Expanding the Repertoire of Spongian-16-One Derivatives in Australian Nudibranchs of the Genus Goniobranchus and Evaluation of Their Anatomical Distribution

Extracts of the mantle and viscera of the Indo-Pacific nudibranchs Goniobranchus aureopurpureus and Goniobranchus sp. 1 afforded 11 new diterpenoids (1–11), all of which possess a tetracyclic spongian-16-one scaffold with extensive oxidation at C-6, C-7, C-11, C-12, C-13, and/or C-20. The structures and relative configuration were investigated by NMR experiments, while X-ray crystallography provided the absolute configuration of 1, including a 2′S configuration for the 2-methylbutanoate substituent located at C-7. Dissection of animal tissue revealed that the mantle and viscera tissues differed in their metabolite composition with diterpenes 1–11 present in the mantle tissue of the two nudibranch species.

The current study, which forms part of our comprehensive study on nudibranchs of the genus Goniobranchus [7][8][9][10][11][12][13][14][15][16][17][18][19], represents the first chemical report on the secondary metabolite profile of the two nudibranch species, Goniobranchus aureopurpureus and Goniobranchus sp. 1 [20], each reassigned from an earlier taxonomic classification as Chromodoris species [21]. The structures and relative configuration of eleven isolated diterpene metabolites were determined by analysis of their two-dimensional NMR spectra as well as where applicable by X-ray crystallography to determine the absolute configuration. Our study also investigated the anatomical location of the terpenes and compared this with the distribution of metabolites in other Goniobranchus species.

Diterpenes from Goniobranchus aureopurpureus
Six specimens of G. aureopurpureus were collected from Nelson Bay (New South Wales, Australia) in March 2016. Specimens were dissected into their mantle and viscera and each body part was finely chopped, extracted with acetone and the extract concentrated under vacuum. The aqueous residues were partitioned with diethyl ether to yield an orange oil from the mantles and a green oil from the visceras. The individual mantle extracts were combined, as were the individual viscera extracts prior to fractionation by silica-flash chromatography. Subsequent normal phase high-performance liquid chromatography (NP-HPLC) yielded terpenes 1-5. The known compounds macfarlandin E [22], aplyviolene [23], polyrhaphin B [24], shahamin C [25], and secoshahamin [26] were also isolated from the mantle extract, while the viscera provided luffarin-X [27], spongian-16one [4,5], 7α-acetoxyspongian-16-one [28], polyrhaphin A [24], 15,16-diacetoxyshahamin B [25], and 12-desacetoxypolyrhaphin A [29]. The known terpenes spongian-16-one, 7αacetoxyspongian-16-one, macfarlandin E, aplyviolene, polyrhaphin B, and secoshahamin were isolated from both tissues. The new spongian diterpenes (1)(2)(3)(4)(5) show varying levels of oxidation, particularly at positions C-6, C-7, C-13 and C-20 ( Figure 1). Metabolite 1 was isolated as a colourless oil and displayed a sodiated ion at m/z 501.2829 [M + Na] + from high-resolution electrospray ionisation mass spectrometry (HRESIMS) for C27H42O7. These data indicated an additional seven carbons and five oxygens when compared to spongian-16-one. The 1 H and 13 C NMR spectroscopic data (Tables 1 and 2; see Supplementary Material) also supported a substituted spongian-16-one skeleton [4,5] but with a methyl singlet at δH 2.04 and a triplet signal at δH 4.85, suggesting additional functionality, namely an acetate group and substitution at C-7, respectively. Doublet and triplet signals at δH 1.15 (J = 6.9 Hz) and δH 0.89 (J = 7.4 Hz), respectively, were attributed to the methyl groups of a 2-methylbutanoate ester, accounting for the remaining five carbon atoms, with gCOSY and HSQC data further validating the CH3(CH3CH2)CHsubstructure. Signals for three ester carbonyls (δC 169.7, 174.0, and 175.4) validated six of the oxygen atoms present in the molecular formula. HMBC correlations from the signals at δH 4.85 (H-7, t, J = 2.8 Hz) and 1.15 d) to the signal at δC 175. 4 -14) to the signal at δC 81.1 (C-13), located the acetate group at C-13. The signal for Me-20 of spongian-16-one [4,5] was replaced by signals for an oxymethylene H2-20 (δH 4.05 and δH 3.92) in 1. These proton chemical shifts were inconsistent with esterification at C-20 [7,30]; therefore, a hydroxy group was located at C-20, identifying the final oxygen atom. NOESY correlations observed between H-7/Me-17 and H2-20/Me-17 confirmed the same relative configuration as spongian-16-one; however, the configuration of the acetate at C-13 and the 2′-methyl in Metabolite 1 was isolated as a colourless oil and displayed a sodiated ion at m/z 501.2829 [M + Na] + from high-resolution electrospray ionisation mass spectrometry (HRES-IMS) for C 27 H 42 O 7 . These data indicated an additional seven carbons and five oxygens when compared to spongian-16-one. The 1 H and 13 C NMR spectroscopic data (Tables 1  and 2; see Supplementary Material) also supported a substituted spongian-16-one skeleton [4,5] but with a methyl singlet at δ H 2.04 and a triplet signal at δ H 4.85, suggesting additional functionality, namely an acetate group and substitution at C-7, respectively. Doublet and triplet signals at δ H 1.15 (J = 6.9 Hz) and δ H 0.89 (J = 7.4 Hz), respectively, were attributed to the methyl groups of a 2-methylbutanoate ester, accounting for the remaining five carbon atoms, with gCOSY and HSQC data further validating the CH 3 (CH 3 -14) to the signal at δ C 81.1 (C-13), located the acetate group at C-13. The signal for Me-20 of spongian-16-one [4,5] was replaced by signals for an oxymethylene H 2 -20 (δ H 4.05 and δ H 3.92) in 1. These proton chemical shifts were inconsistent with esterification at C-20 [7,30]; therefore, a hydroxy group was located at C-20, identifying the final oxygen atom. NOESY correlations observed between H-7/Me-17 and H 2 -20/Me-17 confirmed the same relative configuration as spongian-16-one; however, the configuration of the acetate at C-13 and the 2 -methyl in the 2-methylbutanoate substituent could not be determined by NMR methods. Metabolite 1 was crystallized from 10% EtOAc/hexanes, producing small needle-shaped crystals which were suitable for diffraction. The resulting crystal structure obtained established the overall relative configuration. The absolute configuration was assigned as 5S, 7R, 8R, 9R, 10R, 13S, 14R, 2 S; within naturally-occurring 2-alkylalkanoic acid derivatives, the 2 S configuration is favoured [31]. In 1, the cyclohexane rings A, B, and C each adopt a chair conformation. As a result, adjacent molecules interact through hydrogen bonds O(7)H•••O(4) = 2.17 Å, 167 • , resulting in the formation of an undulating one-dimensional polymeric chain that extends down parallel to the crystallographic a-axis ( Figure 2). The name of compound 1 was assigned as (-)-13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate.    Diterpene 2 was isolated as a colourless oil and found to have the same C27H42O7 molecular formula as 1 inferred from HRESIMS (m/z 501.2824 [M + Na] + ). Examination of the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2) revealed similar signals to those of 1, including a methyl doublet at δH 1.15 (J = 6.9 Hz) and a methyl triplet at δH 0.91 (J = 7.4 Hz) for the methyl groups of a 2-methylbutanoate ester; there was also an acetate methyl singlet at δH 2.03. HMBC correlations from the signals at δH 4.87 (H-7, d, J = 3.2 Hz) and 1.15 d) to the signal at δC 174.9 confirmed the 2-methylbutanoate group at C-7. The configuration of the 2′-methyl in the ester sidechain could not be established further, owing to the small sample size, but was selected as identical to that in 1 on biogenetic considerations. HMBC correlations from H-20a (δH 4.79) and H-20b (δH 4.73) to the signal at δC 170.6 confirmed the position of the acetoxy group at C-20; there were NOESY correlations between H2-20/Me-17 and H-20b/Me-19. The doublet appearance of H-7 (J = 3.2 Hz) was initially considered consistent with an equatorial OH group at C-6; however, the signal for H-5 was a broadened singlet rather than the doublet with a large J value anticipated if H-6 was axial, (cf. aplyroseol-19 from Chromodoris reticulata [33]). The NOESY correlation between H-6/Me-18 supported an equatorial H-6, while the absence of an NOE between H-6 and Me-17, although not diagnostic, was also consistent with the changed configuration at C-6 compared to that in aplyroseol-19 [33]. The NOESY correlation between H-7/Me-17 placed the C-7 ester substituent on the opposite face to Me-17. Compound 2 was assigned the systematic name (-)-20-acetoxy-6β-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate.
Metabolite 3, also isolated as a colourless oil, displayed an adduct ion at m/z 443.2779 [M + Na] + in HRESIMS analysis, which established the molecular formula as C25H40O5 with an additional five carbons and three oxygens compared with spongian-16-one. Due to the small sample quantity (<0.1 mg), a Shigemi tube was employed to increase the sensitivity of NMR signal detection [34]. The spectroscopic data again revealed a 2-methylbutanoate moiety, located at C-7 from the identical HMBC correlations for H-7 to those in 1 and 2. NOESY data could not be obtained, but the similar appearance of the signals for H-6 (δH 4.18, br s) and H-7 (δH 4.84, J = 2.6 Hz) compared to 2 established the axial hydroxy group  [32] representation of the crystal structure of (5S, 7R, 8R, 9R, 10R, 13S, 14R, 2 S)-13-acetoxy-20-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)butanoate (1) shown with 30% probability ellipsoids. Diterpene 2 was isolated as a colourless oil and found to have the same C 27 H 42 O 7 molecular formula as 1 inferred from HRESIMS (m/z 501.2824 [M + Na] + ). Examination of the 1 H and 13 C NMR spectroscopic data (Tables 1 and 2) revealed similar signals to those of 1, including a methyl doublet at δ H 1.15 (J = 6.9 Hz) and a methyl triplet at δ H 0.91 (J = 7.4 Hz) for the methyl groups of a 2-methylbutanoate ester; there was also an acetate methyl singlet at δ H 2.03. HMBC correlations from the signals at δ H 4.87 (H-7, d, J = 3.2 Hz) and 1.15 (7-OCOCHCH 3 CH 2 CH 3 , d) to the signal at δ C 174.9 confirmed the 2-methylbutanoate group at C-7. The configuration of the 2 -methyl in the ester sidechain could not be established further, owing to the small sample size, but was selected as identical to that in 1 on biogenetic considerations. HMBC correlations from H-20a (δ H 4.79) and H-20b (δ H 4.73) to the signal at δ C 170.6 confirmed the position of the acetoxy group at C-20; there were NOESY correlations between H 2 -20/Me-17 and H-20b/Me-19. The doublet appearance of H-7 (J = 3.2 Hz) was initially considered consistent with an equatorial OH group at C-6; however, the signal for H-5 was a broadened singlet rather than the doublet with a large J value anticipated if H-6 was axial, (cf. aplyroseol-19 from Chromodoris reticulata [33]). The NOESY correlation between H-6/Me-18 supported an equatorial H-6, while the absence of an NOE between H-6 and Me-17, although not diagnostic, was also consistent with the changed configuration at C-6 compared to that in aplyroseol-19 [33]. The NOESY correlation between H-7/Me-17 placed the C-7 ester substituent on the opposite face to Me-17. Compound 2 was assigned the systematic name (-)-20-acetoxy-6β-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate.
Metabolite 3, also isolated as a colourless oil, displayed an adduct ion at m/z 443.2779 [M + Na] + in HRESIMS analysis, which established the molecular formula as C 25 H 40 O 5 with an additional five carbons and three oxygens compared with spongian-16-one. Due to the small sample quantity (<0.1 mg), a Shigemi tube was employed to increase the sensitivity of NMR signal detection [34]. The spectroscopic data again revealed a 2methylbutanoate moiety, located at C-7 from the identical HMBC correlations for H-7 to those in 1 and 2. NOESY data could not be obtained, but the similar appearance of the signals for H-6 (δ H 4.18, br s) and H-7 (δ H 4.84, J = 2.6 Hz) compared to 2 established the axial hydroxy group at C-6 and the equatorial ester group at C-7. The 7.8 Hz coupling between H-13 and H-14 assigned the cis C/D ring junction. The name of compound 3 was assigned as (-)-6β-hydroxy-7α-oxyspongian-16-one-7α-(2-methyl)-butanoate.
Diterpene 5 was isolated as a colourless oil and displayed a sodiated molecular ion peak by HRESIMS at m/z 401.2293 [M + Na] + , corresponding to a molecular formula of C 22 H 34 O 5 . The 1 H and 13 C NMR spectroscopic data (Tables 1 and 2) showed an acetoxy methyl singlet at δ H 2.09 and associated carbonyl signal at δ C 169.7, comparable to those in the NMR data of 7α-acetoxyspongian-16-one [28]. HMBC correlations from the signals at δ H 2.09 and 4.84 (H-7, d, J = 3.1 Hz) to the carbonyl at δ C 169.7 confirmed the position of the acetoxy group at C-7. The doublet appearance of H-7 suggested hydroxy substitution at C-6. The relative configuration of 5 was identical to that of 2 from the NOESY correlations between H-6/Me-18, H-7/Me-17, H-5/H-9, and H-9/H-14. Compound 5 was assigned the systematic name (-)-7α-acetoxy-6β-hydroxyspongian-16-one.

Anatomical Distribution of Metabolites
Comparison of individual body parts by 1 H NMR spectroscopy, together with subsequent isolation work, revealed that new metabolites 1-5 were solely isolated from the mantle tissue of G. aureopurpureus. Likewise, new metabolites 6-11 were isolated only from the mantle tissue of Goniobranchus sp. 1. We also found that both species had more chemical diversity of metabolites in the mantle relative to the viscera. A full list of metabolites found in each body part is provided in the Supplementary Material. This pattern of anatomical distribution matches that of four Goniobranchus species that we previously studied (G. tinctorius, G. tasmaniensis, G. collingwoodi, and G. splendidus) [7,35]. These species may accumulate compounds in the mantle as they feed on a variety of sponge species with different chemistry. Compounds in the mantle are thought to be used for defensive purposes, and complex defensive mixtures may provide protection from a range of predators [10]. In contrast, we previously found two species (G. hunterae and G. verrieri) with the same metabolites in the mantle and viscera tissue, and one species (G. daphne) with fewer compounds in the mantle compared to the viscera [35].

Conclusions
In conclusion, the isolation work was conducted on two Goniobranchus species and afforded eleven new spongian diterpenes with oxidation at various positions, such as C-6, C-7, C-11, C-12, C-13, and/or C-20. The X-ray structure of 1 provided insight into the absolute configuration of the parent spongian-16-one [4,5]. Many of these highly oxygenated spongian diterpenes were only isolated from the mantle tissue, where they may play a role in deterring predators.

General Experimental Procedure
Specific rotations were measured at 23 • C on a Jasco P-2000 polarimeter for solutions in CHCl 3 using a 1-millilitre cell (10-centimetre path length). NMR spectroscopic data were recorded on a Bruker Avance 500 spectrometer using a 5-millimetre SEI probe or a Bruker Avance DRX 700 MHz spectrometer with a 5-millimetre TXI Zgrad probe for solutions in CDCl 3 at 298K. Heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) data were acquired using a 1 J C-H of 145 Hz, while HMBC spectra were acquired using n J C-H of 8 Hz. Positive and negative ion electrospray mass spectra were determined using either a Bruker Esquire HCT 3D ion trap instrument for low-resolution electrospray ionization mass spectrometry (LRESIMS) or a MicrOTOF-Q or an Orbitrap Elite instrument for high-resolution electrospray ionization mass spectrometry (HRESIMS) with MeOH as solvent. Normal-phase high-performance liquid chromatography (NP-HPLC) was undertaken using a Waters 515 pump connected to a Gilson 132 series refractive index detector with a Phenomenex Luna (5 µm, 10 × 250 mm) column, using isocratic elution conditions at flow rates between 1-2 mL/ min. Silica gel 60 G and silica TLC plates F 254 were purchased from Merck. Solvents were either distilled or were HPLC grade.

Biological Material
Six individuals of Goniobranchus aureopurpureus were collected from Nelson Bay (#1469-1474), New South Wales in March 2016. Three individuals of Goniobranchus (Chromodoris) sp. 1 were collected from Mudjimba (#1368 and #1563) and Gneerings Reefs (#1575) (Mooloolaba, Queensland) in October 2015 and October 2016. All collections were stored in individual containers at −20 • C until dissection into mantle and gut prior to extraction.

Extraction and Purification
The mantle and viscera tissue of each specimen of G. aureopurpureus and G. sp 1 were extracted in acetone (3 × 2 mL) and sonicated (5 min) separately. The extracts were reduced to aqueous suspensions, extracted with Et 2 O (3 × 3 mL), dried over anhydrous Na 2 SO 4 , and concentrated under N 2 to give an orange oil (mantle tissue) or a green oil (viscera). The 1 H NMR profile of the mantle and viscera extracts were compared between the specimens of each species and showed similar chemistry; for G. aureopurpureus (specimens #1469-1474) the mantle extracts were combined (51.9 mg) and the viscera extracts combined (56.1 mg) to produce two extracts. For G. sp 1 (specimens #1563, 1368 and 1575), the mantle extracts were combined (96.8 mg), as were the viscera extracts (71.2 mg). The extracts were further separated by NP-flash column chromatography with a stepwise solvent gradient from 100% hexanes to 100% MeOH.

X-ray Crystallographic Structure Determination
Full details of X-ray crystallography methods and data are available in the Supplementary Materials.
Crystallographic data for 1: C 27 H 42 O 7 (M =478.60 g/mol): orthorhombic, space group P2 1  Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/md19120680/s1. Figure S1: An image of G. aureopurpureus and Giniobranchus sp. 1; Figure S2 Data Availability Statement: Raw NMR data files are available from the authors on request. All other data is contained within this manuscript.