Cytosporin Derivatives from Arctic-Derived Fungus Eutypella sp. D-1 via the OSMAC Approach

A chemical investigation of the Arctic-derived fungus Eutypella sp. D-1 based on the OSMAC (one strain many compounds) approach resulted in the isolation of five cytosporin polyketides (compounds 1–3 and 11–12) from rice medium and eight cytosporins (compounds 2 and 4–11) from solid defined medium. The structures of the seven new compounds, eutypelleudesmane A (1), cytosporin Y (2), cytosporin Z (3), cytosporin Y1 (4), cytosporin Y2 (5), cytosporin Y3 (6), and cytosporin E1 (7), were elucidated by analyzing their detailed spectroscopic data. Structurally, cytosporin Y1 (4) may be a key intermediate in the biosynthesis of the isolated cytosporins, rather than an end product. Compound 1 contained a unique skeleton formed by the ester linkage of two moieties, cytosporin F (12) and the eudesmane-type sesquiterpene dihydroalanto glycol. Additionally, the occurrence of cyclic carbonate moieties in compounds 6 and 7 was found to be rare in nature. The antibacterial, immunosuppressive, and cytotoxic activities of all compounds derived from Eutypella sp. D-1 were evaluated. Unfortunately, only compounds 3, 6, 8, and 10–11 displayed immunosuppressive activity, with inhibitory rates of 62.9%, 59.5%, 67.8%, 55.8%, and 68.7%, respectively, at a concentration of 5 μg/mL.

The OSMAC approach has emerged as a powerful tool in the field of natural product biodiscovery, stimulating the production of a wider range of new metabolites [10]. During our exploration of structurally diverse bioactive natural products from polar fungi, we discovered a series of terpenoids with unique skeleton characteristics from the talented Arctic fungus strain Eutypella sp. D-1 [5,7,9]. This strain has proven to be a prolific source of metabolites with diverse biological activities [7,9]. To enhance the chemical diversity of Eutypella sp. D-1, we employed the one strain many compounds (OSMAC) strategy, utilizing different culture conditions. Through high-performance liquid chromatography (HPLC) analysis, some structural analogs during fermentation on two distinct media, solid rice medium and defined solid medium, were dramatically discovered. Subsequent chemical investigation led to the isolation of 12 cytosporin derivatives, including seven new cytosporins-eutypelleudesmane A (1), cytosporin Y (2), cytosporin Z (3), cytosporin Y1 (4), cytosporin Y2 (5), cytosporin Y3 (6), and cytosporin E1 (7)-together with five known biogenetic-related analogs-cytosporin X (8), cytosporin E (9), cytosporin L (10), and cytosporins D and F (11)(12) (Figure 1). Herein, we present the detailed purification, structure elucidation, and bioactive evaluation of these compounds.

Results
Eutypelleudesmane A (1) was isolated as a light-brown oil. The molecular formula was determined as C36H56O7 from HRESIMS and NMR data (Table 1), indicating the presence of nine degrees of unsaturation. The IR spectra confirmed the presence of hydroxy (3357 cm −1 ) and carbonyl (1741 cm −1 ) groups [3][4][5]. Additionally, the 13 C NMR analysis revealed one ester carbonyl signal (δC 171.0) and six double-bond carbon signals (δC 121. 2, 124.7, 125.1, 134.1, 135.6, and 136.4), accounting for four degrees of unsaturation. The remaining five degrees of unsaturation were attributed to the pentacyclic ring structure present in the molecule.
Upon comparing the 1D NMR data of compound 1 and cytosporin F (12), it was observed that one set of signals was similar to compound 12, while the remaining signals resembled a derivative of eudesmane-type sesquiterpene, dihydroalanto glycol. By

Results
Eutypelleudesmane A (1) was isolated as a light-brown oil. The molecular formula was determined as C 36 H 56 O 7 from HRESIMS and NMR data (Table 1), indicating the presence of nine degrees of unsaturation. The IR spectra confirmed the presence of hydroxy (3357 cm −1 ) and carbonyl (1741 cm −1 ) groups [3][4][5]. Additionally, the 13 C NMR analysis revealed one ester carbonyl signal (δ C 171.0) and six double-bond carbon signals (δ C 121. 2, 124.7, 125.1, 134.1, 135.6, and 136.4), accounting for four degrees of unsaturation. The remaining five degrees of unsaturation were attributed to the pentacyclic ring structure present in the molecule. Upon comparing the 1D NMR data of compound 1 and cytosporin F (12), it was observed that one set of signals was similar to compound 12, while the remaining signals resembled a derivative of eudesmane-type sesquiterpene, dihydroalanto glycol. By utilizing 2D NMR correlations (Figure 2), these two structural fragments, labeled as A and B, were deduced. The COSY spectrum revealed the presence of seven continuous spin systems: (a) C-3−C-4, (b) C-6−C-7, (c) C-14-C-15-C-16-C-17-C-18-C-19-C-20, (d) C-23-C-24-C-25, (e) C-27-C-28-C-29-C-30-C-31, (f) C-29-C-33-C-34, and (g) C-33-C-35 ( Figure 2). Fragment A, comprising C-2 to C-22, exhibited similarity to compound 12 based on a comparison of their 1D NMR spectra. HMBC correlations from H-4α to C-2, C-5, C-6, and C-10; from H-6 to C-8; from H-7 to C-5, C-8, and C-9; from H-10 to C-5, C-6, C-8, and C-9; from H 3 -11 and H 3 -12 to C-2 and C-3; and from H 2 -13 to C-8, C-9, and C-10 were detected. These correlations, along with the chemical shift of C-2 (δ C 76.6) and C-10 (δ C 67.5), indicated the formation of two six-membered rings by connecting C-5 (δ C 55.7) with C-10 and C-2 with C-10 via an O-atom, as well as the location of the two methyl groups CH 3 -11 and CH 3 -12 both at C-2 and one methylene group CH 2 -13 at C-9. The presence of an oxirane resulting from the conjugation of C-5 and C-6 via O-atom was supported by the downfield shift of C-5 and C-6 (δ C 59.7) [3,4]. Furthermore, the direct linkage between C-8 and C-14 was established by HMBC correlations from H-14 to C-7, C-8, and C-9. An additional acetyl group was identified to be connected to C-13 based on the HMBC correlations from H-13 and H-22 to C-21 and the chemical shift of C-13 (δ C 61.5). Fragment B, spanning from C-23 to C-37, exhibited characteristics of a eudesmane-type sesquiterpene moiety, as deduced from the analysis of the remaining 1 H and 13 C NMR data. HMBC correlations from H-31α and H-31β to C-27 and C-32; from H 2 -23 to C-27 and C-32; and from H 3 -36 to C-23, C-27, C-31, and C-32 confirmed the presence of a linkage of C-23, C-27, and C-31 via the quaternary carbon C-32, and placed the methyl group CH 3 -36 at C-32 as well. The methyl group CH 3 -37 was demonstrated to be connected to C-25 and C-27 via C-26 by the HMBC correlations from H 3 -37 to C-25, C-26, and C-27. The linkage of fragments A with B through C-3 (δ C 73.8) and C-30 (δ C 66.7) via an O-atom was supported by the downfield resonance of C-3 and C-30, along with HMBC correlations from H-30 to C-3. Additionally, the connection of two hydroxyl groups with a downfield carbon shift at C-7 (δ C 64.6) and C-34 (δ C 67.5) were determined to satisfy the molecular formula. Consequently, the planar structure of 1 was established as depicted.
group CH3-37 was demonstrated to be connected to C-25 and C-27 via C-26 by the HMBC correlations from H3-37 to C-25, C-26, and C-27. The linkage of fragments A with B through C-3 (δC 73.8) and C-30 (δC 66.7) via an O-atom was supported by the downfield resonance of C-3 and C-30, along with HMBC correlations from H-30 to C-3. Additionally, the connection of two hydroxyl groups with a downfield carbon shift at C-7 (δC 64.6) and C-34 (δC 67.5) were determined to satisfy the molecular formula. Consequently, the planar structure of 1 was established as depicted.  The relative configuration of 1 was established by analyzing coupling constants and NOESY experiments [9]. The trans configuration of the conjugated C-14/C-15 double bond was inferred based on the large coupling constant (16.0 Hz) and the NOESY correlations of H-14/H 2 -16. The observed similarity in the NMR chemical shift values and NOESY correlations of H-7/H-10, H-10/H 3 -12, H 3 -12/H-4β, H-4α/H-6, and H-3/H 3 -11 indicated that the relative configurations of fragment A in 1 were identical to those of 12 [3][4][5]. Additional NOESY correlations of H-30/H 3 -35, H-30/H-36β, and H-31β/H-36β and those of H-27/H-29, H-27/H-31α, and H-27/H-33 indicated the β-orientation and α-orientation of these protons in fragment B, respectively ( Figure 3). Furthermore, the absence of a NOESY correlation between H-3 and H-30 supported the trans relationship between these two protons [5]. Thus, the relative structure of 1 was determined. Furthermore, the characteristic positive Cotton effect at 242 nm in the CD spectrum of 1 was virtually identical to that of cytosporins D and F (11)(12) (Figure 4) [5], which suggested the absolute configuration of 1 was assigned as 3S,5R,6S,7R,10S,27S,29S,30R,32S,33R.
Cytosporin X (2) was obtained as a light-brown oil and determined to have a molecular formula of C 19 H 30 O 4 based on HRESIMS and NMR data, corresponding to an unsaturation index of 5. The presence of hydroxy functionality was indicated by IR absorption bands at 3359 cm −1 . The 13 C NMR (Table 2) and DEPT spectra revealed the presence of 19 carbons, including six double-bond carbon signals (δ C 117. 3, 124.6, 131.4, 131.6, 135.4, and 135.9) and five oxygenated carbon signals (δ C 57. 4, 59.3, 62.2, 64.3, and 69.5). The COSY spectrum of 2 showed three distinct spin systems: C-2/C-3, C-7/C-8/C-9/C-10/C-11/C-12/C-13, and C-15/C-16 ( Figure 2). HMBC correlations from H-2 to C-4 and C-6; from H-3 to C-1 and C-4; from H-6 to C-1, C-2, and C-4; and from H 2 -14 to C-4, C-5, and C-6, along with the comparison of the chemical shifts of C-1 (δ C 59.3) and C-2 (δ C 57.5) to those of cytosporins D and F [3,4], determined the oxirane-fused cyclohexene moiety with one methylene group (CH 2 -14) attached at C-5. The isoamylene group was connected to C-1 based on the HMBC correlations from H 2 -15 to C-1, C-2, and C-6, as well as from H 3 -18 and H 3 -19 to C-16 and C-17. Further HMBC correlations from H-7 to C-3, C-4, and C-5 established the connectivity of C-4 and C-7. With this assignment secured, each of the three oxygenated carbon at C-3 (δ C 64.3), C-6 (δ C 69.5), and C-14 (δ C 62.2) had to be substituted with a hydroxy group to satisfy the molecular formula. The relative stereocenter of 2 was determined from NOESY correlations and coupling constants in comparison with those Mar. Drugs 2023, 21, 382 5 of 13 of 11 and 12 [3,4]. The conjugated C-7/C-8 double bond was assigned as trans upon its large coupling constant (16.0 Hz). The NOESY correlations of H-2/H 2 -15 and H-6/H 2 -15 in CDCl 3 and 3-OH/H-6 in DMSO-d 6 (Figure S16), combined with the similarity between the calculated and the experimental ECD spectra, confirmed the absolute configurations of 2 as 1R,2S,3R,6R ( Figure 4).  4 . The similarity of the 1 H and 13 C NMR data between 3 and 11 indicated that compound 3 was the derivative of 11. The presence of a pentasubstituted benzene moiety (δ H 6.61 (1H, s); δ C 115.1 (CH), 118.8 (C), 123.6 (CH), 126.8 (C), 144.8 (C), and 146.7 (C)) instead of the oxirane-fused cyclohexene moiety in 11 was suggested by the 1 H and 13 C NMR spectra. This was further confirmed by the further HMBC correlations from H-4β to C-5, C-6, and C-10; from H-6 to C-7, C-8, and C-10; from H 2 -13 to C-8, C-9, and C-10; and from H-14 to C-7, C-8, and C-9 ( Figure 2). Additionally, one hydroxy group was attached to C-7, as evidenced by its chemical shift (δ C 146.7) and the molecular formula. The conjugated C-14/C-15 double bond in 3 was assigned as trans based on the similar 1 H NMR chemical shift values and coupling constants (16.5 Hz)  27/H-29, H-27/H-31α, and H-27/H-33 indicated the β-orientation and α-orientation of these protons in fragment B, respectively ( Figure 3). Furthermore, the absence of a NOESY correlation between H-3 and H-30 supported the trans relationship between these two protons [5]. Thus, the relative structure of 1 was determined. Furthermore, the characteristic positive Cotton effect at 242 nm in the CD spectrum of 1 was virtually identical to that of cytosporins D and F (11)(12) (Figure 4) [5], which suggested the absolute configuration of 1 was assigned as 3S,5R,6S,7R,10S,27S,29S,30R,32S,33R.     Cytosporin X (2) was obtained as a light-brown oil and determin ular formula of C19H30O4 based on HRESIMS and NMR data, correspo uration index of 5. The presence of hydroxy functionality was indicate bands at 3359 cm -1 . The 13 C NMR (Table 2) and DEPT spectra revealed carbons, including six double-bond carbon signals (δC 117.3, 124.6, 131 135.9) and five oxygenated carbon signals (δC 57.4, 59.3, 62.2, 64.3, an spectrum of 2 showed three distinct spin systems: C-2/C-3, C-7/C-8/C-9 13, and C-15/C-16 ( Figure 2). HMBC correlations from H-2 to C-4 and 1 and C-4; from H-6 to C-1, C-2, and C-4; and from H2-14 to C-4, C-5, a the comparison of the chemical shifts of C-1 (δC 59.3) and C-2 (δC 57 sporins D and F [3,4], determined the oxirane-fused cyclohexene moi ylene group (CH2-14) attached at C-5. The isoamylene group was con on the HMBC correlations from H2-15 to C-1, C-2, and C-6, as well as 19 to C-16 and C-17. Further HMBC correlations from H-7 to C-3, C-4, Cytosporin Y1 (4), in the form of a light-yellow oil, had a molecular formula of C 19 H 32 O 5 based on HRESIMS (m/z 363.2139 [M + Na] + ), which is larger than that of cytosporin Y (2) by 18 amu. The NMR data of 4 (Table 3) were nearly identical to those of 2, indicating the same carbon skeleton. Considering the degrees of unsaturation of 4, the observed downfield shift of one quaternary carbon (δ C 59.3) and one methine (δ H /δ C 3.29/57.5) in 2 to δ C 74.3 and δ H /δ C 3.76/75.0 in 4, respectively, suggested that 4 was the oxirane ring-opening product of 2. This hypothesis was further supported by further COSY and key HMBC correlations, as shown in Figure 2. The E-geometry of the ∆ 7,8 double bond was deduced from a NOESY correlation between H-7 and H 2 -9, as well as the coupling constants (16.5 Hz). Additional NOESY correlations of H-2/H 2 -15, H-2/H-6, H-3/H-6, and H-6/H 2 -15 indicated the same orientation of these protons. Furthermore, the comparison of the calculated and the experimental ECD spectra confirmed the absolute configurations of 4 as 1S,2R,3R,6S ( Figure 5).  Cytosporin Y2 (5) was obtained as a light-yellow oil. Extensive NMR analyses and HRESIMS data (m/z 389.1928 [M + Na] + ) led to the determination of its molecular formula as C20H30O6. The overall NMR data of 5 indicated a structure similar to 4, with the notable difference of an additional quaternary carbon. This carbon was identified as a carbonate moiety based on the strong IR absorption at 1647 cm −1 and the diagnostic 13 C NMR signal at δC 154.8 [4]. Another significant difference was observed for C-1 and C-2, resonating at δC 74.3 and 75.0 in compound 4, whereas in compound 5, these signals resonated at δC 84.4 and 71.3, respectively (Table 3). This observation, along with the key HMBC correlations from H-2 to C-20, led to the linkage of the carbonyl to both oxygen atoms at C-1 and C-2 to form a cyclic carbonate moiety. The relative configurations of 5 were determined via a detailed analysis of the NOESY correlations of H-2/H-15a, H-3/H-15b, H-6/H-15a, H-6/H-15b, and H-7/H2-9, as well as the coupling constants (16.5 Hz) of H-7/H2-9. Furthermore, the calculated ECD spectrum of 5 exhibited a close resemblance to the experimental one, confirming the absolute configuration as 1R,2R,3R,6S ( Figure 5).
Cytosporin Y3 (6) was isolated as a light-yellow oil. Its molecular formula was determined to be C20H30O6, the same as that of 5, based on HRESIMS data. A comparison of the IR, UV, and NMR data ( Table 4) of 6 with those of 5 suggested that 6 was an isomer of 5. Cytosporin Y2 (5) was obtained as a light-yellow oil. Extensive NMR analyses and HRESIMS data (m/z 389.1928 [M + Na] + ) led to the determination of its molecular formula as C 20 H 30 O 6 . The overall NMR data of 5 indicated a structure similar to 4, with the notable difference of an additional quaternary carbon. This carbon was identified as a carbonate moiety based on the strong IR absorption at 1647 cm −1 and the diagnostic 13 C NMR signal at δ C 154.8 [4]. Another significant difference was observed for C-1 and C-2, resonating at δ C 74.3 and 75.0 in compound 4, whereas in compound 5, these signals resonated at δ C 84.4 and 71.3, respectively (Table 3). This observation, along with the key HMBC correlations from H-2 to C-20, led to the linkage of the carbonyl to both oxygen atoms at C-1 and C-2 to form a cyclic carbonate moiety. The relative configurations of 5 were determined via a detailed analysis of the NOESY correlations of H-2/H-15a, H-3/H-15b, H-6/H-15a, H-6/H-15b, and H-7/H 2 -9, as well as the coupling constants (16.5 Hz) of H-7/H 2 -9. Furthermore, the calculated ECD spectrum of 5 exhibited a close resemblance to the experimental one, confirming the absolute configuration as 1R,2R,3R,6S ( Figure 5). Cytosporin Y3 (6) was isolated as a light-yellow oil. Its molecular formula was determined to be C 20 H 30 O 6 , the same as that of 5, based on HRESIMS data. A comparison of the IR, UV, and NMR data (Table 4) of 6 with those of 5 suggested that 6 was an isomer of 5. Further analysis of the 13 C NMR chemical shift of C-2 (δ C 79.2) and C-3 (δ C 75.2), along with the unambiguous HMBC correlations from H-2 and H-3 to C-20 of 2, revealed that the cyclic carbonate moiety was fused with C-2-C-3 in 6. The relative configuration and E-geometry of the ∆ 7,8 double bond in 6 were determined from the NOESY correlations of H-2/H-3, H-2/H-6, H-3/H-6, H-2/H-15b, H-6/H-15a, H-6/H-15b, and H-7/H 2 -9. The absolute configuration of 6 was subsequently determined to be 1R,2S,3S,6R based on the opposite CD spectra ( Figure 6)       Cytosporin E1 (7) was also purified as a light-yellow oil and exhibited a HRESIMS ion peak at m/z 407.2034 [M + Na] + , consistent with the molecular formula C 20 H 32 O 7 with five degrees of unsaturation. The 1 H and 13 C NMR data of 7 (Table 4) closely resembled those of the known compound cytosporin E (9), except for two additional methylenes (δ C /δ H 31.0/2.26 and 30.0/1.49) in 7 and the absence of two olefinic methines (δ C /δ H 123.5/6.39 and 137.1/6.03) in 9. These observations indicated that C-8 in 7 was substituted by a heptane subunit instead of the 1-heptene part in 9, which was also confirmed by COSY correlations of H 2 -14 (δ H 2.26 2 -14 to C-7 (δ C 78.5) and C-8 (δ C 133.3). The relative configuration of 7 was inferred to be different from that of compound 9 through a comparison of the 13 C NMR data between 7 (C-6 δ C 81.3 and C-7 δ C 78.5) and 9 (C-6 δ C 81.0 and C-7 δ C 75.3), as well as the analysis of NOESY correlations of H-3/H 3 -11, H-4α/H-6, H-4α/H-7, H-4β/H-10, H-4β/H 3 -12, and H-10/H 3 -12 in MeOD-d 4 and 3-OH/H-10 and 5-OH/H-10 in DMSO-d 6 ( Figure S75). The absolute configurations of 7 were subsequently assigned as 3S,5R,6S,7S,10S based on the similarity of its calculated and the experimental ECD spectra ( Figure 6).
Structurally, considering the close relationship in biosynthesis among compounds 1-12, a biosynthetic pathway different from the previous literature for these compounds is proposed (Figure 7) [3]. The possible precursor originated from phenylmethanol [3]. The subsequent addition of an isoprenyl unit, followed by hydroxylation and the addition of an aliphatic chain, would give the intermediate i. The hydroxylation of the C-1/C-6 double bond in i gave rise to the key intermediate 4. Compound 2 was derived from 4 via a dehydration cyclization reaction. Compound 3 was generated from i via the epoxidation of the C-16/C-17 double bond and a cyclization reaction. Compounds 5 and 6 were derived from the dehydration reaction of compound 4 with carbonic acid by different attack directions and substitution positions, respectively. Compounds 7, 10, and 11 were obtained from 6, 4, and 2 via the same cyclization reaction as 3, respectively. Compound 9 was derived from 10 via the carbonic acid substitution, while compound 8 was formed through the hydrogenation of 11. The cyclization of compound 2, followed by an acetylation reaction, resulted in the formation of compound 12. Another possible precursor, the eudesmane-type sesquiterpene dihydroalanto glycol, was generated from farnesyl pyrophosphate with two steps of cyclization, dehydrogenation, and hydroxylation reaction [15]. Then, 1 was formed from the above two precursors, ii and 12, via a condensation reaction.

Fungal Material
The fungus Eutypella sp. D-1 (GenBank accession number FJ430580) was separated from the sample collected near London Island of Kongsfjorden in the Ny-Ålesund District of the Arctic area and recognized based on 18S rDNA gene sequence analysis. The strain

Fungal Material
The fungus Eutypella sp. D-1 (GenBank accession number FJ430580) was separated from the sample collected near London Island of Kongsfjorden in the Ny-Ålesund District of the Arctic area and recognized based on 18S rDNA gene sequence analysis. The strain (No. D-1) was deposited in the Department of Marine Biomedicine and Polar Medicine, Naval Medical Center of PLA, Naval Medical University.