Amino Acid-Coupled Bromophenols and a Sulfated Dimethylsulfonium Lanosol from the Red Alga Vertebrata lanosa

Vertebrata lanosa is a red alga that can commonly be found along the shores of Europe and North America. Its composition of bromophenols has been studied intensely. The aim of the current study was therefore to further investigate the phytochemistry of this alga, focusing more on the polar components. In total, 23 substances were isolated, including lanosol-4,7-disulfate (4) and the new compounds 3,5-dibromotyrosine (12), 3-bromo-5-sulfodihydroxyphenylalanine (13), 3-bromo-6-lanosyl dihydroxyphenylalanine (14), 3-(6′-lanosyl lanosyl) tyrosine (15) and 5-sulfovertebratol (16). In addition, 4-sulfo-7-dimethylsulfonium lanosol (7) was identified. While, in general, the dimethylsulfonium moiety is widespread in algae, its appearance in bromophenol is unique. Moreover, the major glycerogalactolipids, including the new ((5Z,8Z,11Z,14Z,17Z)-eicosapentaenoic acid 3′-[(6′’-O-α-galactopyranosyl-β-D-galactopyranosyl)]-1-glycerol ester (23), and mycosporine-like amino acids, porphyra-334 (17), aplysiapalythine A (18) and palythine (19), were identified.


Introduction
Vertebrata lanosa (L.) T.A. Christensen, formerly known as Polysiphonia lanosa (L.) Tandy, is a red alga belonging to the family of Rhodomelaceae. It is distributed commonly around the Atlantic shores of Europe and North America and the Baltic Sea [1], where it grows as an epiphyte on Ascophyllum nodosum [2]. Due to its characteristic aroma, it can be used as a spice [2], but also, the application of extracts as cosmetic ingredients is increasingly popular [3]. According to its widespread occurrence and usage, several studies have been conducted to investigate the phytochemical composition of V. lanosa, most of which focused on bromophenols. A series of different hydroxylated bromophenol derivatives, particularly lanosol (2,3-dibromo-4,5-dihydroxybenzyl alcohol) and similar benzyl alcohols or benzaldehydes, has been isolated [4][5][6][7]. These also included dimeric [6,7] or even tetrameric structures [7], as well as bromophenols with less common scaffolds, such as rhodomelol and methylrhodomelol [6,8], or vertebratol, which is composed of a dibromodihydroxybenzyl moiety linked to ornithin via an ureido group [6].
Regarding their bioactivities, selected lanosol derivatives [5,6] and dimeric bromophenols [6] were assayed for their antimicrobial properties against different Gram-positive and Gram-negative bacteria, but the inhibition of bacterial growth was either rather weak or absent [5,6]. Cytotoxic effects have been observed for certain bromophenolic compounds enriched in the dichloromethane fraction of a methanolic extract [9] but not for all bromophenols from V. lanosa in general [7].
Despite the numerous phytochemical investigations, much less is known about the more polar secondary metabolites of the alga. For example, only one sulfated bromophenol, namely 2,3-dibromo-5-hydroxybenzyl-1 ,4-disulfate, has been reported to be present in a hydroethanolic extract from V. lanosa [10] and was isolated as dipotassium salt [11,12].
Further, the galactan sulfate of the red alga has been characterized as a polysaccharide of the agar class, containing methylated and sulfated units of D-and L-galactose [13]. The high quantities of the polysaccharide [13] could possibly contribute to the use of V. lanosa extracts in cosmetics as a moisturizing component [3,14]. Moreover, the presence of mycosporine-like amino acids (MAA) [15], which are known for their very efficient absorbance of UV radiation [16,17], might have an additional value in the seaweed extract for cosmetical purposes. Similar to the bromophenols [18], seasonal variations of MAA patterns in red algae [15] have to be considered. The composition of MAA in V. lanosa has been investigated using LC-ESI-qTOF-MS, resulting in the identification of several known compounds, such as porphyra-334 or palythine. In addition, the presence of MAAs previously undescribed for this species was also indicated [15], however, no MAAs have been isolated from V. lanosa for structure elucidation until now.
The aim of the current study was, therefore, to further characterize the phytochemical composition of V. lanosa, with a focus on the more polar compounds in particular, including the major MAA of the alga. Partitioning of a methanol-water extract between water and ethyl acetate led to an enrichment of sulfated bromophenols and MAAs, with a high amount of polysaccharides. Thirteen compounds were isolated from the aqueous phase, including three new sulfated bromophenols and one bromophenol carrying a unique sulfonium moiety. Their structures were elucidated by means of mass spectrometry and NMR spectroscopy. Furthermore, one new galactolipid was obtained from the ethyl acetate partition, together with eleven known bromophenols and galactolipids.
The fragmentation of 7 in positive-and negative-mode HR- , suggesting a sulfonium moiety in the side chain. The presence of two bromine atoms could also be deduced from the isotopic pattern in the intensity ratio of 1:2:1. An analysis of the 1 H-, 13 C-and 2D-NMR spectra (Table 1 and Figure 2b) also supported a sulfonium substructure by the shifts of the benzylic carbon (δ C 50.16) and the methyl groups (δ C 25.28, δ H 2.97); as in the literature, similar shifts have been reported for sulfonium moieties [20]. HMBC correlations from the benzylic position to methyl groups C-8, C-9 and the aromatic carbons C-2 and C-6 confirmed the connection between the two subunits. The position of the sulfate moiety was assigned due to a stronger signal intensity of the aromatic proton to C-4 (δ C 141.40) in the HMBC spectrum in comparison to C-5 (δ C 151.20) and the shift similarity of the aromatic signals in the known metabolite 4 (Supplementary Materials) [19]. The structure of 7 was therefore established as 4-sulfo-7-dimethylsulfonium lanosol.   An analysis of the 1 H-, 13 C-and 2D-NMR data of 12-15 showed that all of them possess carboxylic carbons (δ C approx. 173) adjacent to a α-carbon with an amino function (δ C approx. 55) and a benzylic β-carbon (δ C approx. 35) ( Table 2), indicating a phenylalanine or tyrosine scaffold.  10 79 Br 2 NO 3 + ) suggested 12 to be a dibromotyrosine derivative, which was corroborated by the 1 H-NMR data being consistent with the literature values for synthesized 3,5-dibromo-L-tyrosine [21]. The configuration of α-carbon was assigned by the optical rotation ([α] 20 D = −67.7) in comparison to the literature data of tyrosine [22]. This is the first report of this compound being isolated from its natural source. HMBC correlations of the aromatic and the benzylic protons were used to elucidate the substitution of the phenyl ring: the β-protons showed intense HMBC correlations with the unsubstituted aromatic carbons (δ C 132.12 (C-2) and 123.92 (C-6)). H-2, in turn, strongly correlated to β-carbon; C-3; C-4 and C-6 (δC 35.37, 111.84, 146.47 and 123.92, resp.) and H-6 to β-carbon; C-2; C-4 and C-5 (δ C 35.37, 132.12, 146.47 and 140.47, resp.). Additionally, both protons showed weak four-bond correlations to the respective carbons. The sulfate moiety was determined to be attached to C-5 by the relative upfield shift of the respective carbon in comparison to unsubstituted phenolic carbons. The configuration of 13 was also determined by optical rotation ([α] 20 D = −10.4) and comparison to the literature data of tyrosine [22]. Hence, the chemical structure of 13 was found to be 3-bromo-5-sulfo-L-dihydroxyphenylalanine. Both 14 and 15 showed substituted aromatic carbons with similar shifts (C-6, δ C 128.62 and C-3, δ C 126.47, resp.) in their 13 C-NMR spectra. For both of these carbons, compared to unsubstituted tyrosine, the expected upfield shift in the ortho-position to the hydroxyl group was not apparent, suggesting aliphatic or benzylic substituents attached to the tyrosine scaffold. Further analysis of the HMBC correlations revealed the presence of either one (14) or two (15) lanosyl moieties attached to C-6 (14) or C-3 (15). The isotopic pattern of the [M -H] − ion of 14 (m/z 557.8233/555.8243/553.8265/551.8285) in the relative abundance of 1:3:3:1 indicated the presence of three bromine atoms in the molecule, suggesting a bromine substitution in the dihydroxyphenylalanine scaffold in addition to the lanosyl moiety. The position of the third bromine-substituted carbon was deduced from the strong upfield shift and the HMBC correlations from H-2 to C-3 (δ C 109.97) to be at position 3. Thus, the chemical structure of 14 was determined to be 3-bromo-6-lanosyl dihydroxyphenylalanine.
The second lanosol residue of 15 is connected to C-6 via its benzylic carbon. The assignment of certain carbons (δ C 113.94, C-3 and δ C 143.33, C-4 ) was not possible from HMBC correlations, since there are no protons in the molecular vicinity. Based on the substitution of the 2 ,3 -dibromo-4 ,5 -dihydroxybenzyl moiety and since phydroxybenzaldehyde is the biogenetic origin of the lanosol scaffold [23], we propose the reported assignments (see Table 2) and, therefore, the structure of 15 to be 3-(6 -lanosyl lanosyl)-tyrosine. The structures of compounds 10-15 are given in Figure 3. , which corresponds to vertebratol [6]. NMR data were mostly consistent with those of vertebratol [6], except for benzylic carbon (δ C 75.85), which was shifted about 30 ppm downfield, and the brominated aromatic carbons (δ C 114.34 and 112.70, see also Table 1). Once again, the position of the sulfate moiety was assigned by HMBC correlations of H-6 to C-4 and C-5 and, additionally, by comparison of the simulated spectra of the respective 4-and 5-sulfo derivatives. Thus, the structure of 16 was found to be 5-sulfovertebratol (Figure 2c). Due to the low yield, the configuration of 16 could not be determined.

Mycosporine-Like Amino Acids
The presence of several MAAs in V. lanosa has been reported recently, however, not all MAAs could be unambiguously identified by LC-MS [15]. Therefore, the aqueousphase W2 was further fractionated to obtain pure MAAs. Briefly, W2 was first depleted of polysaccharides by ethanolic precipitation and, subsequently, fractionated via cation exchange chromatography on Amberlite™ IR120 (H) resin. The fraction containing the highest amount of MAA (Amb9) was finally purified by preparative HPLC to obtain 17 (5 mg), 18 (1 mg) and 19 (1 mg). The structures of these MAAs are given in Figure 4a. To assess the purity of the compounds and effectiveness of the fractionation procedure, a HPLC method using a HILIC stationary phase was developed to facilitate the targeted isolation of the desired MAA. The isolated compounds correspond to porphyra-334 (17), palythine (18) and aplysiapalythine A (19), respectively, as determined by the comparison of HR-ESI-MS and NMR data (see Supplementary Materials) with the literature [24,25]. Compound 19 was distinguished from its isomer palythinol by the HMBC correlation of methylene protons H-1 (δ H 3.48 and 3.45) to C-1 (δ C 162.72) in the cyclohexenimine ring [24].

Discussion
Rhodomelaceae, especially the genus Vertebrata (syn. Polysiphonia), are known to frequently contain haloaryl derivatives [32], and a series of bromophenolic compounds was isolated from a hydroalcoholic extract of V. lanosa. Lanosol (1), representing the simplest structure, has been isolated from various red algae previously and is reported to possess cytotoxic [9], antiviral [33], antioxidant [34] and glucosidase-inhibiting [35] activity in vitro [32,36]. Similarly, the methyl ether of lanosol (2) showed cytotoxic [9] and antimicrobial [27] effects [32,36], although it might be an isolation artifact formed during methanolic extraction [5]. This might also be the case for 3, which has not been isolated from algae before, unlike the native phenyl acetic acid derivative found in Rhodomela confervoides [37]. Compared to simple lanosols, dimeric bromophenols, such as 5, seem to enhance the observed in vitro effects [6,7,[38][39][40][41] and, additionally, showed antifungal [42] and anti-inflammatory [43] activity. Further, 5 and, to a lesser extent, also 6 have been reported to inhibit glucose-6-phosphate dehydrogenase [40,44]. It should, however, be mentioned that the observed biological activities of bromophenols were often relatively weak-in particular, the antimicrobial effects [6,32,36]-and, with the exception of some compounds such as 8, rather unselective [36]. Kurihara et al. pointed out that enzyme inhibition could be the result of an o-quinone addition to the respective proteins [35], and as Baell emphasized, compounds with a variety of reported moderate bioactivities should be considered as potential drug leads only with caution, as, particularly, molecules with catechol substructures are prone to interfering with multiple in vitro assays [45].
In addition to the more frequently occurring bromophenols, two new brominated tyrosine derivatives (12 and 13) and two amino acids coupled to lanosyl moieties (14 and 15) were also isolated. A bromotyrosine has previously been found in Rhodomela confervoides [46], as well as derivatives of lanosol coupled to pyroglutamate [47]. So far, Ltyrosine (11) has been considered the main precursor of bromophenols, with the respective hydroxy benzoic acid derivatives being brominated at later steps in biosynthesis [23]. On the other hand, as also suspected by Ma et al. [46], the presence of 12 and 13 suggests that bromination occurs at a much earlier stage of biosynthesis. Whether bromoperoxidases in V. lanosa, unlike other macroalgae [48], are capable of directly converting L-tyrosine remains a topic for further research.
Another intriguing finding with respect to the biosynthesis is that, for Vertebrata species, typically, 2,3-dibrominated benzyl moieties have been reported (Vertebrata decipiens (syn. Polysiphonia decipiens) [38] and V. lanosa [6]). This is well in line with most of the compounds isolated in this study (1-7 and 14-16); however, 12 is an exception from this substitution pattern. The 3,5-dibrominated benzyl moiety, as in 12, is usually found in members of the Polysiphonia genus (Polysiphonia stricta (syn. P. urceolata) [49] and P. morrowii [50]), and the occurrence of 12 in V. lanosa might therefore be a hint for the presence of two regioselective bromoperoxidases in this alga.
Along with the amino acid-coupled compounds, sulfated bromophenols (4, 7, 14 and 16) were also isolated. Lanosol-4,7-disulfate (4) is one of the major secondary metabolites within the extract, and its occurrence in V. lanosa has been known for a long time [11,12]. Probably due to its high quantity, it has previously even been assumed to be the only sulfated compound in extracts from this alga [10]. On the other hand, the presence of sulfates could depend on parameters like the extraction procedure, as suggested by Weinstein et al., who suspected lanosol and its derivatives to be artifacts [51]. Despite this, Barreto and Meyer still found the lanosol disulfate ester in Osmundaria serrata after an extraction procedure of one week [52]. Taking into account that salt, unlike the unsulfated lanosols, was inactive as a feeding deterrent [53], they proposed bromophenols to be stored in algae as inactive salts and the release of lanosols upon injury [52]. On the other hand, Ma et al. did not find a significant difference in the cytotoxicity of a certain bromophenol versus its sulfate ester [46]. Within the current study, we also found the sulfate ester of vertebratol (16), a bromophenol that was just recently isolated from V. lanosa [6], but we cannot infer from our data which compound is genuine to the plant.
In contrast, compound 7 represents a unique structure containing a dimethyl sulfonium group. Generally, methyl sulfonium moieties, particularly dimethyl sulfonioproionate (DMSP), are widely distributed and also present in marine algae [54], including V. lanosa [55,56], where it acts as an antioxidant [57], cryoprotectant and osmolyte [54,58,59]. DMSP is biosynthesized in several steps via methionine [59] and degraded enzymatically to dimethylsulfide by DMSP lyases [56]. In the case of 7, the C-3 alkyl side chain is formally replaced by a dibromo dihydroxy benzyl moiety. Whether the sulfur atom is derived from an amino acid, e.g., cysteine, similar to the biosynthesis of methionine [60], and at what stage of lanosol formation remains a subject for further research. Interestingly, a bromophenol containing a similar sulfoxide structure was previously isolated from Rhodomela confervoides [47]. It could be regarded as an oxidation product of 7 comparable to dimethyl sulfoxide (DMSO) produced from DMSP in bacteria and microalgae [61,62]. Accordingly, 7 could be a precursor of the respective sulfoxide reported by Zhao et al. [47], and it would be interesting to explore if there are further sulfoxonium intermediates that could suggest a route of oxidation similar to the biosynthesis of DMSO from DMSP via dimethyl sulfoxonium propionate (DMSOP) [62]. However, the conversion of DMSOP to DMSO has been experimentally confirmed in bacteria [62], and the enzymatic cleavage of DMSP in V. lanosa is reported to yield mainly dimethyl sulfide [56]. On the other hand, Lee and de Mora summarized their findings supporting the hypothesis of intracellular production of DMSO by algae [61]. If such a biosynthetic route is applicable to a structurally distinct substrate such as 7 remains, of course, highly speculative.
Apart from bromophenols, several glycerogalactolipids (20-22 and the new compound 23) were isolated. They represent a class of fatty acid derivatives attached to a glycerogalactoside moiety and are very common in algae [29,63], but they have not been reported for V. lanosa until now. Typically, glycerogalactolipids are constituents of membranes in plant plastids and other organisms performing photosynthesis [64]. Apart from their role in photosynthesis, they may also be involved in radical scavenging and cryoprotection [65] and exert haemolytic activity in organisms like oysters or fish [29,30].
Finally, the major MAAs in V. lanosa were identified, confirming the presence of porphyra-334 (17), the most abundant MAA in this alga, and palythine (19) [15]. Compound 18, which was suspected to be palythinol based on its HPLC and UV data [15], was instead found to be aplysiapalythine A.

Plant Material and Chemicals
Algal material of V. lanosa was collected, identified and kindly provided as a gift by Nutramara Ltd. in March (VL1, 700 g) and October (VL2, 900 g) 2020 from Dungloe Bay, West Donegal, Ireland. Voucher specimens of both collection series (IPBP524 and IPBP528, respectively) were deposited at the Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Germany. Both batches of algae were kept separately during extraction and fractionation.
If not stated otherwise, all chemicals were purchased from VWR (Darmstadt, Germany). The solvents used for the analytical and preparative work were of analytical grade.
Thin-layer chromatography (TLC) for analytical purposes was performed on silica gel plates 60 F 254 (0.2 mm; Merck, Darmstadt, Germany) using ethyl acetate/water/formic acid (90:5:5 v/v/v) as the standard mobile phase. Visualization of the compounds was achieved under UV light (254 nm or 365 nm, resp.) and at daylight after spraying with thymol/sulfuric acid reagent, followed by heating the plate to approximately 105 • C for bromophenolic compounds and for MAA by spraying with ninhydrin.
Analysis by UPLC-qTOF-MS was carried out as follows: Separation was performed on a Dionex Ultimate 3000 RS Liquid Chromatography System (Thermo Fisher Scientific, Waltham, MA, USA) over a Dionex Acclaim RSLC 120, C18 column (2.1 × 100 mm, 2.2 µm) with a binary gradient (A: water with 0.1% formic acid; B: acetonitrile with 0.1% formic acid) at 0.4 mL/min: t 0min 5% B, t 0.4min 5% B, t 9.9min 100% B, t 15min 100% B, t 15.1min 5% B and t 20min 5% B. The injection volume was 2 or 5 µL. Eluted compounds were detected using a Dionex Ultimate DAD-3000 RS over a wavelength range of 200-400 nm and a Bruker Daltonics micrOTOF-QII time-of-flight mass spectrometer equipped with an Apollo electrospray ionization source in positive or negative mode (depending on the respective substance) at 3 Hz over a mass range of m/z 50-1500 using the following instrument settings: nebulizer gas nitrogen, 3.5 bar; dry gas nitrogen, 9 L/min, 180 • C (positive mode) or 200 • C (negative mode); capillary voltage, 4500 V; end plate offset, −500 V; collision energy, +3 eV or −8 eV; transfer time, 100 µ and pre-pulse storage, 6 µs; the collision energy and collision RF settings were combined to the single spectrum of 1650 (positive mode) or 2483 (negative mode) summations. MS/MS scans were triggered by AutoMS2 settings within a range of m/z: 200-1500. Internal dataset calibration (HPC or enhanced quadratic mode) was performed for each analysis using the mass spectrum of a 10-mM solution of sodium formiate in isopropanol-water-formic acid-1M NaOH solution (50 + 50 + 0.2 + 1) that was infused during LC re-equilibration using a divert valve equipped with a 20-µL sample loop.
Simulation of NMR spectra and generation of IUPAC names was performed with ChemDraw Ver. 21.0, PerkinElmer Informatics Inc. 2022 (Waltham, MA, USA).
Optical rotations were measured using Autopol V (Rudolph analytical research) at 20 • C and 589 nm. The compound concentrations ranged from 0.1 to 0.6 g/100 mL, and the solvents used were methanol, water and methanol/water (1:1 v/v).

Capillary Zone Electrophoresis (CZE)
The derivatization of the carbohydrates for analysis by CZE electrophoresis was conducted as described by Noe and Freissmuth [68].

Extraction and Fractionation
Air-dried and ground algae were extracted five times for 20 min using methanol/water (8:2, v/v) (3.75 L/500 g) in an ultrasonic bath. The crude extract was filtered and lyophilized after evaporation of the organic solvent, yielding M1 (122.52) and M2 (148.07 g). Subsequent defatting by stirring five times with 1.25 L petroleum ether/100 g of crude extract for 10 min yielded the defatted extracts Md1 (96.11 g) and Md2 (142.21 g). All extracts and fractions were stored at −20 • C.

Solvent Partitioning of the Extracts
Twenty grams of defatted extract were sequentially partitioned between 1 L water and 1 L ethyl acetate. Each partitioning step was repeated four times with fresh ethyl acetate, yielding the ethyl acetate fractions E1 (1.184 g) and E2 (1.767 g) and the aqueous fractions W1 (80.18 g) and W2 (131.44 g).

Sugar Precipitation and Ion Exchange Chromatography
To obtain an MAA-enriched fraction, the polysaccharides were precipitated in the first step. Therefore, 50 g of W2 dissolved in 500 mL of water were added dropwise to 2 L of ice-cold ethanol while stirring. After stirring overnight and centrifugation (3000× g, 15 min), the residual polysaccharide fraction WP2 (9.6 g) and the polysaccharide-depleted supernatant WM2 (38.5 g) were obtained.