2.1. Structure Elucidation
The molecular formula of
1 was established to be C
15H
22O
3 from a HRESIMS peak at
m/
z 273.1461 [M + Na]
+ and by
13C NMR analyses. A close inspection of
1H NMR,
13C NMR, DEPT, and HSQC data of
1 (
Table 1 and
Table 2) revealed the presence of two methyl (δ
H 1.26, δ
C 23.2 and δ
H 1.03, δ
C 18.4) groups, four methylenes (δ
C 39.9, 32.7, 28.7), including one oxygen-bearing (δ
H 4.11, 3.89, δ
C 60.9), and seven methines (δ
H 1.89, δ
C 37.5, δ
H 1.41, δ
C 40.2, δ
H 1.65, δ
C 48.6, δ
H 2.09, δ
C 62.8, δ
H 6.24, δ
C 130.1, δ
H 5.69, δ
C 131.6), together with one oxygen-bearing (δ
H 2.92, δ
C 77.9), one carbonyl carbon (δ
C 210.2), and one oxygenated quaternary carbon (δ
C 74.7). These data and five unsaturated degrees from the molecular formula suggested that compound
1 possessed three rings. COSY-45 data and HMBC correlations (
Figure 2) from H-4 (δ
H 2.09) to C-5 (δ
C 37.5), C-10 (δ
C 48.6), C-14 (δ
C 23.2), from H-5 (δ
H 1.89) to C-4 (δ
C 62.8), C-6 (δ
C 28.7), C-10, C-13 (δ
C 74.7), from H
2-6 (δ
H 1.28, 1.14) to C-5, C-7 (δ
C 32.7), C-8 (δ
C 40.2) and C-10, from H-7a (δ
H 1.78) to C-5, C-6, C-8 and C-15 (δ
C 18.4), from H-7b (δ
H 1.08) to C-9 (δ
C 77.9), from H-8 (δ
H 1.41) to C-7, C-9 and C-10, from H
3-15 (δ
H 1.03) to C-7, C-8 and C-9, from H-9 (δ
H 2.92) to C-10, C-11 (δ
C 130.1), C-15, from H-10 (δ
H 1.65) to C-11, C-12 (δ
C 131.6), from H-11 (δ
H 6.24) to C-13, from H-12 (δ
H 5.69) to C-4, C-13, and C-14 revealed the presence of a decalin moiety and established a Δ
11 double bond and the location of methyl groups at C-8 and C-13 in
1.
HMBC correlations from H-1a (δ
H 4.11) to C-2 (δ
C 39.9), C-3 (δ
C 210.2) and C-13, from H-2a (δ
H 2.64) to C-1 (δ
C 60.9), C-3, from H-2b (δ
H 2.21) to C-3, C-4 established a saturated γ-pyrone derivative moiety in
1. NOESY cross-peaks H
3-14 (δ
H 1.26)/H-4 (δ
H 2.09), H-1β (δ
H 3.89); H
3-15 (δ
H 1.03)/H-9 (δ
H 2.92); H-5/H-2β (δ
H 2.64), H-6β (δ
H 1.28), H-7β (δ
H 1.08), H-9; H-10 (δ
H 1.65)/H-6α (δ
H 1.14), H-8 (δ
H 1.41), H-4; H-9/H
3-15, H-5 and H-4/H-6α, H-10, H
3-14 indicated a
trans-ring fusion of the A and B rings,
cis-ring fusion of the B and C rings, the α-orientation of H
3-14 and 9-OH, and the β-orientation of H
3-15. The planar structure and relative configuration of
1 were unequivocally confirmed by X-ray analysis, which was carried out for a single crystal obtained by recrystallization from acetonitrile–water (
Figure 2 and
Figure S9).
Esterification of
1 with (
R)- and (
S)-MTPA chloride occurred at the C-9 hydroxy group to give the (
S)- and (
R)-MTPA esters
1a and
1b, respectively. The observed chemical shift differences Δδ (δ
S − δ
R) (
Figure 3) indicated the 9
R configuration. These data and observed NOESY correlations determined the absolute stereostructure of
1 as 4
R, 5
S, 8
S, 9
R, 10
R, and 13
S. Compound
1 was named zosteropenilline A.
The molecular formula of 2 was established to be C15H22O3 from a HRESIMS peak at m/z 273.1474 [M + Na]+ and by 13C NMR analyses. The general features of the 13C NMR of 2 resembled those of 1 with the exception of the C-9–C-11 and C-15 carbon signals. COSY and HSQC spectra of 2 revealed the partially connectivity sequence of the protons in the A ring as CH2(15)-CH(8)-CH2(9)-CH(10). These data and HMBC correlations from H-9b (δH 0.93) to C-7 (δC 29.1), C-8 (δC 40.5), C-10 (δC 41.4), C-11 (δC 134.4), and C-15 (δC 68.1); and from H2-15 (δH 3.50, 3.47) to C-7, C-8 and C-9, established the A ring structure, lacking the hydroxy group at C-9, and indicated the presence of a hydroxymethyl group at C-8 in 2. The NOESY cross-peaks H-4 (δH 2.07)/H-10 (δH 1.74), H3-14 (δH 1.26); H-8 (δH 1.63)/H-9α (δH 1.91), H-10; H-5 (δH 1.78)/H-9β (δH 0.93); H-9β/H2-15 (δH 3.50, 3.47) indicated the trans-ring fusion of the A and B ring, cis-ring fusion of the B and C ring, and the β-orientation of hydroxymethyl group at C-8. Compound 2 was named zosteropenilline B.
The molecular formula of
3 was established to be C
15H
22O
3 from a HRESIMS peak at
m/
z 273.1463 [M + Na]
+ and by
13C NMR analyses. The
1H and
13C NMR spectra (
Table 1 and
Table 2) for this compound were very similar to those obtained for zosteropenilline B (
2) with the exception of the C-6–C-10 and C-15 carbon and proton signals. The HMBC correlations H-9a (δ
H 1.75)/C-8 (δ
C 69.7), C-10 (δ
C 36.9); H
3-15 (δ
H 1.25)/C-7 (δ
C 24.9), C-8, C-9 (δ
C 44.5) established the structure of
3 including the hydroxy and methyl groups at the C-8 position. The relative configuration of
3 was defined based on the observed NOESY correlations (
Figure S27). Compound
3 was named zosteropenilline C.
The molecular formula of
4 was established to be C
15H
22O
2 from a HRESIMS peak at
m/
z 257.1510 [M + Na]
+ and by
13C NMR analyses. The
1H and
13C NMR data (
Table 1 and
Table 2) observed for
4 closely resembled those obtained for zosteropenilline B (
2) with the exception of the C-7–C-9 and C-15 carbon and proton signals. The mutual correlations from H
3-15 to C-7 and C-9 established the structure of the A ring and location methyl group at C-8. Compound
4 was named zosteropenilline D.
The absolute configurations of zosteropenillines B–D (
2–
4) were determined on the basis of the ECD spectroscopy data. The geometry of the stable conformations of compounds
1–
4 were optimized using general procedure, described in the experimental section of the paper. The internal rotations of the hydroxyl groups as well as the inversions of the six-membered rings A and C were accounted for during fulfilled conformational analysis. We found that for all these compounds ring C can exist in two stable conformations: “chair” and “boat”. The “chair” conformation is more stable for about ΔE
# (chair → boat) ≈ 2 kcal·mol
−1 and the barrier for interconversion between conformations is ΔE
# (chair → boat) ≈ 6 kcal·mol
−1. Analogously, ring A can also exist in “chair” and “boat” conformations. The “chair” conformation is more stable for about ΔE
# (chair → boat) ≈ 10 kcal·mol
−1 and the barrier for interconversion between conformations is ΔE
# (chair → boat) ≈ 14 kcal·mol
−1 (
Figure 4 and
Figure 5).
To obtain theoretical ECD spectra the excitation energies and the rotatory strengths for
1–
4 were calculated using time-dependent density functional theory (TDDFT) with PBE1PBE exchange-correlation functional and cc-pvTz basis set [
8]. A comparison of statistically averaged ECD spectra for
1–
4 with corresponding experimental spectra is presented in
Figure 6. The comparison of all theoretical spectra with the experimental ones showed that all spectra are qualitatively similar in the region λ ≥ 200 nm, where the pronounced Cotton effects occur, thus proving 4
R, 5
S, 8
S, 9
R, 10
R, 13
S absolute configuration for
1; 4
R, 5
S, 8
S, 10
R, 13
S for
2; 4
R, 5
S, 8
R, 10
R, 13
S for
3; 4
R, 5
S, 8
S, 10
R, 13
S for
4.
The molecular formula of
5 was established to be C
15H
24O
3 from a HRESIMS peak at
m/
z 283.1538 [M − H]
− and by
13C NMR analyses. The COSY-45 data and HMBC correlations from H-4 (δ
H 2.11) to C-5 (δ
C 38.5), C-6 (δ
C 29.0), C-10 (δ
C 53.5), from H-5 (δ
H 1.95) to C-6, C-10, from H-6a (δ
H 1.33) to C-4 (δ
C 63.7), C-7 (δ
C 31.8) and C-10, from H-7a (δ
H 1.73) to C-5, C-9 (δ
C 77.2), from H
3-15 (δ
H 1.06) to C-7, C-8 (δ
C 39.2), C-9, from H-9 (δ
H 3.29) to C-5, C-11 (δ
C 67.3), from H-10 (δ
H 1.61) to C-5, C-9, C-11, from H-11 (δ
H 4.38) to C-12 (δ
C 72.7), and from H
3-14 (δ
H 1.36) to C-4, C-12, C-13 (δ
C 77.5) revealed the presence of a decalin moiety in
1 and established the location of methyl groups at C-8 and C-13 and hydroxy groups at C-9, C-11 and C-12. HMBC correlations from H-1a (δ
H 4.22) to C-2 (δ
C 37.9), C-3 (δ
C 206.0) and C-13, from H-2a (δ
H 2.67) to C-1 (δ
C 60.9), C-3, from H-4 (δ
H 2.11) to C-2 and C-3 established a saturated γ-pyrone derivative in
5 (
Figure S40). The relative configuration of
5 was assigned based on NOESY cross-peaks (
Figure 7) and
1H-
1H coupling constants (
Table 2). Observed NOESY correlations and magnitudes of the vicinal coupling constants between H-4 and H-5; H-5 and H-10; H-9 and H-10; H-10 and H-11; H-11 and H-12 indicated a
trans-ring fusion of the A and B rings,
cis-ring fusion of the B and C rings, the
α-orientation of H
3-14, 9-OH, 11-OH groups and the β-orientation of H
3-15 and 12-OH groups. Compound
5 was named zosteropenilline E.
The molecular formula of
6 was established to be C
15H
24O
4 from a HRESIMS peak at
m/
z 291.1579 [M + Na]
+ and by
13C NMR analyses. The
13C NMR data of
6 matched those for
1 with the exception of the C-1–C-3 and C-13–C-14 carbon signals. The structure of the decalin moiety and location of the methyl groups at C-8 and C-13 and oxygen functions at C-9 and C-13 were established as for zosteropenilline A (
1) based on COSY and HMBC correlations. The COSY-45 data and HMBC correlations from H
2-1 (δ
H 3.91) to C-2 (δ
C 48.6) and C-3 (δ
C 217.6), from H
2-2 (δ
H 2.86, 2.76) to C-1 (δ
C 57.6) and C-3, and from H-4 (δ
H 2.59) to C-3 indicated the presence of a 3-hydroxy-1-oxopropyl residue (C-1–C-3 residue numbering) at C-4. The relative configuration of
6 was assigned on the basis of a NOESY experiment and
1H-
1H coupling constants (
Table 2). Compound
6 was named zosteropenilline F. Zosteropenilline F is an epimer of the pallidopenilline A (
13) which was earlier isolated from the
P. thomii associated with the brown alga
Sargassum pallidum [
7]. The
1H and
13C NMR data observed for
6 closely resembled those obtained for pallidopenilline A (measured in CDCl
3) (
Figures S58–S68) with the exception of the C-4, C-13 and C-14 carbon and proton signals The molecular formula of
7 was established to be C
15H
24O
3 from a HRESIMS peak at
m/
z 275.1621 [M + Na]
+ and by
13C NMR analyses. The
13C NMR data (
Table 3) observed for
7 closely resembled those obtained for zosteropenilline D (
4) with the exception of the C-1–C-3 and C-13–C-14 carbon signals. The structure of the decalin moiety, location of the methyl groups at C-8 and C-13, oxygen functions at C-13 and 3-hydroxy-1-oxopropyl residue at C-4 were established by HMBC and COSY correlations (
Figures S74 and S75). The relative configuration of
7 was assigned based on NOESY correlations H-4 (δ
H 2.89)/H-10 (δ
H 1.84); H-10/H-8 (δ
H 1.46) and H-5 (δ
H 1.48)/H
3-14 (δ
H 1.20) and H
3-15 (δ
H 0.89) recorded in CDCl
3 and 13-OH (δ
H 4.95)/H-4 correlation in spectrum of
7 recorded in DMSO-
d6 (
Figure S77). Compound
7 was named zosteropenilline G.
The molecular formula of
8 was established to be C
15H
24O
4 from a HRESIMS peak at
m/
z 291.1571 [M + Na]
+ and by
13C NMR analyses. The
1H NMR,
13C NMR, DEPT and HSQC spectra of
8 (
Table 3 and
Table 4) displayed obvious signals for two methyl (δ
H 1.60, δ
C 20.9 and δ
H 1.02, δ
C 17.8) groups, four methylenes (δ
C 43.6, 30.5, 31.9) including one oxygen-bearing (δ
H 3.85, 2H, δ
C 57.9), seven methines (δ
H 1.69, δ
C 37.9, δ
H 1.47, δ
C 39.2, δ
H 1.28, δ
C 49.6, δ
H 2.92, δ
C 61.7, δ
H 3.25, δ
C 82.3, δ
H 4.32, δ
C 73.2, δ
H 5.53, δ
C 129.0,) including two oxygen-bearing, one carbonyl carbon (δ
C 214.0) and one
sp3 quaternary carbon (δ
C 131.5). The structure of a 1,2,3,4-tetrasubstituted cyclohexane ring and side chain in
8 were found by extensive NMR spectroscopy (
1H and
13C NMR, COSY, HSQC and HMBC) as for zosteropenillines E (
5) and F (
6). HMBC correlations from H-4 (δ
H 2.92) to C-5 (δ
C 37.9), C-12 (δ
C 129.0), C-13 (δ
C 131.5), from H-10 (δ
H 1.28) to C-5, C-11 (δ
C 73.2), C-12, from H-11 (δ
H 4.32) to C-12, C-13, and from H
3-14 (δ
H 1.60) to C-4 (δ
C 61.7), C-12, C-13 established a B ring structure, Δ
12 double bond and the location of the hydroxy group at C-11. The relative configuration of
8 was assigned based on NOESY cross-peaks H-4/H-10; H-5 (δ
H 1.69)/H-9 (δ
H 3.25), H-11, H
3-15 (δ
H 1.02); H-8 (δ
H 1.47)/H-10; H-9/H-11 and
1H-
1H coupling constants (
Table 4). Compound
8 was named zosteropenilline H.
The molecular formula of 9 was established to be C15H22O3 from a HRESIMS peak at m/z 273.1462 [M + Na]+ and by 13C NMR analyses. Structures of an A ring and side chain in 9 were established as for zosteropenillines E (5) and F (6). Observed long-range COSY correlations H-4 (δH 3.15)/H2-14 (δH 4.99, 4.59), H-12 (δH 6.20)/H-14b. and HMBC correlations from H-4 (δH 3.15) to C-13 (δC 140.8), C-14 (δC 112.8), from H-9 (δH 2.89) and H-10 (δH 1.85) to C-11 (δC 130.6) and from H2-14 (δH 4.99, 4.59) to C-4 (δC 59.6), C-12 (δC 129.3), C-13 indicated the presence of a diene system at the C-11 and C-13(14) in 9. The relative configuration of 9 was assigned based on the NOESY correlations H-4/H-10; H-8 (δH 1.43)/H-10 and H-5 (δH 1.68)/H-9, H3-15 (δH 1.05). Compound 9 was named zosteropenilline I.
The molecular formula of
10 was established to be C
15H
24O
4 from a HRESIMS peak at
m/
z 291.1573 [M + Na]
+ and by
13C NMR analyses. The
13C NMR data (
Table 3) observed for
10 matched the data reported for zosteropenilline G (
7) with the exception of the C-7–C-9 and C-15 carbon signals. These data, together with the molecular mass difference of 16 mass units between
7 and
10, indicated the presence of the hydroxymethyl group at C-8 in
10 instead of a methyl group. NOESY cross-peaks H-4 (δ
H 2.92)/H-6b (δ
H 0.95), H-10 (δ
H 1.87); H-5 (δ
H 1.52)/H-7b (δ
H 1.02), H-9b (δ
H 0.82), H
3-14 (δ
H 1.20); H-7b/H-9b, H
2-15 (δ
H 3.47, 3.45); H-8 (δ
H 1.61)/H-6b, H-10 indicated the
trans-ring fusion of the A and B rings, a β-orientation of the side chain, H
3-14 and hydroxymethyl group at C-8. Compound
10 was named zosteropenilline J.
The molecular formula of 11 was established to be C15H22O5 from a HRESIMS peak at m/z 305.1370 [M + Na]+ and by 13C NMR analyses. The UV spectrum exhibits a λmax at 242 nm (log ε 3.46), consistent with the enone system in 11. The COSY and HSQC spectra of 11 revealed the partial connectivity sequences of the protons in the A ring as CH(5)-CH2(6)-CH2(7)-CH(8) and CH2(15)-CH(8)-CH2(9). These data and HMBC correlations from H-9a (δH 2.60) to C-7 (δC 27.9), C-8 (δC 41.1) and C-15 (δC 67.4), from H-9b (δH 2.01) to C-10 (δC 166.3), from H2-15 (δH 3.58, 3.54) to C-7, C-8 and C-9 (δC 38.3) established the structure of A ring. Long range correlations from H-4 (δH 3.01) to C-5 (δC 39.4), C-10, C-12 (δC 200.3) and C-13 (δC 74.2), from H-11 (δH 5.90) to C-5 and C-13 and from H3-14 (δH 1.19) to C-4 (δC 61.6), C-12, C-13 determined the structure of B ring and indicated the 10-en-12-one position for the enone chromophore in 11. The structure of side chain in 11 was found by NMR spectroscopy as for zosteropenilline F (6). Observed NOESY correlations H-5 (δH 2.89)/H-9b (δH 2.01), H3-14; H2-15/H-9b indicated these protons to be on the same side of the molecule. These data and the magnitude of the coupling constant of the H-4 signal (δH 3.01, J = 9.8 Hz) indicated H-4 and 13-OH to be on the other side of molecule. Compound 11 was named zosteropenilline K.
The molecular formula of
12 was established to be C
15H
24O
5 from a HRESIMS peak at
m/
z 307.1501 [M + Na]
+ and by
13C NMR analyses. The structure of the A ring in
12 was established based on COSY and HMBC correlations as for zosteropenilline K (
11). HMBC correlations from H-11 (δ
H 5.60) to C-5 (δ
C 40.3), C-9 (δ
C 37.2), C-12 (δ
C 68.0), C-13 (δ
C 72.4), from H-12 (δ
H 4.36) to C-4 (δ
C 57.7), C-10 (δ
C 144.5), C-11 (δ
C 118.8), C-13, and from H
3-14 (δ
H 1.20) to C-4, C-12, C-13 established the structure of B ring, location oxygenated functions at C-12, C-13 and methyl group at C-13 in
12. The relative configuration of
12 was defined based on NOESY correlations H-5/H
3-14; H
3-14/H-12; H-4/H-6b; H-6b/H-8 and the magnitude of the coupling constant of the H-4 signal (δ
H 3.09,
J = 10.0 Hz). Compound
12 was named zosteropenilline L. Besides the new zosteropenillines (
1–
12), a known pallidopenilline A (
13) was also isolated from this fungus. For the first time, pallidopenilline A was found in fungus
P. thomii KMM 4675 [
7].