D. orbita*, have a much higher saturated fatty acid and sterol content than gastropods with gelatinous egg masses, although the later may require unsaturated fatty acids to maintain fluidity in the gelatinous matrix.
