(5S)-5-[(4aR,8aS,9E,11S,13R,14S,16R,17R,19S)-11,19-Dihydroxy-8,10,13,16-tetramethyl-18-methylidene-3,4,5,6,8a,11,12,13,14,15,16,17,18,19,20,21-hexadecahydro-2H-14,17-epoxybenzo[2,3]cyclohexadeca[1,2-b]pyridine-7-yl]-3-methylfuran-2(5H)-one (12-Methylgymnodimine B)

Abstract

A new member of the gymnodimine class of spiroimine toxins has been isolated from a laboratory culture strain of Alexandrium ostenfeldii. Extensive one-dimensional (1D) and two-dimensional (2D) NMR data analysis was used to elucidate its structure as 12-methylgymnodimine B.

1. Introduction

The threat from marine harmful algal blooms (HABs) to the world’s oceans and to the seafood industry is increasing as the rate of toxic bloom detection is on the rise. In HAB toxin research, the dinoflagellate species Alexandrium ostenfeldii is an important member because it has repeatedly been shown to produce members of both the saxitoxin family and the spiroimine toxin groups, gymnodimines and spirolides [1,2,3,4]. HAB spiroimine toxins exert their toxicity primarily through the inhibition of nicotinic acetylcholine receptors [5]. Although still relatively uncommon, the occurrence of both spirolides and gymnodimines in the same organism is not surprising as they appear to share a closely linked biosynthetic pathway [1,6]. There have been 13 members of the spirolide class of toxins fully characterized and several more suggested based on mass spectral data [7,8,9]. In contrast, only five gymnodimines are reported, the most recent being gymnodimine D, recorded last year [1,10,11,12,13]. While analyzing the extracts of cultured A. ostenfeldii cells originating from a re-occurring bloom off the coast of North Carolina for the continued production of 12-methylgymnodimine (2) and 13-desmethylspirolide C, we detected the presence of a minor component at m/z 538 (1) which appeared to be a new gymnodimine congener. Detailed NMR, and high resolution mass spectral analyses allowed us to assign the structure of this new analog as 12-methylgymnodimine B (1) (Figure 1).

2. Results

During the purification of 12-methylgymnodimine (2) and 13-desmethyl spirolide C from A. peruvianum [2], an additional congener (0.9 mg from 100 g wet weight cell pellet vs. 2.6 mg of (2)) of gymnodimine was tentatively identified by UV-LC-MS, displaying an M + H parent ion at m/z 538. This corresponds to the molecular formula of a gymnodimine analog containing an additional oxygen compared with 12-methylgymnodimine (2) [1] or, alternatively, the addition of an extra methyl to either gymnodimine B or C (3 or 4) [11,12]. The latter proposal was confirmed by the HR-ESIMS data (M + H = 538.3531; calculated = 538.3532; Δ = −0.2 ppm) which accounted for a molecular formula of C₃₃H₄₇NO₅. Subsequent one-dimensional (1D) and two-dimensional (2D) NMR experiments further confirmed this as a new member of the gymnodimine class of spiroimines. The planar structure of this new compound was assembled using ¹H, ¹³C, HSQC, COSY, TOCSY, and HMBC data (

Table 1. NMR data for 12-methylgymnodimine B in pyridine-d₅.

Position	δH (mult, J in Hz)	δC (mult)	COSY	HMBC	ROESY
1		175.3 C(q)
2		130.2 C(q)
3	7.15 (br s)	148.8 CH	4,25	1,2,4,25	25
4	5.95 (br s)	81.3 CH	3,25	2, 3,5, 6, 24	26
5		125.0 C(q)
6		134.1 C(q)
7	3.66 (m)	46.1 CH	8	6,8,9,22	20a,26,27
8	5.34 (d, 11.0)	125.3CH	7	7,10, 27,22	10, 26, 27, 31a,32a
9		142.9 C(q)
10	4.52 (d, 10.5)	77.2 CH	11a,b	8,11, 27	8, 11b, 12, 28
11a	2.81 (t, 12.0)	39.9 CH2	10, 11b, 12	9, 10,12, 13, 26	27
11b	1.47 (m)		10, 11a, 12	9, 10,12, 26	10, 13
12	1.40 (m)	35.7 CH	11a,b, 28	11,13, 28	10, 15, 18
13	3.71 (m)	83.6 CH	12, 14a,b	11,15,16, 28	11b,14b, 16, 28
14a	2.15 (m)		13,15	15,16, 29	15,18
14b	1.58 (m)	38.4 CH2	13,15	15,16, 29	13, 16, 28, 29
15	2.94 (m)	36.8 CH	14 a,b, 16, 29	14,16,17, 29	12, 14a,18,19a
16	4.08 (d, 8.5)	92.1 CH	15	15,17, 18, 29, 30	13, 14b, 29
17		156.3 C(q)
18	4.35 (d, 10.0)	70.9 CH	19a,b	16, 17, 19,20,30	12,14a,15,19a
19a	2.58 (m)		18, 19b, 20a,b	18, 20,21	15, 18
19b	2.04 (m)	34.6 CH2	18, 19a, 20a,b	18, 20,21
20a	2.98 (m)	31.1 CH2	19a,b, 20b	18, 19,21	7
20b	2.37 (m)		19a,b, 20a	18, 19,21
21		174.3 C(q)
22		41.9 C(q)
23a	1.71 (m)	32.9 CH2	23b, 24 a,b	5,7,24,31
23b	1.27 (m)		23a	5,7,21,22,24,31
24a	2.04 (m)	19.8 CH2	23a, 24b
24b	1.59 (m)		23a, 24a	5,6,22
25	2.00 (s)	11.0 CH3	3,4	1, 2, 3, 4	3
26	1.66 (s)	17.0 CH3		6, 7, 8	4,7
27	2.14 (s)	12.0 CH3		7, 8, 9, 10	7,11a
28	0.96 (d; 6.5)	17.3 CH3	12	11,12,13	10,13,14b
29	1.06 (d; 6.5)	17.6 CH3	15	14,15,16	14b, 16
30a	5.55 (m)	112.5 CH2	30b	16,17,18
30b	5.11 (m)		30a	16,17,18
31a	1.83 (m)	26.4 CH2	31b, 32a,b	7, 21, 22,23	8
31b	1.35 (m)		31a, 32a,b	7, 21, 22,23
32a	1.58 (m)	21.3 CH2	31a, 32b, 33a,b	22,33
32b	1.44 (m)		31a, 32a, 33a,b	22,33
33a	3.64 (m)	49.5 CH2	32a,b, 33b	21, 31
33b	3.45 (m)		32a,b, 33a	21, 31

). Comparison with the known spectral data for 2 confirmed that it is a very closely related congener, also containing methylation at the C-12 position (

Table 2. Proton and carbon assignments in pyridine-d₅ for 12-methylgymnodimine B (1) and 12-methylgymnodimine (2).

Position	12-Methyl Gym B δH	12-Methyl Gym B δC	12-Methyl Gym δH	12-Methyl Gym δC
1		175.3		175.4
2		130.2		130.3
3	7.15	148.8	7.13	148.7
4	5.95	81.3	5.95	81.4
5		125.0		125.3
6		134.1		134.1
7	3.66	46.1	3.63	46.6
8	5.34	125.3	5.34	126.2
9		142.9		141.7
10	4.52	77.2	4.58	77.3
11a	2.81	39.9	2.92	40.3
11b	1.47		1.61
12	1.40	35.7	1.52	37.1
13	3.71	83.6	3.84	83.7
14a	2.15	38.4	1.78	36.0
14b	1.58		1.52
15	2.94	36.8	2.14	38.6
16	4.08	92.1	4.12	89.8
17		156.3		134.5
18	4.35	70.9	5.40	126.8
19a	2.58	34.6	2.77	22.8
19b	2.04		2.11
20a	2.98	31.1	2.65	31.4
20b	2.37		2.48
21		174.3		171.8
22		41.9		41.8
23a	1.71	32.9	1.71	34.4
23b	1.27		1.32
24a	2.04	19.8	2.06	20.0
24b	1.59		1.59
25	2.00	11.0	2.00	11.1
26	1.66	17.0	1.69	17.0
27	2.14	12.0	2.10	11.7
28	0.96	17.3	0.92	17.0
29	1.06	17.6	1.06	20.6
30a	5.55	112.5	1.54	14.9
30b	5.11
31a	1.83	26.4	1.89	27.4
31b	1.35		1.32
32a	1.58	21.3	1.54	21.4
32b	1.44		1.47
33a	3.64	49.5	3.75	50.6
33b	3.45		3.40

). Analysis of the NMR data allowed us to determine that the region differentiating this new congener from 2 corresponded to additional oxygenation at C-18 in conjunction with double bond isomerization, resulting in an exomethylene group (C-17 and C-30) (Table 2). Specifically, the resonances at position 18 in 12-methylgymnodimine (2) δ_H 5.40 δ_X 126.8) now appeared much further upfield δ_H 4.35, δ_C 70.9) in the new compound. Additionally, the resonance for C-17 was shifted downfield from 134.5 ppm in 2 to 156.3 ppm in 1 and the C-30 methyl of 12-methylgymnodimine (2) is now present as an exomethylene (δ_H 5.55, 5.11; δ_X 112.5). This oxygenation and isomerization is also observed in gymnodimines B and C (3 and 4) which differ from each other only in the configuration of the C-18 stereocenter.

Extensive ROESY analysis allowed us to determine the relative configuration of all stereocenters in 12-methylgymnodimine B with the exception of C-4 (Table 1, Figure 2). Based on biosynthetic considerations, it is likely that this center retains the same configuration as was determined for gymnodimine by X-ray analysis [14]. However, sample limitations precluded us from definitively assigning this center. Importantly, ROESY correlations between H-12 and H-10 and H-15 allowed us to confirm that the relative configuration of 12-methylgymnodimine B (1) corresponds with that established for 12-methylgymnodimine (2) at position 12. Additional ROESY correlations from H-18 to H-12, H-14a, and H-15 correspond to the configuration assigned to gymnodimine B (3) at position 18 (Figure 2) [1,11,12]. While the optical rotations of gymnodimines B and C were not reported, the optical rotation for (1) was determined to be ${\left[\mathsf{\alpha }\right]}_D^{26}$ = −30 (c. 0.2, MeOH) which is similar in value and sign to that reported for (2) [1].

3. Experimental Section

A clonal culture of Alexandrium ostenfeldii AP0411 (previously called A. peruvianum) was grown and harvested according to our previously reported protocol [2,6].

The A. peruvianum cells were filtered onto glass microfiber (VWR, 691-, Radnor, PA, USA) paper and the media was extracted with XAD resin to collect the remaining organics. The organics were then rinsed from the resins with 100% MeOH and 100% acetone washes. The cells were extracted with 2× 400 mL 80% MeOH and 1× 400 mL 100% MeOH. All organic extracts were combined and dried to HP20 resins for desalting and primary fractionation. The extract-bound resins were washed with 100% water and the remaining de-salted extract was eluted with increasing concentrations of acetone. The dried desalted extract was then fractionated on a 20 g C₁₈ SPE cartridge with a stepwise elution of MeCN and H₂O (0.1% TFA). LC-MS monitoring indicated that the spiroimines eluted in the 40% and 60% MeCN fractions. The spiroimine-containing fractions were then purified by reversed phase HPLC (semi-prep Gemini 5 µm C₁₈ 110A 250 × 10 mm column) under isocratic conditions (37% MeCN:63% H₂O (0.05% formic acid)) 2 mL/min.

Optical rotations were measured with a Rudolph Research Analytical Autopol^® III automatic polarimeter (Hackettstown, NJ, USA) in methanol. NMR analyses were performed using a Bruker® Avance 1 500 MHz system (Billerica, MA, USA) run by TopSpin version 2.0. HPLC isolation was performed using a Waters Breeze HPLC system (Waters, Manchester, UK) with a Waters dual wavelength detector. LC-MS monitoring was performed on a Waters Micromass ZQ mass spectrometer tandem to an Agilent 1600 high performance liquid chromatography (HPLC) instrument (Santa Clara, CA, USA). All solvents were of HPLC grade and were used without further purification. The HRMS spectrum was obtained on Waters UPLC i-Class system couples to a Waters Xevo-G2XS QToF-MS mass spectrometer. The system was operated in electrospray positive mode (ESI⁺) with the capillary voltage set at 2.5 kV, source and desolvation temperatures at 100 °C and 550 °C, respectively, and desolvation gas flow at 800 L/h. The MS-MS data was obtained with the same instrument under the same conditions with a set mass of 538.4 and a collision energy of 35 eV. All solvents were of mass spectrometry grade and were used without further purification.

4. Discussion

This new congener of the gymnodimine class of spiroimine toxins further expands the structural variety of this group and hence needs to be incorporated into any shellfish monitoring program. In addition to the fast acting toxicity profile for spiroimine toxins, recent literature indicates potential therapeutic value for these compounds in combating neurodegenerative diseases such as Alzheimer’s disease, including the closely related gymnodimine C [15,16] as well as the spirolides [17,18]. Increasing the library of structurally diverse spiroimines available for this research is important for enhancing drug discovery efforts.