Bioactive Aliphatic Sulfates from Marine Invertebrates

The occurrence of sulfated steroids and phenolics in marine organisms is quite widespread, being typically reported from Echinoderms. In contrast, alkane and alkene aliphatic sulfates are considerably rarer with examples being reported from a diverse array of organisms including echinoderms, sponges and ascidians. While no ecological roles for these metabolites have been proposed, they do exhibit a diverse array of biological activities including thrombin inhibition; the ability to induce metamorphosis in larvae; antiproliferative, antibacterial and antifungal properties; and metalloproteinase inhibition. Of particular interest and an avenue for future development is the finding of antifouling properties with low or nontoxic effects to the environment. This review focuses on alkyl sulfates and related sulfamates, their structures and biological activities. Spectroscopic and spectrometric techniques that can be used to recognize the presence of sulfate groups are also discussed, data for which will enhance the ability of researchers to recognize this class of chemically- and biologically-interesting marine natural products.


Introduction
Sulfur is the sixth most abundant element and its metabolism is critically important to global biogeochemical cycles. Sulfate is a universal electron acceptor in marine environments because of its high abundance and stability in seawater [1]. Sulfated compounds belonging to the classes of triterpenes, steroids and phenolics are widely distributed in marine organisms with sulfated steroids constituting the largest subset of these secondary metabolites [2,3]. The echinoderms including ophiuroids, sea stars, sea cucumbers and sea urchins are a rich source of sulfated metabolites, especially sulfated saponins (steroidal and triterpenoid) and sulfated aglycone steroids. Ophiuroids are characterized by containing polar sulfated steroidal polyols and a lack of saponins [4,5] while, in marine sponges, these metabolites are frequently found as steroidal and phenolic sulfates [6]. Therefore, the distribution of these compounds is extremely different among the phyla, namely Porifera, Echinodermata, Mollusca and Urochordata, where they have been described [7].
However, sulfated alkanes and alkyls are rarer, with only a few aliphatic sulfates having been reported from marine organisms [4,6,8]. These compounds have been isolated from echinoderms, summarized. It is anticipated that these data will help facilitate dereplication of these natural product lipids (Tables 2-14). With the information compiled in this review, our goal is to encourage researchers in the area of marine natural products in search of these rare compounds with a prior knowledge of their biological and ecological potential to extend their research to other biological activities not yet investigated for these metabolites.  Asterias forbesi [32] No activity Dissulfato 5 (2) Asterias forbesi [32]

Biological Activity of Aliphatic Sulfates Compounds
Concerning the biological potential of the set of substances analyzed in this review, it was observed that 45% of them showed metamorphosis-inducing activity, highlighting the important role of these substances as kairomones in the interaction with other species. This is followed by cytotoxicity (25%) and antimicrobial (14%) activities ( Figure 3). In total, 14 sulfates were tested for in vitro toxicity and the cytotoxicity potential of aliphatic sulfates was expressive against several types of carcinoma. The panel of strains could be expanded and the remaining compounds remain to have an undisclosed cytotoxic potential. The knowledge of the antimicrobial potential of aliphatic sulfates is still insipient, since few strains were investigated and the method used was restricted to zones of growth inhibition measurement. Unfortunately, sulfates 1, 2, 23 and 24 have not been tested for any biological activity and, therefore, their biological potential remains unknown. The biological activities of aliphatic sulfates mentioned in this review are outlined in more detail in the following section. of carcinoma. The panel of strains could be expanded and the remaining compounds remain to have an undisclosed cytotoxic potential. The knowledge of the antimicrobial potential of aliphatic sulfates is still insipient, since few strains were investigated and the method used was restricted to zones of growth inhibition measurement. Unfortunately, sulfates 1, 2, 23 and 24 have not been tested for any biological activity and, therefore, their biological potential remains unknown. The biological activities of aliphatic sulfates mentioned in this review are outlined in more detail in the following section.

Thrombin Inhibition
The enzyme thrombin is an important target for the treatment of thrombosis and related diseases, which are responsible for deaths and incapacity [36]. The toxadocials A-C and toxadocic acid A (3-6) were isolated from the marine sponge Toxadocia cyhdrica. Toxadocials (3-6) are a rare class of natural products with one carbaldehyde group in the middle of an alkyl chain. The biosynthesis of these compounds is proposed to involve an aldol condensation of two units of hydroxylated or sulfated aldehydes followed by dehydration and reduction. All four natural products exhibited inhibition of thrombin with IC50 of 6.5, 4.6, 3.2 and 2.7 µg/mL, respectively. The authors suggested that the activity was associated with the presence of the sulfate esters. [17,33].

Morphological Inducing Defense
Some sulfated compounds seem to mediate interspecific interactions among aquatic species. According to Yasumoto et al. [9][10][11][12], different sulfated alkanes and alkenes (7, 9, 11-12, 19-22, and 26-40) can induce a morphological defense of phytoplankton. These compounds are known as kairomones since their production by marine grazers works as a semiochemical that induces morphological changes in phytoplankton species including the unicellular Scenedesmus subspicatus [37]. When the grazer Daphnia magna is present in the water, S. subspicatus changes its reproductive mode, forming colonies of two, four and eight cells, which, in addition to the production of spines, are strategies that can increase the chances of S. subspicatus escaping consumption.

Thrombin Inhibition
The enzyme thrombin is an important target for the treatment of thrombosis and related diseases, which are responsible for deaths and incapacity [36]. The toxadocials A-C and toxadocic acid A (3)(4)(5)(6) were isolated from the marine sponge Toxadocia cyhdrica. Toxadocials (3-6) are a rare class of natural products with one carbaldehyde group in the middle of an alkyl chain. The biosynthesis of these compounds is proposed to involve an aldol condensation of two units of hydroxylated or sulfated aldehydes followed by dehydration and reduction. All four natural products exhibited inhibition of thrombin with IC 50 of 6.5, 4.6, 3.2 and 2.7 µg/mL, respectively. The authors suggested that the activity was associated with the presence of the sulfate esters. [17,33].

Morphological Inducing Defense
Some sulfated compounds seem to mediate interspecific interactions among aquatic species. According to Yasumoto et al. [9][10][11][12], different sulfated alkanes and alkenes (7, 9, 11-12, 19-22, and 26-40) can induce a morphological defense of phytoplankton. These compounds are known as kairomones since their production by marine grazers works as a semiochemical that induces morphological changes in phytoplankton species including the unicellular Scenedesmus subspicatus [37]. When the grazer Daphnia magna is present in the water, S. subspicatus changes its reproductive mode, forming colonies of two, four and eight cells, which, in addition to the production of spines, are strategies that can increase the chances of S. subspicatus escaping consumption.
Life-history changes are also induced in the sea-flea Daphnia pulex when exposed to kairomones produced by a predatory midge larvae of the genus Chaoborus. Individuals exposed to kairomones produced more defensive structures and released neonates more quickly than control individuals [38]. It is interesting to note that these induced responses were impaired by the presence of waterborne copper 2 + ions. Weiss at al. (2018), in their studies of Chaoborus kairomone chemicals inducing defenses in Daphnia, found infochemicals from active digestion, consisting of fatty acids conjugated to the amino group of glutamines via the N-terminus. These cues are involved in Chaoborus digestive processes, which explains why they are consistently released despite the disadvantage for its emitter [27].

Cytotoxicity
Marine natural products can exhibit high levels of cytotoxic activities. In general, alkyl sulfates isolated from marine ascidians have simple structures derived from polyketides and, in some cases, derived from isoprene, with frequently associated cytotoxic and antiproliferative activities [15]. The normoterpenoid 7, and the sulfates 16 and 17 have been described as cytotoxic towards the murine fibrosarcoma cell line (WEHI) with IC 50 20.9, 15.0 and 12.2 µg/mL, respectively [14]. Compounds 13, 14 and 15 were evaluated against different types of carcinoma, such as human melanoma (IGR-1), murine macrophage (J774), murine fibrosarcoma (WEHI 164) and murine leukemia (P388) and presented only weak cytotoxicity with IC 50 between 50 and 370 µg/mL [6,16]. Other sulfate alkenes, such as 25, were weakly active against leukemia cells (P388) [5]. Furthermore, the sulfated alkanes 45 and 46 were active in BALB/c murine macrophages cells (J774A.1) at 100 µM [15]. The presence of a sulfate group at C-18 and/or the hydroxyl group at the end of the chain of 45 and 46 seems to be essential for the cytotoxic activity, since compounds without these groups did not exhibit biological activity. The chain length also seems to influence activity since compound 45 (C 19 ) was more active than compound 44 (C 20 ) against cells J774A.1 [15]. In the same way, the sulfates 41-43 and 30 isolated from sea cucumber Apostichopus japonicus exhibited pronounced cytotoxicity against lung adenocarcinoma human cells line A549 with IC 50 0.063, 0.062, 0.064 and 0.065 µM, respectively [13]. The sulfated alkenes (5Z)-decenyl sulfate (43) and (3E)-decenyl sulfate (30) were isolated from the sea cucumber Apostichopus japonicus and showed potent activity against osteosarcoma cells (MG63) with IC 50 values of 0.064 and 0.057 µM, respectively [13].

Antimicrobial Activity
Antibacterial and antifungal activities were reported for 7-10, isolated from the hepatopancreas of the ascidian Halocynthia roretzi, with zones of growth inhibition (12 mm and 10 mm) for all the compounds at concentration of 0.2 mg/disk against Vibrio alginoliticus and Mortierella ramaniana, respectively [8]. The sulfates 41-43 and 30 isolated from sea cucumber Apostichopus japonicus also showed potent antibacterial action against Escherichia coli at 0.05 µg/disk, with compounds 43 and 30 exhibiting zones of growth inhibition of 13 and 15 mm, respectively [13]. Thus, indicating a selective action to Gram-negative bacteria, which has a double phospholipid membrane that protects the cell wall, which rendered the entry to the cell more difficult [39] Additionally, compounds 43 and 30 presented a growth inhibition zone of 8 mm when evaluated against the fungal Septoria trici. Those data led to the conclusion that the double bond at the carbon chain improved the biological activity in the case of compounds 43 and 30, when compared mainly with 42, which has the same number of carbons.

Metalloproteinase Inhibitor
Metalloproteinases are correlated to various physiological processes, such as extra-cellular matrix degradation and tissue remodeling. Problems with the regulation of these enzymes can lead to pathological conditions, such as cancers, cardiac, cartilage, and neurological problems, making metalloproteinase inhibitors of interest [40]. Sodium 1-(12-hydroxy) octadecanyl sulfate (18) was isolated from a marine tunicate of the family Polyclinidae and is structurally related to the forbesins [32].

Other Activities
The alkyl sulfates 7-10 were isolated from the hepatopancreas extract of the ascidian Halocynthia roretzi, suggesting that they may play some physiological role in the digestive system of these ascidians. Studies regarding the marine sponge Callyspongia truncata led to the isolation of compounds callyspongins A (11) and B (12). These polyacetylene sulfated compounds inhibited the fertilization of starfish gametes. Compound 11 was particularly potent, inhibiting sperm mobility at 50 µM and prevented fertilization at 6.3 µM. Compound 12 was weaker in its biological activity, inhibiting sperm mobility at 100 µM and blocking fertilization at 50 µM, highlighting the ecological role of these compounds in the interaction with other species [35]. No mode of action studies of 11 and 12 have been reported.

Chemical Characterization of Aliphatic Sulfates
To date, 38 natural alkyl sulfates and eight related sulfamates have been reported from marine invertebrates. In this review, the 1 H and 13 C NMR data reported for these natural products are tabulated (Tables A1-A13) to make it easier for research groups to recognize the presence of these metabolites in marine samples. The chemical shifts of hydrogen and carbons near the point of sulfation are characteristically different in sulfated and non-sulfated compounds, allowing the distinction between sulfated compounds and their respective alcohols.
Compounds containing sulfate groups typically have a strong absorption in the IR spectrum at 1350-1450 cm −1 . In addition, the presence of the sulfate group can be determined by the sodium rhodizonate test. Sodium rhodizonate gives an orange-red colored complex with the Ba 2+ ion. The absorbance of the red-colored complex is measured at 520 nm. In the presence of sulfate, BaSO 4 precipitate forms, and, consequently, the color and the absorbance value decrease [41]. Mass spectrometry can also be useful for detection of sulfate, with the presence of the [M − H] − = m/z 97 ion detected in negative ion mode ESIMS, suggesting the presence of the sulfate group in the structure.
Another method used to confirm the presence of sulfate substitution is solvolysis to afford the corresponding alcohol [16]. One method for this is to heat the sulfate in dioxane at 100 • C for 4 h [24] or to heat the sulfate in dioxane-piridine mixture (1:1) at 130 • C for 3 h [14]. Subsequent analysis of the alcohol-containing product, particularly concerning adjacent methylene protons, can lead to evidence of the presence of a sulfate group in the original natural product. Methylene groups bearing the sulfate moiety exhibited an increased 1 H NMR chemical shift when compared to the chemical shifts of the corresponding alcohol. The combination of this downfield shift of methylene 1 H NMR signals and a higher than expected polarity of the original sample may be indicative of the presence of a primary alcohol esterified by sulfuric acid [4].
Considering the unsaturated compounds, 10 of them have a single double bond (5, 15, 17, 19, 28,  30-32, and 43), as well as the sulfamate 33. Eight structures pointed to having two double bonds in their skeletons, these being the sulfates 4, 8-9, 20, and 23-25, including the sulfamate 34. Compounds 11 and 12 are distinguished by the presence of five triple bonds and are polyacetylene sulfates.
On the other hand, the chemical structures of compounds 2-6, 11-14, and 44-45 called attention to the presence of more than one sulfate group in the main carbon chain.
By comparing the lengths of the carbon chains, compounds 16 and 41 are found to have the smallest chain-containing eight carbons atoms-contrasting with the longer carbon chains with 50 C-atoms present in compounds 2-4.
Compounds 23-25, isolated from the Echinus Temnopleurus hardiwickii, present the peculiar presence of both sulfate and ammonium ions; while sulfated alkenes are common in marine invertebrates, the occurrence of complex counterions are rare [5]. The trimethylammonium moiety in 23 was confirmed by HRESIMS. The E configuration of the double bond in C-3 was assigned on the basis of the H-3/H-4 coupling constant (15.4 Hz) and the 13 C NMR chemical shifts of C-2 (δ 32.6) and C-5 (δ 36.2). The absolute configuration of 23 was determined through oxidative degradation.
The peculiar polyacetylene sulfates, callyspongins A (11) and B (12), were isolated from the marine sponge Callyspongia truncate. The presence of an ene-diyne unit was suggested by the UV spectrum that showed absorption maxima at 267 and 287 nm (ε = 8900 and 7100, respectively). The absolute configuration of C-2 in 11 was determined to be R, after hydrolysis and comparison of the optical rotation data and spectroscopic data of the product obtained with siphonodiol, isolated from Siphonochalina truncata [35].
On the other hand, the toxadocials are distinguished by the presence of multiple sulfate units in their chemical structures, as supported by the IR band (1250 cm −1 ), by positive sodium rhodizonate test and by a deshielded methine signal for H-7, H-17, H-31 and H-41 (δ 4.31, quint, J = 7 Hz; δ C 81.0) in toxadocial A (3), for example. Toxadocial A was also hydrolyzed to afford the respective tetraol.
In addition to the multiple sulfates, the toxadocials have the aldehyde function (δ H 9.51, d, δ C 207.6) together with the alkyl chains [17].
As presented, the chemistry of marine invertebrates, especially the ascidians, has drawn attention due to the peculiarity of its metabolites. Considering sulfated aliphatic molecules, some of these compounds occur in relatively remarkable quantities in ascidians, being a sign of the importance of these metabolites for these organisms and among living organisms in aquatic ecosystems. Consequently, their distribution in a marine environment, quantitative studies involving the biosynthesis of these compounds, the evaluation of the biological potential and the participation of these compounds as kairomones are approaches that need to be better understood, since the contribution of sulfate groups seems to be determinant of the biological activity and interactions between species.

Conslusions
Alkyl sulfates constitute an interesting group of marine natural products that have been reported from organisms from a diverse range of Phyla (Echinodermata, Porifera, Chordata and Mollusca). The natural products cover a structural diversity that encompasses differences in stereochemistry, chain length, the presence of unsaturation and number of sulfates present in the molecular structure. The association of different biological activities with this structural class make them of particular interest. Among these properties, the ability of alkyl sulfates to exhibit ecologically-relevant morphological, metamorphosis-inducing and antifouling activities makes them attractive for further study. The relative ease of detection of the presence of the sulfate substituent(s), via negative ion ESI MS and IR spectroscopy, combined with microscale hydrolysis to the corresponding alcohol and subsequent 1H NMR chemical shift comparative analysis, means these rare marine natural products should be more readily recognized in chromatography column fractions by natural product chemists. In this respect, the compilation of NMR data may be valuable for a quick confirmation of the presence of these compounds in marine species. The discovery of new examples of alkyl sulfate natural products, combined with synthesis, will accelerate the ability to undertake further studies and perhaps uncover new biological functions for this specific class of metabolites.
Author Contributions: All authors contributed equally to the analysis of data from the references and the preparation of the manuscript. All authors approved the final text.

Conflicts of Interest:
The authors declare no conflict of interest.

Appendix A
a Signals with identical letters may be interchanged.