Oxygenated Sesquiterpenes From the Indo-Pacific Nudibranch Ardeadoris rubroannulata : Structure Revision of Pu’ulenal

Abstract

Seven oxygenated sesquiterpenes 1 to 7 each with a drimane framework were isolated from an organic extract of the nudibranch Ardeadoris rubroannulata collected from Eastern Australia. The structure of pu’ulenal 2 was revised by 1D NOESY, providing a 9Z configuration, while isopu’ulenal 3 has the 9E configuration previously ascribed to pu’ulenal.

Introduction

The drimane sesquiterpene polygodial (1) is a distinctive and bioactive natural product isolated from plants^1-4 and from marine molluscs.^5,6 In addition to anti-infective activity, this hot, peppery tasting bicyclic dialdehyde displays antifeedant activity linked to adduct formation with biological amines.^7-9 Although nudibranch molluscs may acquire drimane sesquiterpenes from dietary marine sponges, some species of nudibranchs have been shown to produce polygodial via de novo biosynthesis.^6,10-12 Herein, we report a study on the chemistry of a single specimen of Ardeadoris rubroannulata, a brightly-coloured nudibranch collected from Hancock Reef, S.E. Queensland, Australia, the mantle of which was found to contain polygodial. We also report six other oxygenated terpenes, including pu’ulenal (2) for which a structure revision was undertaken following the isolation of the double bond isomer isopu’ulenal (3). To our knowledge, this work constitutes the first chemical investigation of A. rubroannulata.

Results and Discussion

An organic extract from a single specimen of A. rubroannulata (#1649) collected near Coolum, S.E. Queensland, Australia, provided pu’ulenal 2 ¹³ and isopu’ulenal 3 (Figure 1) along with the known metabolites polygodial 1,^1-6,14 drimenin 4,^15,16 isodrimenin 5,^17,18 cinnamolide 6,^3,16 and euryfuran 7. ¹⁹ The isolated metabolites were separated by normal phase HPLC (20-30% EtOAc in hexanes) following silica flash chromatography. Known compounds were identified through comparison of mass spectrometric data, literature and in-house NMR data. The structures of pu’ulenal 2 and isopu’ulenal 3 were defined following detailed spectroscopic analysis and led to a revision of the C-9/C-11 configuration for 2.

Figure 1.

Structures of sesquiterpenes 1 to 7.

The oxygenated sesquiterpene pu’ulenal (2) was isolated as an amorphous white solid and displayed a sodiated peak in the HRESIMS at m/z 299.1611 [M  +  Na]⁺. The corresponding molecular formula of C₁₇H₂₄O₃, as well as an acetoxy methyl singlet (11-OCOCH₃, δ 2.09) in the ¹H NMR spectrum of 2 suggested there was an acetate group. Three singlet methyl signals at δ_H 0.88 (Me-13), δ_H 0.93 (Me-14) and δ_H 0.98 (Me-15) confirmed the presence of the trimethyl substituted cyclohexane moiety. The 500 MHz HSQC and ¹H NMR spectra of 2 revealed a singlet aldehyde proton (H-12, δ_H 9.62) and signals for two alkenes at δ_H 6.76, δ_C 143.9 (H-7) and δ_H 7.13, δ_C 129.6 (H-11). In addition, the HMBC spectrum of 2 revealed two quaternary alkene carbons (C-8 δ_C 136.0 and C-9 δ_C 127.9), indicating two separate alkenes in the structure (Table 1, see Supplemental Material). There were also HMBC correlations from H-5 to C-3 and C-7, H-6 to C-7 and C-8, H-7 to C-9, H-11 to C-8, C-9, and the acetate carbonyl, while the aldehyde proton H-12 gave HMBC correlations to C-7, C-8, and C-9 (Figure 2).

Figure 2.

Selected HMBC and NOESY correlations observed for pu’ulenal 2 and isopu’ulenal 3.

Table 1.

¹H and ¹³C NMR Data of Terpenes 2 to 3.

	Pu’ulenal (2)		Isopu’ulenal (3)
Position	δ1Ha,b,c	δ13C d	δ1Ha,b,e	δ13C d
1	1.81, br d (12.5)	36.5, CH2	2.65, m	37.8, CH2
	1.53, m	-	1.65, m	-
2	1.62, m	18.5, CH2	1.63, m	18.9, CH2
	1.62, m	-	1.54, m	-
3	1.48, m	42.0, CH2	1.45, m	41.6, CH2
	1.21, td (13.2, 4.1)	-	1.23, m	-
4	-	33.4, C	-	33.4, C
5	1.48, m	48.5, CH	1.43, m	48.2, CH
6	2.51, dt (21.0, 4.9)	25.6, CH2	2.56, m	25.5, CH2
	2.24, ddd (21.0, 11.5, 3.4)	-	2.30, ddd (20.3, 12.2, 2.7)	-
7	6.76, dd (4.9, 3.4)	143.9, CH	6.79, dd (5.9, 2.7)	154.0, CH
8	-	136.0, C	-	136.2, C
9	-	127.9, C	-	125.4, C
10	-	36.0, C	-	39.0, C
11	7.13, s	129.6, CH	8.28, s	133.9, CH
12	9.62, s	191.4, CH	9.47, s	193.4, CH
13	0.88, s	32.4, CH3	0.92, s	33.2, CH3
14	0.93, s	21.4, CH3	0.94, s	22.0, CH3
15	0.98, s	19.3, CH3	1.05, s	19.1, CH3
11- OAc	-	167.8, C	-	167.1, C
11- OAc	2.09, s	20.7, CH3	2.17, s	20.9, CH3

^a

Chemical shifts referenced to ¹H at δ_H 7.27 and δ_C 77.16.

^b

Coupling constant in Hz.

^c

At 500 MHz.

^d

Values taken from 2D NMR data.

^e

At 700 MHz.

The marine natural products literature contains a single report of the isolation of pu’ulenal from the nudibranch Chromodoris albonotata by Schulte and Scheuer. ¹³ Their study reported that the C-9/C-11 alkene of pu’ulenal possessed an E-geometry based on observations following the addition of an NMR shift reagent (Eu[fod]₃). However, when we conducted an 1D NOESY experiment in which H-11 was irradiated, NOE correlations to H-1a, H-1b and Me-15 were revealed, suggesting that pu’ulenal instead possessed a Z-geometry (Figure 2). This was further supported through a 1D NOESY correlation observed to the acetoxy methyl (11-OCOCH₃) on irradiation of the aldehyde proton H-12. Fortuitously, crystallization of a sample of 2 was achieved from 10% EtOAc/hexanes and a suitable crystal then subjected to x-ray crystallographic analysis at the Australian Synchrotron. Processing of this data produced the crystal structure shown in Figure 3, confirming that the C-9/C-11 alkene of pu’ulenal possesses a Z-geometry, and thereby correcting the initial erroneous assignment of the configuration by Schulte and Scheuer. ¹³ In addition, the data allowed for the assignment of the absolute configuration of 2 as 5S,10S.

Figure 3.

ORTEP representation of the crystal structure of pu’ulenal 2 shown with 50% probability ellipsoids.

Isopu’ulenal (3) was isolated as a white amorphous solid and displayed an adduct ion at m/z 299.1618 [M  +  Na]⁺ from HRESIMS, corresponding to a molecular ion of C₁₇H₂₄O₃. The ¹H NMR spectrum of 3 was closely analogous to that of 2, except that the H-11 alkene signal appeared at δ_H 8.28 as opposed to δ_H 7.13 in pu’ulenal. HSQC, HMBC and COSY data acquired at 700 MHz allowed for the chemical shift assignments of all protonated and quaternary carbons in 3, revealing again close similarities to the data obtained for 2. The combination of these results led to the conclusion that 3 possessed the same planar structure as pu’ulenal, differing only in its geometry across the C-9/C-11 alkene, and thereby matching the structure originally proposed for pu’ulenal by Schulte and Scheuer. ¹³ To confirm the C-9/C-11 alkene E-geometry of 3, 1D NOESY experiments were performed, irradiating H-7, H-11 and H-12. Unexpectedly the only NOE correlation observed from these experiments was between H-7 and H-12, while the anticipated correlation between H-11 and H-12 was not observed (Figure 2). This suggested that the s-trans conformer of the C-7/C-8/C-12 α,β-unsaturated aldehyde in 3 is favoured over its s-cis conformer, a phenomenon that has been previously observed in polygodial (1), which possesses a similar structural motif. ¹⁴

The conformation of 3 was explored using molecular modelling and DFT calculations. This study suggested that isopu’ulenal (3) possesses two major conformers (Figure 4), in one of which the aldehyde adopts the s-trans form (∼99%), while the other conformer adopts the s-cis orientation (∼1%). The preference for the s-trans conformer could arise due to a number of different effects such as secondary orbital interactions of the complex conjugated system present in 2 and 3 or through intramolecular attractions between H-11 and the carbonyl oxygen of C-12. This significant preference for the s-trans form also explains the large ¹H chemical shift difference between the H-11 protons of 3 (δ_H 8.28) and 2 (δ_H 7.13). In 3, H-11 is directly in the plane of the C-12 carbonyl group and would be subject to the effects of its deshielding cone.

Figure 4.

Transoid (A) and cisoid (B) conformers of isopu’ulenal 3.

Fortuitously both drimenin 4 and isodrimenin 5 crystallized after HPLC isolation, and their absolute configurations were therefore determined by x-ray crystallographic analysis and found as expected to be (5S, 10S) (Figures 5 & 6). Since the original structural studies on isodrimenin 4^17,18 reported ¹H NMR data alone, we provide below the ¹³C NMR data to supplement the NMR assignment.

Figure 5.

ORTEP representation of the crystal structure of drimenin 4 shown with 50% probability ellipsoids.

Figure 6.

ORTEP representation of the crystal structure of isodrimenin 5 shown with 50% probability ellipsoids.

Experimental

General: Specific rotations were measured at 23 °C on a Jasco P-2000 polarimeter for solutions in CHCl₃ using a 1 mL cell (10 cm path length). NMR data were measured on a Bruker Avance 500 MHz or a Bruker Avance III HD700 MHz spectrometer (5 mm TCI inverse cryoprobe) for solutions in CDCl₃ at 298 K. HSQC and HMBC data were acquired using a ¹J_C−H of 145 Hz, while HMBC spectra were acquired using ⁿJ_C−H of 8 Hz. Low resolution electrospray ionization mass spectrometry (LRESIMS) was performed on a Thermo LCQ Fleet ion trap spectrometer in positive mode. High resolution electrospray ionization mass spectrometry (HRESIMS) was performed on an Orbitrap Elite instrument with a standard ESI source (sodium formate) with MeOH as solvent. Normal phase high performance liquid chromatography (NPHPLC) was undertaken using a Waters 515 pump connected to a Gilson 132 series refractive index detector with a Waters μPorasil (10 μm, 7.8  ×  300 mm) or a Phenomenex Luna (5 μm, 10  ×  250 mm) column, and using isocratic elution conditions at flow rates between 1 to 2 mL/ min. Silica gel 60 G and silica TLC plates F₂₅₄ were purchased from Merck. Solvents were either distilled or were of HPLC grade.

Animal material: The nudibranch specimen A. rubroannulata (#1649) was collected from Hancock Shoal, near Coolum, S.E. Qld in January 2017 and stored at −20 °C until extraction.

Extraction and isolation of metabolites: The frozen nudibranch (5.3 g) was dissected into its mantle (2.1 g), gut (1.3 g) and mantle rim (1.9 g). The individual body parts were finely chopped then extracted with acetone (6  ×  3 mL) and sonicated (5 min). Each extract was filtered through cotton wool and concentrated to an aqueous suspension before partitioning between H₂O (2 mL) and EtOAc (7  ×  3 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered through cotton wool and evaporated under N₂ to yield a light orange oil for the mantle (49.6 mg), a dark green oil for the gut (15.7 mg) and a dark orange oil for the mantle rim (256.1 mg). NP flash column chromatography, with a stepwise solvent gradient (hexanes: hexanes/DCM: DCM: DCM/EtOAc: EtOAc: MeOH) was used to further separate the mantle extract. Fractions eluting from hexanes (100%) provided euryfuran 7 (0.8 mg). Fractions eluting from hexanes/DCM (1:1) and hexanes/DCM (1:4) were separated by NP HPLC (20% EtOAc in hexanes) to give drimenin 4 (1.2 mg), isodrimenin 5 (2.6 mg), cinnamolide 6 (0.6 mg), polygodial 1 (1.7 mg), pu’ulenal 2 (4.3 mg) and isopu’ulenal 3 (0.8 mg).

Pu’ulenal (2)

White crystals. [α]_D: − 48 (c 0.18 CHCl₃). HR-ESI-MS m/z [M  +  Na]⁺ calcd. for C₁₇H₂₄NaO₃: 299.1618; found: 299.1611. ¹H NMR (CDCl₃, 500 MHz) and ¹³C NMR (CDCl₃, 500 MHz) are presented in Table 1.

Isopu’ulenal (3)

Colorless oil. [α]_D: − 45 (c 0.02 CHCl₃). HR-ESI-MS m/z [M  +  Na]⁺ calcd. for C₁₇H₂₄NaO₃: 299.1618; found: 299.1618. ¹H NMR (CDCl₃, 700 MHz) and ¹³C NMR (CDCl₃, 700 MHz) are presented in Table 1.

¹³C NMR data for isodrimenin (5) (CDCl₃, 700 MHz) δ 172.3 (C-11), 159.0 (C-8), 135.6 (C-9), 70.4 (C-12), 52.3 (C-5), 41.7 (C-3), 34.9 (C-10), 34.4 (C-1), 33.4 (C-13), 33.1 (C-4), 25.2 (C-7), 21.3 (C-14), 18.2 (C-2), 18.0 (C-6).

Crystal Structure Determination of Pu’ulenal 2, Drimenin 4 and Isodrimenin 5

Data were collected at the MX1 beamline of the Australian Synchrotron with Silicon Double Crystal monochromated radiation at 100(2) K (λ  =  0.7108 Å) ²⁰ . Data integration and reduction were undertaken with XDS. ²¹ Subsequent computations were carried out using Olex2. ²² Structures were solved with ShelXT ²³ and refined and extended with ShelXL. ²⁴ Carbon-bound hydrogen atoms were included in idealised positions and refined using a riding model. The Flack ²⁵ parameter unambiguously confirmed the absolute structures. Crystallographic data is summarized below, and the CIFs have been deposited at the Cambridge Crystallographic Data Centre with CCDC numbers 2107013 to 2107015 for 2, 4 and 5, respectively. These data are available free of charge from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1 EZ, UK; fax: ( + 44) 1223-336-033; or e-mail: [e-mail]

Crystal data for C₁₇H₂₄O₃ (M  =  276.36 g/mol): orthorhombic, space group P2₁2₁2₁ (no. 19), a  =  6.2730(12) Å, b  =  11.436(2) Å, c  =  20.720(4) Å, V  =  1486.4(5) ų, Z  =  4, T  =  100.15 K, μ(MoKα)  =  0.083 mm⁻¹, Dcalc  =  1.235 g/cm³, 27 409 reflections measured (3.932° ≤ 2Θ ≤ 63.486°), 4489 unique (R_int  =  0.0603, R_sigma  =  0.0357) which were used in all calculations. The final R₁ was 0.0473 (I > 2σ[I]) and wR₂ was 0.1302 (all data).

Crystal data for C₁₅H₂₂O₂ (M  =  234.32 g/mol): orthorhombic, space group P2₁2₁2₁ (no. 19), a  =  7.2850(15) Å, b =  7.8310(16) Å, c  =  22.516(5) Å, V  =  1284.5(5) ų, Z  =  4, T  =  100(2) K, μ(synchrotron)  =  0.078 mm⁻¹, Dcalc  =  1.212 g/cm³, 21 314 reflections measured (5.508° ≤ 2Θ ≤ 56.566°), 3106 unique (R_int  =  0.0363, R_sigma  =  0.0198) which were used in all calculations. The final R₁ was 0.0411 (I > 2σ[I]) and wR₂ was 0.1123 (all data)

Crystal data for C₁₅H₂₂O₂ (M  =  234.32 g/mol): orthorhombic, space group P2₁2₁2₁ (no. 19), a  =  7.0990(14) Å, b =  7.7390(16) Å, c  =  23.307(5) Å, V  =  1280.5(4) ų, Z  =  4, T  =  100(2) K, μ(synchrotron)  =  0.078 mm⁻¹, Dcalc  =  1.216 g/cm³, 18 090 reflections measured (5.548° ≤ 2Θ ≤ 56.566°), 2919 unique (R_int  =  0.0396, R_sigma  =  0.0229) which were used in all calculations. The final R₁ was 0.0342 (I > 2σ[I]) and wR₂ was 0.0815 (all data).

Supplemental Material

sj-docx-1-npx-10.1177_1934578X211055025 - Supplemental material for Oxygenated Sesquiterpenes From the Indo-Pacific Nudibranch Ardeadoris rubroannulata: Structure Revision of Pu’ulenal

Supplemental material, sj-docx-1-npx-10.1177_1934578X211055025 for Oxygenated Sesquiterpenes From the Indo-Pacific Nudibranch Ardeadoris rubroannulata: Structure Revision of Pu’ulenal by Patrick T. Narbutas, Gregory K. Pierens, Jack K. Clegg and Mary J. Garson in Natural Product Communications

Supplemental Material

sj-cif-2-npx-10.1177_1934578X211055025 - Supplemental material for Oxygenated Sesquiterpenes From the Indo-Pacific Nudibranch Ardeadoris rubroannulata: Structure Revision of Pu’ulenal

Supplemental material, sj-cif-2-npx-10.1177_1934578X211055025 for Oxygenated Sesquiterpenes From the Indo-Pacific Nudibranch Ardeadoris rubroannulata: Structure Revision of Pu’ulenal by Patrick T. Narbutas, Gregory K. Pierens, Jack K. Clegg and Mary J. Garson in Natural Product Communications