New Isomalabaricane-Derived Metabolites from a Stelletta sp. Marine Sponge

In continuation of our studies on a Vietnamese collection of a Stelletta sp., sponge we have isolated two new isomalabaricane triterpenoids, stellettins Q and R (1 and 2), and four new isomalabaricane-derived nor-terpenoids, stellettins S-V 3–6, along with previously known globostelletin N. Among them, compound 3 contains an acetylenic fragment, unprecedented in the isomalabaricane family and extremely rare in other marine sponge terpenoids. The structures and absolute configurations of all new compounds were established by extensive NMR, MS, and ECD analyses together with quantum-chemical modeling. Additionally, according to obtained new data we report the correction in stereochemistry of two asymmetric centers in the structures of two known isomalabaricanes, 15R,23S for globostelletin M and 15S,23R for globostelletin N.


Introduction
Malabaricanes are rather a small group of tricarbocyclic triterpenoids found in different tropical flowering terrestrial plants [1][2][3]. Isomalabaricanes, which differ from malabaricanes in the configuration of C-8 asymmetric center and have an α-oriented CH 3 -30, are known as metabolites of four genera of marine sponges-Stelletta, Jaspis, Geodia and Rhabdastrella-belonging to the class Demospongiae. Some of them are highly cytotoxic against tumor cells [4]. Since the first isolation of three yellow highly conjugated isomalabaricane-type triterpenoids from the marine sponge Jaspis stellifera in 1981 [5] more than 130 isomalabaricanes and related natural products have been reported from the abovementioned sponge genera. It was noticed that Stelletta metabolites are quite different depending on the collection. Indeed, isomalabaricane triterpenoids were mainly found as very complex mixtures in tropical sponge samples, while boreal and cold-water sponges contain mostly alkaloids and lipids. From a chemo-ecological point of view, this indicates that studied sponges are able to produce different types of secondary metabolites in order to adapt to the various living conditions [6]. In confirmation, our attempt to find isomalabaricanes in a cold-water Stelletta spp., collected in 2019 in the Sea of Okhotsk, was unsuccessful, as the characteristic yellow pigments were not detected by thin layer chromatography in the extracts of these sponges.
Additionally, in result of the chemical investigation of the sponge Stelletta tenuis, Li et al. identified two naturally occurring α-pyrones, namely gibepyrones C and F, along with three isomalabaricane-type triterpenoids [7]. These α-pyrones were supposed to be the oxidation products of the co-occurring stellettins [6]. Gibepyrone F had previously been isolated from the fungal plant pathogen Gibberella fujikuroi [8], as well as from the sponge Jaspis stellifera [9]. These findings allow to presume that symbiotic microorganisms in the corresponding sponges are involved in the generation of some metabolites.
Diverse isomalabaricane-type nor-terpenoids, containing less than 30 carbon atoms in their skeleton systems, have been found together with isomalabaricanes several times [10][11][12]. Their presence could be explained either by oxidative degradation of C 30 metabolites or by precursor role of nor-terpenoids in the biosynthesis of these compounds [12,13]. However, the biogenesis of isomalabaricane compounds in sponges remains to be mysterious so far.
Recently, we have reported the isolation of two isomalabaricane-type nor-terpenoids, cyclobutastellettolides A and B, and series of known isomalabaricanes from a Stelletta sp. [14] We suppose that new data on structural variety of isomalabaricane derivatives supported with strong evidence on stereochemistry could someday shed light on their origin.
In the present work, an investigation of the chemical components of a Stelletta sp. from Vietnamese waters was continued. Herein, we report the isolation and structural elucidation of six new compounds 1-6 and known globostelletin N [15].

Results and Discussion
The frozen sample of a marine sponge Stelletta sp. was finely chopped and extracted with EtOH, then the extract was concentrated under reduced pressure and subjected to Sephadex LH-20 and silica gel column chromatography followed by normal-and reversedphase HPLC procedures ( Figure S73) to afford new stellettins Q-V 1-6 together with known globostelletin N [15] (Figure 1).
Molecules 2021, 26, x FOR PEER REVIEW 2 of 12 been isolated from the fungal plant pathogen Gibberella fujikuroi [8], as well as from the sponge Jaspis stellifera [9]. These findings allow to presume that symbiotic microorganisms in the corresponding sponges are involved in the generation of some metabolites. Diverse isomalabaricane-type nor-terpenoids, containing less than 30 carbon atoms in their skeleton systems, have been found together with isomalabaricanes several times [10][11][12]. Their presence could be explained either by oxidative degradation of C30 metabolites or by precursor role of nor-terpenoids in the biosynthesis of these compounds [12,13]. However, the biogenesis of isomalabaricane compounds in sponges remains to be mysterious so far.
Recently, we have reported the isolation of two isomalabaricane-type nor-terpenoids, cyclobutastellettolides A and B, and series of known isomalabaricanes from a Stelletta sp. [14] We suppose that new data on structural variety of isomalabaricane derivatives supported with strong evidence on stereochemistry could someday shed light on their origin.
In the present work, an investigation of the chemical components of a Stelletta sp. from Vietnamese waters was continued. Herein, we report the isolation and structural elucidation of six new compounds 1-6 and known globostelletin N [15].

Results and Discussion
The frozen sample of a marine sponge Stelletta sp. was finely chopped and extracted with EtOH, then the extract was concentrated under reduced pressure and subjected to Sephadex LH-20 and silica gel column chromatography followed by normal-and reversed-phase HPLC procedures ( Figure S73) to afford new stellettins Q-V 1-6 together with known globostelletin N [15] (Figure 1). Stellettin Q (1) was isolated as a yellow oil with molecular formula C32H44O6 deduced by HRESIMS ( Figure S3). The NMR data of 1 (Table 1; Figures S4 and S5) were closely related to the spectral characteristics of isomalabaricane globostelletin K (Figures 1 and  S55 and S56) initially found in the marine sponge Rabdastrella globostellata [15] and also coisolated from the studied Stelletta sp. [14]. Stellettin Q (1) was isolated as a yellow oil with molecular formula C 32 H 44 O 6 deduced by HRESIMS ( Figure S3). The NMR data of 1 (Table 1; Figures S4 and S5) were closely related to the spectral characteristics of isomalabaricane globostelletin K (Figure 1, Figures S55 and S56) initially found in the marine sponge Rabdastrella globostellata [15] and also co-isolated from the studied Stelletta sp. [14]. The detailed analysis of 2D spectra (COSY, HSQC, HMBC etc.) of 1 supported the main structure ( Figure 2 and Figures S6-S9). The signals of methyl group at δ H 2.06, s; δ C 21.2 and acetate carbonyl at δ C 171.0, together with the HMBC correlation of axial proton H-3 at δ H 4.54, dd (11.7, 5.2) to that carbonyl, revealed the O-acetyl substitution at C-3 in the ring A. Moreover, the signal of C-3 at δ C 80.8 instead of ketone signal at δ C 219.2 in 13 C NMR spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, while the 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH 3 -28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with the signal of CH 3 -18 at δ H 1.79, s and its ROESY correlation with CH 3 -30. As well as E configuration of 24(25)-double bond was found from the W-path COSY correlation of protons H-24/CH 3 -27. Moreover, the signal of C-3 at δC 80.8 instead of ketone signal at δC 219.2 in 13 C NMR spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, while the 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH3-28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with the signal of CH3-18 at δH 1.79, s and its ROESY correlation with CH3-30. As well as E configuration of 24(25)-double bond was found from the W-path COSY correlation of protons H-24/CH3-27. The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD curve ( Figure S10) were analogous to those of globostelletin K [15] ( Figure S57) suggesting the same stereochemistry of the side chain. This assignment was in a good agreement with the computational ECD results performed using density functional theory (DFT) with the nonlocal exchange-correlation functional B3LYP [16], the polarization continuum model (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 package of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13Z,24E geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group fully satisfies the similarity of the experimental and theoretical ECD spectra of 1 ( Figure 3). In detailes, statistically avereged curve ( Figure 3) follows the shape of the experimental one, although even more close coincidence was indicated for theoretically less probable conformer ( Figure S67). In addition, we could conclude that the presence of 3-acetoxy or 3-oxo functions in the structures of the corresponding compounds insignificantly affects the shape of their ECD curves. According to obtained new data we pose the same 15R,23S stereochemistry for globostelletin K ( Figure S69).   Moreover, the signal of C-3 at δC 80.8 instead of ketone signal at δC 219.2 in 13 C NMR spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, while the 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH3-28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with the signal of CH3-18 at δH 1.79, s and its ROESY correlation with CH3-30. As well as E configuration of 24(25)-double bond was found from the W-path COSY correlation of protons H-24/CH3-27. The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD curve ( Figure S10) were analogous to those of globostelletin K [15] ( Figure S57) suggesting the same stereochemistry of the side chain. This assignment was in a good agreement with the computational ECD results performed using density functional theory (DFT) with the nonlocal exchange-correlation functional B3LYP [16], the polarization continuum model (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 package of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13Z,24E geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group fully satisfies the similarity of the experimental and theoretical ECD spectra of 1 ( Figure 3). In detailes, statistically avereged curve ( Figure 3) follows the shape of the experimental one, although even more close coincidence was indicated for theoretically less probable conformer ( Figure S67). In addition, we could conclude that the presence of 3-acetoxy or 3-oxo functions in the structures of the corresponding compounds insignificantly affects the shape of their ECD curves. According to obtained new data we pose the same 15R,23S stereochemistry for globostelletin K ( Figure S69).  ), HMBC ( Moreover, the signal of C-3 at δC 80.8 instead of ketone signal at δC 219.2 in 13 C NMR spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, while the 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH3-28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with the signal of CH3-18 at δH 1.79, s and its ROESY correlation with CH3-30. As well as E configuration of 24(25)-double bond was found from the W-path COSY correlation of protons H-24/CH3-27. The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD curve ( Figure S10) were analogous to those of globostelletin K [15] ( Figure S57) suggesting the same stereochemistry of the side chain. This assignment was in a good agreement with the computational ECD results performed using density functional theory (DFT) with the nonlocal exchange-correlation functional B3LYP [16], the polarization continuum model (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 package of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13Z,24E geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group fully satisfies the similarity of the experimental and theoretical ECD spectra of 1 ( Figure 3). In detailes, statistically avereged curve ( Figure 3) follows the shape of the experimental one, although even more close coincidence was indicated for theoretically less probable conformer ( Figure S67). In addition, we could conclude that the presence of 3-acetoxy or 3-oxo functions in the structures of the corresponding compounds insignificantly affects the shape of their ECD curves. According to obtained new data we pose the same 15R,23S stereochemistry for globostelletin K ( Figure S69).  ) and ROESY ( Moreover, the signal of C-3 at δC 80.8 instead of ketone signal at δC 219.2 in 13 C NMR spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, while the 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH3-28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with the signal of CH3-18 at δH 1.79, s and its ROESY correlation with CH3-30. As well as E configuration of 24(25)-double bond was found from the W-path COSY correlation of protons H-24/CH3-27. The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD curve ( Figure S10) were analogous to those of globostelletin K [15] ( Figure S57) suggesting the same stereochemistry of the side chain. This assignment was in a good agreement with the computational ECD results performed using density functional theory (DFT) with the nonlocal exchange-correlation functional B3LYP [16], the polarization continuum model (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 package of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13Z,24E geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group fully satisfies the similarity of the experimental and theoretical ECD spectra of 1 ( Figure 3). In detailes, statistically avereged curve ( Figure 3) follows the shape of the experimental one, although even more close coincidence was indicated for theoretically less probable conformer ( Figure S67). In addition, we could conclude that the presence of 3-acetoxy or 3-oxo functions in the structures of the corresponding compounds insignificantly affects the shape of their ECD curves. According to obtained new data we pose the same 15R,23S stereochemistry for globostelletin K ( Figure S69).   The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD curve ( Figure S10) were analogous to those of globostelletin K [15] ( Figure S57) suggesting the same stereochemistry of the side chain. This assignment was in a good agreement with the computational ECD results performed using density functional theory (DFT) with the nonlocal exchange-correlation functional B3LYP [16], the polarization continuum model (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 package of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13Z,24E geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group fully satisfies the similarity of the experimental and theoretical ECD spectra of 1 ( Figure 3). In detailes, statistically avereged curve ( Figure 3) follows the shape of the experimental one, although even more close coincidence was indicated for theoretically less probable conformer ( Figure S67). In addition, we could conclude that the presence of 3-acetoxy or 3-oxo functions in the structures of the corresponding compounds insignificantly affects the shape of their ECD curves. According to obtained new data we pose the same 15R,23S stereochemistry for globostelletin K ( Figure S69). 28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with th signal of CH3-18 at δH 1.79, s and its ROESY correlation with CH3-30. As well as E configuration of 24(25)-double bond was found from the W-path COSY correlation o protons H-24/CH3-27. The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD curv ( Figure S10) were analogous to those of globostelletin K [15] ( Figure S57) suggesting th same stereochemistry of the side chain. This assignment was in a good agreement with the computational ECD results performed using density functional theory (DFT) with th nonlocal exchange-correlation functional B3LYP [16], the polarization continuum mode (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 packag of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13Z,24E geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group fully satisfies the similarity of the experimental and theoretical ECD spectra of 1 ( Figure 3). In detailes, statistically avereged curve ( Figure 3) follows the shape of the experimental one although even more close coincidence was indicated for theoretically less probabl conformer ( Figure S67). In addition, we could conclude that the presence of 3-acetoxy o 3-oxo functions in the structures of the corresponding compounds insignificantly affect the shape of their ECD curves. According to obtained new data we pose the same 15R,23S stereochemistry for globostelletin K ( Figure S69).    [15] and co-isolated by us. Moreover, the ECD spectrum of 2 ( Figure S18) displayed the same curve and peaks as those published for globostelletin M ( Figure S61).
However, structure modeling as well as calculation of ECD spectra for possible stereoisomers of 2 demonstrated a good agreement between experimental and theoretical spectra for 15R,23S absolute configuration (Figure 4) quite differ from 15S,23S reported for globostelletin M [15]. This inconsistence encouraged us to re-investigate the stereochemistry of co-isolated globostelletins M and N. We have obtained NMR and ECD spectra of the both compounds (Figures S59-S65) and they were identical to those provided as supplementary data by Li et al. [15]. At the same time, our computational results suggested globostelletin M to possess the same 15R,23S absolute configuration ( Figure S70) of cyclopentene unit as 2, while globostelletin N has 15S,23R stereochemistry ( Figure S71). Based on the data we believe that previously published research comprises some inaccuracies and the stereochemistry of these centres in corresponding isomalabaricanes should be revised. It was noted that the isomalabaricane-type terpenoids undergo a photoisomerization of the side chain 13-double bond during the isolation and storage [19,20]. We consider compounds 1 and 2 to be the 13Z/E pair of the same 15R,23S isomer. However, structure modeling as well as calculation of ECD spectra for possible stereoisomers of 2 demonstrated a good agreement between experimental and theoretica spectra for 15R,23S absolute configuration (Figure 4) quite differ from 15S,23S reported for globostelletin M [15]. This inconsistence encouraged us to re-investigate the stereochemistry of co-isolated globostelletins M and N. We have obtained NMR and ECD spectra of the both compounds ( Figures S59-S65) and they were identical to those provided as supplementary data by Li et al. [15]. At the same time, our computationa results suggested globostelletin M to possess the same 15R,23S absolute configuration ( Figure S70) of cyclopentene unit as 2, while globostelletin N has 15S,23R stereochemistry ( Figure S71). Based on the data we believe that previously published research comprises some inaccuracies and the stereochemistry of these centres in corresponding isomalabaricanes should be revised. It was noted that the isomalabaricane-type terpenoids undergo a photoisomerization of the side chain 13-double bond during the isolation and storage [19,20]. We consider compounds 1 and 2 to be the 13Z/E pair of the same 15R,23S isomer. The molecular formula C19H28O3 of stellettin S (3) calculated from HRESIMS data ( Figure S19) showed 3 to be a rather smaller molecule then classical C30-isomalabaricanes intriguing due to the lack of a significant part in the molecule,when compared with the majority of known isomalabaricanes and their derivatives. The 13 C-and DEPT NMR spectra ( Table 2; Figures S21 and S22) exhibited 19 resonances, including those of carbony carbon at δC 216.5 (C-3) and carboxyl carbon at δC 178.8 (C-12) as well as two down-shifted quaternary carbons at δC 88.1 (C-13), 77.8 (C-14). 1 H-and 13 C-NMR spectra ( Figures S20  and S21) revealed five methyls, two methylene, two methine groups and seven quaternary carbons, suggesting an isoprenoid nature. In the HSQC spectrum ( Figure S23) four methy singlets (δH 1.06, 1.08, 1.25, and 1.62) correlated with carbon signals at δC 21.6 (CH3-29) 25.9 (CH3-28), 30.8 (CH3-30) and 23.3 (CH3-19), respectively, while singlet of one more methyl group at δH 1.80 gave a cross-peak with high field signal at δC 3.7 (CH3-18). The further inspection of 2D spectra ( Figures 5 and S23-S26) revealed the bicyclic framework resembling the core of globostelletin A ( Figure 5), isolated from the sponge Rhabdastrella globostellata [13].
This was confirmed by the key long-range HMBC correlations from gem-dimethyl group (CH 3 -28 and 29) to C-3, C-4 and C-5; from H-5 to C-1, C-4, C-6, C-9 and C-10; from methyl CH 3 -19 to C-1, C-9 and C-10; from methyl CH 3 -30 to C-7, C-8 and C-9 as well as from the methylene of carboxymethyl group (CH 2 -11) to C-8, C-9, C-10 and carboxyl carbon C-12 ( Figure 5 and Figure S24). The empirical formula, besides bicyclic system and two carbonyls, required two additional degrees of unsaturation which were accounted for an acetylenic bond in a short side chain. The NMR signals of two quaternary carbons at δ C 88.1 (C-13), 77.8 (C-14) and methyl (CH 3 -18) at δ H 1.80, δ C 3.7 were finally attributed to the methylacetylenic substituent at C-8, that was confirmed by HMBC correlations from CH 3 -30 to C-8 and C-13, along with that from CH 3 -18 to C-7, C-8, C-9, C-13, C-14 and CH 3 -30. Analogous methylacetylenic substituent was characterized previously with similar chemical shifts in a series of synthetic alkynes [21].
Molecules 2021, 26, x FOR PEER REVIEW 6 of 12 30 to C-8 and C-13, along with that from CH3-18 to C-7, C-8, C-9, C-13, C-14 and CH3-30. Analogous methylacetylenic substituent was characterized previously with similar chemical shifts in a series of synthetic alkynes [21]. Interestingly, the NMR signal of CH3-19 (δH 1.62, s) was notably downfield shifted in comparison with that in a number of isomalabaricanes and their derivatives spectra. We explained it by the joint influence of the methylacetylene and carboxymethyl groups. The quantum chemical calculations ( Figure S66) of the chemical shifts for structure 3 confirmed the down-shifted position of the proton signal of CH3-19 and afforded its theoretical chemical shift value of δH 1.69 ppm.
The relative stereochemistry of 3 was determined by ROESY experiment (Figures 6  and S26). A trans-fusion of the bicyclic system was shown by key NOE interactions. The correlations between CH3-19/CH3-29, CH3-28/H-5, H-5/Ha-11, CH3-19/H-9, and CH3-30/Hb-11 showed the β-orientations of CH3-19 and H-9, whereas H-5, CH2-11, and CH3-30 were α-oriented. The chair conformation of the ring B with equatorial positions of H-9 and CH3-30 corresponded to the long-range COSY correlation between H-9 and Hβ-7 ( Figures  5 and S25) together with ROESY correlations H-5/Ha-11 and Hα-7/Hb-11. Taking into consideration the relative stereochemistry of the compound 3 along with above mentioned absolute stereochemistry of the C30 congeners 1 and 2 as well as the fact of coisolation of cyclobutastellettolides A and B [14] with the same absolute configurations we suggested the 5S,8R,9R,10R absolute stereochemistry of stellettin S (3).  The detailed analysis of 2D spectra (COSY, HSQC, HMBC etc.) of 1 supported th main structure (Figures 2 and S6-S9). The signals of methyl group at δH 2.06, s; δC 21.2 an acetate carbonyl at δC 171.0, together with the HMBC correlation of axial proton H-3 at δ 4.54, dd (11.7, 5.2) to that carbonyl, revealed the O-acetyl substitution at C-3 in the ring A Moreover, the signal of C-3 at δC 80.8 instead of ketone signal at δC 219.2 in 13 C NM spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, while th 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and CH 28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement with th signal of CH3-18 at δH 1.79, s and its ROESY correlation with CH3-30. As well as configuration of 24 (25) The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD curv ( Figure S10) were analogous to those of globostelletin K [15] ( Figure S57) suggesting th same stereochemistry of the side chain. This assignment was in a good agreement wit the computational ECD results performed using density functional theory (DFT) with th nonlocal exchange-correlation functional B3LYP [16], the polarization continuum mod (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 packag of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13Z,24 geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group ful satisfies the similarity of the experimental and theoretical ECD spectra of 1 (Figure 3). I detailes, statistically avereged curve (Figure 3) follows the shape of the experimental on although even more close coincidence was indicated for theoretically less probab conformer ( Figure S67). In addition, we could conclude that the presence of 3-acetoxy o 3-oxo functions in the structures of the corresponding compounds insignificantly affec the shape of their ECD curves. According to obtained new data we pose the same 15R,23 stereochemistry for globostelletin K ( Figure S69).  ) and HMBC ( The detailed analysis of 2D spectra (COSY, HSQC, HMBC etc.) of 1 supporte main structure (Figures 2 and S6-S9). The signals of methyl group at δH 2.06, s; δC 21. acetate carbonyl at δC 171.0, together with the HMBC correlation of axial proton H-3 4.54, dd (11.7, 5.2) to that carbonyl, revealed the O-acetyl substitution at C-3 in the ri Moreover, the signal of C-3 at δC 80.8 instead of ketone signal at δC 219.2 in 13 C spectrum of globostelletin K also demonstrated the 3-acetoxy-tricyclic core in 1, whi 3β-orientation of acetoxy group was confirmed by strong correlations of H-3/H-5 and 28 observed in the ROESY spectrum. The 13Z geometry in 1 was in agreement wit signal of CH3-18 at δH 1.79, s and its ROESY correlation with CH3-30. As well configuration of 24 (25) The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD ( Figure S10) were analogous to those of globostelletin K [15] ( Figure S57) suggestin same stereochemistry of the side chain. This assignment was in a good agreement the computational ECD results performed using density functional theory (DFT) wi nonlocal exchange-correlation functional B3LYP [16], the polarization continuum m (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 pa of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13 geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group satisfies the similarity of the experimental and theoretical ECD spectra of 1 (Figure detailes, statistically avereged curve (Figure 3) follows the shape of the experimenta although even more close coincidence was indicated for theoretically less pro conformer ( Figure S67). In addition, we could conclude that the presence of 3-aceto 3-oxo functions in the structures of the corresponding compounds insignificantly a the shape of their ECD curves. According to obtained new data we pose the same 15 stereochemistry for globostelletin K ( Figure S69).   Interestingly, the NMR signal of CH 3 -19 (δ H 1.62, s) was notably downfield shifted in comparison with that in a number of isomalabaricanes and their derivatives spectra. We explained it by the joint influence of the methylacetylene and carboxymethyl groups. The quantum chemical calculations ( Figure S66) of the chemical shifts for structure 3 confirmed the down-shifted position of the proton signal of CH 3 -19 and afforded its theoretical chemical shift value of δ H 1.69 ppm.
The relative stereochemistry of 3 was determined by ROESY experiment (Figure 6 and Figure S26). A trans-fusion of the bicyclic system was shown by key NOE interactions. The correlations between CH 3 -19/CH 3 -29, CH 3 -28/H-5, H-5/H a -11, CH 3 -19/H-9, and CH 3 -30/H b -11 showed the β-orientations of CH 3 -19 and H-9, whereas H-5, CH 2 -11, and CH 3 -30 were α-oriented. The chair conformation of the ring B with equatorial positions of H-9 and CH 3 -30 corresponded to the long-range COSY correlation between H-9 and H β -7 ( Figure 5 and Figure S25) together with ROESY correlations H-5/H a -11 and H α -7/H b -11. Taking into consideration the relative stereochemistry of the compound 3 along with above mentioned absolute stereochemistry of the C 30 congeners 1 and 2 as well as the fact of co-isolation of cyclobutastellettolides A and B [14] with the same absolute configurations we suggested the 5S, 8R, 9R, 10R absolute stereochemistry of stellettin S (3).
Molecules 2021, 26, x FOR PEER REVIEW 6 of 12 30 to C-8 and C-13, along with that from CH3-18 to C-7, C-8, C-9, C-13, C-14 and CH3-30. Analogous methylacetylenic substituent was characterized previously with similar chemical shifts in a series of synthetic alkynes [21]. Interestingly, the NMR signal of CH3-19 (δH 1.62, s) was notably downfield shifted in comparison with that in a number of isomalabaricanes and their derivatives spectra. We explained it by the joint influence of the methylacetylene and carboxymethyl groups. The quantum chemical calculations ( Figure S66) of the chemical shifts for structure 3 confirmed the down-shifted position of the proton signal of CH3-19 and afforded its theoretical chemical shift value of δH 1.69 ppm.
The structures of stellettins U (5) and V (6) corresponded to the same C 19 H 30 O 5 molecular formula deduced from HRESIMS ( Figures S36 and S45). In comparison with co-isolated metabolites, the spectral data of compounds 5 and 6 revealed bicyclic core with keto group at C-3, gem-dimethyl group at C-4 and two angular methyls at C-8 and C-10 ( Table 2). Additionally, 1 H-and 13 C-NMR spectra of compound 5 (Figures S37 and S38) demonstrated signals of two carbonyls (δ C 173.7 and 183.8) and one ethoxy group (δ H 4.15, q (7.1); δ C 60.8 and δ H 1.26, t (7.1); δ C 14.1). HMBC experiment (Figure 7 and Figure S41) allowed to place the carboxy group at C-8 and ethyl ester at C-11 on the basis of congruous correlations from methylenes -CH 2 -CH 3 (δ H 4.15, q (7.1), 2H) and CH 2 -11 (δ H 2.41, dd (17.7, 5.3) and 2.30, m) to carboxyl C-12 (δ C . 173.7) and also from methyl CH 3 -30 (δ H 1.17, s) to carboxyl C-13 (δ C 183.8). The relative stereochemistry of 5 was determined by ROESY spectral analysis ( Figure S43). Correlation between H-9 (δ H 2.78, br t (5.0)) and CH 3 .1)). The key HMBC correlations (Figure 7) satisfied the proposed structure of 6. However, since the values of carboxyl carbons shifts for 6 are close, distinguishing their correlations and direct ester positioning without data for isomer 5 brought some uncertainty. To avoid future difficulties with structurally related esters we calculated carbon chemical shift values for two isomers 5 and 6 ( Figure S66). It was shown, that theoretical δC C-13 (5)   The 1 H-and 13 C-NMR signals of the side chain of 1 as well as the form of ECD cu ( Figure S10) were analogous to those of globostelletin K [15] (Figure S57) suggesting same stereochemistry of the side chain. This assignment was in a good agreement w the computational ECD results performed using density functional theory (DFT) with nonlocal exchange-correlation functional B3LYP [16], the polarization continuum mo (PCM) [17] and split-valence basis sets 6-31G(d), implemented in the Gaussian 16 pack of programs [18] ( Figure S66). The 15R,23S absolute configuration, providing 13Z,2 geometry and trans−syn−trans-fused tricyclic moiety with 3β-oriented acetoxy group fu satisfies the similarity of the experimental and theoretical ECD spectra of 1 (Figure 3) detailes, statistically avereged curve (Figure 3) follows the shape of the experimental o although even more close coincidence was indicated for theoretically less proba conformer ( Figure S67). In addition, we could conclude that the presence of 3-acetoxy 3-oxo functions in the structures of the corresponding compounds insignificantly affe the shape of their ECD curves. According to obtained new data we pose the same 15R, stereochemistry for globostelletin K ( Figure S69). ) correlations of 5 and 6.
Both compounds were supposed to be the half-ester derivatives of the hypothetical dicarboxylic acid. The anhydrous form of the acid was reported by Ravi et al. [5] as a product of ozonolysis of isomalabaricane precursor [22,23]. Moreover, Ravi et al. obtained dimethyl and monomethyl esters of the acid and did not point the place of esterification in the case of the latter.
Among isolated new compounds 1-6, we find stellettin S (3) the most intriguing, since occurrences of acetylene-containing isoprenoids are rare and not so far reported in the isomalabaricane series. To date, several biosynthetic pathways leading to the alkyne formation in natural products has been supported with identified and characterized gene clusters. In the first case, acetylenases, a special family of desaturases, catalyze the dehydrogenation of olefinic bonds in unsaturated fatty acids to afford acetylenic functionalities [24,25]. Next, acetylenases are also used to form the terminal alkyne in polyketides [26]. One more biosynthetic route results in a terminal alkyne formation in acetylenic amino acids and involves consequent transformations by halogenase BesD, oxidase BesC and lyase BesB [27]. Finally, two recent papers describe the molecular basis for the formation of alkyne moiety in acetylenic prenyl chains occurring in a number of meroterpenoids [28,29]. The abovementioned reports highlight hot trends in a scientific search for enzymatic machineries leading to the biologically significant and synthetically applicable acetylene bond in natural compounds. We believe that isolation of the new terpenoidal alkyne 3 could inspire further investigations of the Stelletta spp. sponges and associated microorganisms through genome mining.
According to obtained new data we also report the correction in stereochemistry of two asymmetric centers in globostelletins M ( Figure S70) and N ( Figure S71). Really, their ECD and NMR spectra in comparison with those of globostelletin K and stellettins Q and R ( Figures S59-S71) clearly show rather 15R,23S configuration for globostelletin M instead of previously reported 15S,23S [15] as well as 15S,23R stereochemistry for globostelletin N instead of 15R,23R [15].