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Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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An Acetylenic Lipid from the New Zealand Ascidian Pseudodistoma cereum: Exemplification of an Improved Workflow for Determination of Absolute Configuration of Long-Chain 2‑Amino-3-alkanols A. Norrie Pearce,† Cameron A. E. Hill,‡ Michael J. Page,§ Robert A. Keyzers,⊥ and Brent R. Copp*,† †

School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Swinburne Senior Secondary College, Hawthorn, Victoria 3122, Australia § National Institute of Water & Atmospheric Research (NIWA) Ltd, PO Box 893, Nelson 7010, New Zealand ⊥ Center for Biodiscovery and School of Chemical and Physical Sciences, Victoria University of Wellington, PO Box 600, Kelburn, Wellington 6140, New Zealand

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‡

S Supporting Information *

ABSTRACT: An acetylenic 2-amino-3-alcohol, distaminolyne B (2), isolated from the New Zealand ascidian Pseudodistoma cereum, is reported. The isolation and structure elucidation of 2 and assignment of 2S,3S absolute configuration (AC) using the exciton coupled circular dichroism technique are described. Using a methodologically facile workflow, the same AC was also established by analysis of specific rotation, terminal methyl C-1 δC chemical shift, and NH δH and J values of the N,O-diacetate derivative.

S

presence of both 1-AA and 2-AA compounds in the same collection of a specific organism.3,6,9 We recently reported the acetylenic 1-AA distaminolyne A (1) from a New Zealand ascidian, Pseudodistoma opacum.20 A 2S absolute configuration (AC) was assigned to the natural product by application of the dibenzoate exciton coupled electronic circular dichroism (ECCD) technique. Shortly after, two syntheses of distaminolyne A were reported, with one disputing the 2S AC21 and the second supporting the original assignment.22 In order to clarify these discrepancies and ultimately verify that the original assignment of 2S AC was in fact correct,19,23 further samples of 1 were purified from both P. opacum and the related ascidian P. cereum. During the chromatographic purification of fractions from P. cereum, the presence of a second, closely related natural product was detected. Herein we report the characterization of 2-AA distaminolyne B (2) and assignment of AC by ECCD. An alternative method of AC assignment using specific rotation and 13C NMR chemical shift of the terminal C-1 methyl group and 1H NMR NH δH and J values of the corresponding diacetate derivative is also exemplified and shown to be applicable to all previously reported marine-sourced 2-AAs. Specimens of the ascidian P. cereum were collected from Princes Island, Northland, New Zealand. A MeOH extract of freeze-dried sample was initially fractionated by C18 reversedphase flash column chromatography, with the 75% MeOH/ H2O (+0.05% trifluoroacetic acid (TFA)) fraction being found to be rich in lipids. Repeated C18 chromatography of this

phingolipids, particularly 1-deoxy variants of D-erythrosphingosine,1 have been reported from a diverse range of marine organisms, including ascidians (genera Pseudodistoma2−6 and Clavelina),7−9 sponges (genera Xestospongia10,11 and Haliclona),12 a brown alga (Xiphophora chondrophylla),13 and a clam (Spisula polynyma).14 Structurally, these long-chain 2-amino-3-alkanols (2-AAs)15 incorporate variations in carbon chain length (C11−C18 vs C18 for sphingosine) and often include the presence of chain (poly)unsaturation. A number of different biological activities have been reported for this class of compounds, including inhibition of tumor cell proliferation,2,6,8,9,14,16,17 antimicrobial2,3,5,6,10−13 and antihelminth11 properties, and the ability to inhibit reverse transcriptase.11 Biosynthetically, 2-AAs and the structurally related 1-amino-2alkanols (1-AAs) are thought to be the result of a fumonisinmycotoxin-related pathway (Figure 1) whereby condensation of a polyketide thioester with either glycine or alanine followed by decarboxylation affords an aminoketone that undergoes reduction.18,19 Given the proposed biosynthetic pathway it is perhaps surprising that only three studies to date have noted the

Figure 1. Proposed biosynthesis of 1-AA (R2 = H) and 2-AA (R2 = Me) marine lipids. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 29, 2019

A

DOI: 10.1021/acs.jnatprod.9b00504 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

137.8, δH 5.85; δC 116.3, δH 5.08, 5.02), through two methylenes (δC 33.7, δH 2.25, m; δC 19.6, δH 2.33, t), to a diyne fragment (δC 78.0, 77.1, 66.7, and 66.3), the presence of which was supported by UV−vis spectrum analysis,20 and through a further seven contiguous methylenes to the 2,3amino alcohol terminus. We thus concluded the structure of the new natural product to be the trifluoroacetate salt of 2aminooctadeca-17-ene-11,13-diyn-3-ol, distaminolyne B (2). Assignment of absolute configuration was achieved using the dibenzoate exciton coupled circular dichroism method.24 Reaction of 2 with benzoic acid, coupling agent N-(3dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)·HCl, and 4-dimethylaminopyridine (DMAP) in CH2Cl219,23 for 20 h gave, after diol-bonded normal-phase chromatography, N,O-dibenzoyl derivative 3 in 96% yield. The ECD spectrum of 3 exhibited a bisignate curve with a positive first Cotton effect (Δε +1.9, 239 nm; 0, 232; −3.5, 222) (Figure 2).

fraction using acidified solvents led to the purification of the trifluoroacetate salts of the previously reported 1-AA distaminolyne A (1)19,20,23 and the new 2-AA distaminolyne B (2) as a weakly optically active yellow gum. A molecular formula of C18H29NO for 2 was deduced from a protonated molecule detected in the (+)-HR ESI mass spectrum. It was noted that this formula, which requires five degrees of unsaturation, represented a methylene homologue of co-metabolite distaminolyne A (1). Inspection and comparison of the 1H NMR data (CD3OD) observed for both distaminolynes A and B (see Supporting Information) identified the metabolites to be closely related, with major differences being associated with the presence of a new methyl doublet in 2 (δH 1.27, d, J = 6.7 Hz) and changes in the number of protons and their multiplicities associated with the amino-alcohol terminus of the molecule. All other resonances were essentially equivalent. A similar locus of change was observed in the combination of COSY, 13C, and multiplicity edited-HSQC NMR data, with the C-1 to C-4 fragment now being observed as a CH3 (δC 16.0, δH 1.27, d)−CH (δC 53.5, δH 3.09, m)−CH (δC 73.1, δH 3.44, m)−CH2 (δC 34.6, δH 1.56, m)− fragment. Combined analysis of COSY, HSQC, and HMBC NMR data (Table 1) linked the terminal alkene (δC Table 1. NMR Data (CD3OD) for Distaminolyne B (2) position

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

16.0, CH3 53.5, CH 73.1, CH 34.6, CH2 26.2, CH2 30.5,c CH2 30.1,c CH2 29.8,c CH2 29.5,c CH2 19.6, CH2 78.0, C 66.3,d C 66.7,d C 77.1, C 19.6, CH2 33.7, CH2 137.8, CH 116.3, CH2

δH, mult (J in Hz)

Figure 2. Plot of ECD spectra (MeOH) of 3 (red) and model compounds erythro-2S,3R-4 (blue) and threo-2S,3S-5 (black).

Comparison of the ECD spectrum of 3 with those reported for stereochemically pure model compounds 4 (erythro 2S,3R) and 5 (threo 2S,3S)25,26 revealed an excellent match between 3 and the latter model compound (Figure 2), establishing the AC of 3, and hence that of the natural product 2, as being threo 2S,3S. In their initial report on the synthesis of 4 and 5, Nicholas and Molinski noted that the 1H NMR chemical shift and coupling constant (CDCl3) observed for the benzamide NH proton were indicators of relative configuration, with erythro 4 (δΗ 6.97, br d, J = 7.8 Hz) being noticeably different from the threo 5 (δΗ 6.39, br d, J = 9.0 Hz) diastereomer.25 In the present study, the NH signal of 3 was observed at δΗ 6.39 (br d, J = 9.4 Hz), corroborating the threo relative configuration.

a

HMBCb

1.27, 3.09, 3.44, 1.56, 1.40, 1.36, 1.41, 1.41, 1.51, 2.25,

d (6.7) m m m m m m m m m

2, 1, 1, 3, 6 7

3 3, 4 2, 4, 5 6

2.33, 2.25, 5.85, 5.02, 5.08,

t (6.2) m ddt (17.1, 10.3, 6.7) m ddt (17.1, 1.8, 1.3)

13, 13, 15, 16 16,

7 7, 10, 11 8, 9, 11, 12

14, 16, 17 14, 15, 17, 18 16

As we have previously reported, distaminolyne A isolated from P. cereum is not stereochemically pure, being isolated as a scalemic mixture with 49% ee favoring an S-configuration.19 The enantiopurity of 2 was evaluated by 1H NMR analysis of the bis(S)-MPA derivative 6, prepared from the natural product by reaction with S-(+)-α-methoxyphenylacetic acid [(S)-MPA] and coupling agent EDC·HCl and DMAP base in

17

a1 H (500 MHz), 13C (125 MHz). bHMBC correlations, optimized for 8.3 Hz, reported from the proton resonance to the indicated carbon resonance(s). c,dAssignments are interchangeable.

B



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CH2Cl2. The 1H NMR spectrum of the crude reaction product identified only one set of MPA CαH resonances [δH 4.84 (1H, s), 4.47 (1H, s)] and one set of MPA OMe resonances [δH 3.46 (3H, s), 3.37 (3H, s)], consistent with the presence of only one enantiomer.27 It is interesting to contrast the difference in fidelity of reduction of the putative aminoketone intermediates leading to 1-AA (1 49% ee)19 and 2-AA (2 100% de) natural products in the same specimen of ascidian. Analysis of methods used by other researchers to assign AC to 2-AAs reported from the marine environment identified several intriguing comments regarding the possibility that AC can be determined simply by measurement of the specific rotation and the 13C NMR chemical shift of the terminal C-1 methyl group of the corresponding diacetate derivative. A comprehensive literature analysis, described below, provides compelling support for this relationship, enabling a convenient and straightforward workflow for determining AC of this class of marine natural products. The assignment of AC to distaminolyne B is used to illustrate this method. Including the present study, 44 examples of 2-AAs that contain no other stereogenic centers have been reported from marine sources. These structures (7−49) can be grouped by configuration (determined absolute or relative as indicated) at the C-2 and C-3 positions (Figures 3−5). Table 2 summarizes

Figure 4. Structures of 2S,3R 2-AA marine natural products.

Figure 5. Structures of 2R,3S and 2R,3R 2-AA marine natural products.

(e.g., α-methoxy-α-trifluoromethylphenylacetic acid (MTPA)4−6,13 or MPA12) of the natural product or an Nprotected derivative, and NMR analysis. To date, examples of all four diastereomers of 2-AAs have been reported as natural products, with a dominant number of these bearing the 2S absolute configuration [(2S,3S) nine examples 7−13, 15, 16 (Figure 3), (2S,3R) 12 (Figure 4), (2R, 3S) 11 and (2R,3R) 1 (Figure 5)].32 In two separate accounts, Mori et al. reported the synthesis and correction of AC for two 2-AAs reported by Gulavita and Scheuer10,28 and assigned AC to (+)-xestoaminol C.11,29 Of note were the observations by Mori et al.28,29 concerning the diacetyl derivatives of 2-AAs: 1. Diacetyl derivatives of 2-AAs exhibited specific rotations of higher magnitude than the natural products themselves, and (2R)-derivatives are dextrorotatory, while (2S) are levorotatory. 2. The 13C NMR chemical shift (CDCl3) of the C-1 methyl group of diacetyl 2-AAs is diagnostic of relative configuration, with δC ∼15 observed for erythro

Figure 3. Structures of 2S,3S 2-AA marine natural products.

this set of 2-AAs and lists, where reported, the natural product [α]D, AC and the method(s) used for determination, and optical rotatory and NMR properties of the corresponding N,O-diacetyl derivative (if prepared). Assignment of AC to 2AAs is not trivial, arising from the typically weak specific rotation values observed for this class of compound. As a consequence, a number of different methods have been developed and implemented including total synthesis,17,28−31 chemical degradation,2 conversion to N,O-dibenzoyl derivative with ECD analysis,6−8 and a sequence typified by conversion to an oxazolidinone, for determination of relative configuration,2−6,10−13 followed by chiral auxiliary derivatization C



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Table 2. Summary of Spectroscopic Data Observed for Isolated and Synthesized 2-AA Marine Natural Products or Their Diastereomers and Their N,O-Diacetyl Derivatives entry

cmpd

2-AA [α]D (conc, solvent)

ACa 2S,3S 2S,3S

E

2S,3S 2S,3S 2S,3S

O,M S O,N B,A B,A B,A B,A O,B M O,B O,E,M E2

1

2

−4.4 (0.75, MeOH)

2 3 4

7

−6.2 (0.42, CHCl3)

8

n.r.

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

9 10 11 12 13 13 14 15 16 17 18 19 20 ent-49 2S,3S-34

n.r. n.r. n.r. n.r. n.r. n.r. n.r. −4.4 (0.4, MeOH) +5.1 (0.5, MeOH) +2.7 (0.5, MeOH) +2.4 (0.45, MeOH) +3.4 (0.5, MeOH) +1.4 (0.5, MeOH) −3.8 (0.24, CHCl3/MeOH, 5:1) n.r.

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

21 22 23 24 25 26 27 28 29 30 31 32 33 34 34 34 34 Z-Δ4-34 ent-48

+1.7 (0.044, CH2Cl2) +2.1 (0.056, CH2Cl2) +1.9 (0.025, CH2Cl2) +5.0 (0.14, MeOH) n.r. n.r. n.r. n.r. n.r. +4.5 (0.22, MeOH) +24.9 (1.0, CHCl3) +7.7 (0.26, MeOH) +6.0 (0.06, MeOH) +7.0 (0.14, MeOH) n.r. n.r. n.r. n.r. +9.1 (0.17, CHCl3, MeOH, 5:1)

39 40 41 42 43 44 45 46

35 36 37 38 39 40 41 42

−4.25 (0.0094, MeOH) −5 (0.001, MeOH) +11.4 (0.0022, MeOH)e +8 (0.0013, MeOH)e +3 (0.0012, MeOH)e −4 (0.004, MeOH)f +10.45 (0.0005, MeOH)g +12.17 (0.0009, MeOH)h

2S,3S 2S,3S 2S,3S 2S,3S 2S*,3S* 2S,3S 2S*,3S* 2S,3S 2S,3S 2S*,3S* 2S*,3S* 2S*,3S* 2S*,3S* 2S,3S 2S,3S 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S*,3R* n.r. 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2S,3R 2R,3S 2R,3S 2R,3S 2R,3S 2R,3S 2R,3S 2R,3S 2R,3S 2R,3S

47 48 49 50 51 52

43 44 45 46 47 48

+9.05 (0.0005, MeOH)e +6.6 (0.0014, MeOH)e n.r. n.r. n.r. n.r.

2R,3S 2R,3S 2R,3S 2R,3S 2R,3S 2R,3Sj

di-Ac [α]D (conc, solvent)

methodb

S S

−19.8 (0.48, MeOH) −19.1 (0.35, CHCl3) −23 (0.1, CHCl3) −22.6 (0.5, CHCl3) −24.7 (0.1, CHCl3) −20.3 (0.03, MeOH) −16.8 (0.2, CHCl3) −28.0 (0.1, CHCl3) −12.1 (0.3, CHCl3) −15.2 (0.2, CHCl3) −20 (0.83, CHCl3) −19.1 (0.12, CHCl3) +14 (0.7, CHCl3) n.r. n.r. n.r. n.r. n.r. n.r. −20.6 (0.32, MeOH) −38.7 (0.34, MeOH)

O,P O,B O,B O,M X X X X X E S O,B B B M S S S S E B C C2 C2 C C C C2 C2 B B O,D D

di-Ac NH [δH (J in Hz)]c

di-Ac Me-1 (δC)c

ref

5.54 (9.3)

18.6

5.53 (9.3) 5.53 (9.2) 5.51 (9.0)

18.7 18.5 18.5

this work this work 13 31 5

5.53 (9.0) 5.51 (9.0) 5.50 (8.8) 5.50 (9.0) n.r.d 5.51 (obsc) n.r. n.r. n.r. n.r. n.r. n.r. n.r. 5.53 (9.3) 5.52 (7.8)

18.5 18.5 18.5 18.5 n.r. 18.5 n.r. n.r. n.r. n.r. n.r. n.r. n.r. 18.5 18.5

5 5 5 5 3 5 3 6 6 6 6 6 6 6 29

−30 (0.003, MeOH) −20 (0.003, MeOH) −32 (0.003, MeOH) −23.3 (0.78, MeOH) −21.1 (0.65, MeOH) −24.5 (0.83, MeOH) −21.6 (0.44, MeOH) n.r. −19.2 (0.71, MeOH) n.r. n.r. +15.0 (0.06, MeOH) n.r. n.r. −21.8 (0.40, MeOH) −22.1 (0.17, MeOH) −22.7 (0.6, MeOH) −14.9 (0.8, CHCl3) −52.6 (0.31, MeOH)

n.r. n.r. n.r. 5.86 (8.0) 5.82 (m) 5.84 (8.1) 5.82 (7.7) 5.82 (7.7) 5.81 (m) n.r. n.r. 12.13 n.r. n.r. 5.80 (7.4) 5.84 (7.8) 5.90 (8.2) 5.51−5.60 (m) 5.74 (7.9)

14.8 16.2 13.9 14.9 14.7 14.7 14.7 14.7 14.7 n.r. n.r. 13.9 n.r. n.r. 14.7 14.7 14.8 14.5 14.9

12 12 12 4 4 4 4 4 4 7 14 11 11 11 4 29 30 29 28

+18.8 (0.0008, MeOH) n.r. n.r. n.r. n.r. same as cmpd 35 +10.45 (0.0005, MeOH) +12.17 (0.0009, MeOH)h n.r. n.r. +36 (0.53, MeOH) +36 (0.26, MeOH) +45 (0.84, MeOH) +50.8 (0.21, MeOH)

5.78 (7.4) n.r. n.r. n.r. n.r.

14.8 n.r. n.r. n.r. n.r.

5.78 (7.2) 5.88 (6.5)h

14.8 14.8h

8 8 8 8 8 8 9 9

n.r. n.r. 5.59 5.75 5.73 5.73

n.r. n.r. 15.6 14.8 14.8 n.r.

9 9 2 2 2 10

D

(7.5)i (7.5) (8.5) (7.8)



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Table 2. continued entry

cmpd

di-Ac [α]D (conc, solvent)

di-Ac Me-1 (δC)c

ACa 2R,3S

S

+55.2 (0.94, MeOH)

n.r.

n.r.

28

2R,3R 2R,3Rk 2R,3R

O,D S

+16.7 (0.28, MeOH) +20.7 (1.66, MeOH)

5.53 (9.3) n.r.

n.r. n.r.

10 28

53

48

−11.9 (0.37, CHCl3/MeOH, 5:1)

54 55

49 49

n.r. +8.6 (0.22, CHCl3/MeOH, 5:1)

methodb

di-Ac NH [δH (J in Hz)]c

2-AA [α]D (conc, solvent)

ref

a

Absolute configuration, unless indicated as relative. bMethod used to determine AC: D (degradation), S (total synthesis), O (oxazolidinone), M (Mosher’s analysis of N-Ac derivative), P (MPA analysis of N-Ac derivative), E (ECCD of di-Bz derivative), E2 (hydrogenation and then E), B (biogenetic grounds), N (diacetate C-1 shift giving threo/erythro), A (sign of specific rotation of diacetate gives C-2 position AC), X (diacetate derivative hydrogenation, hydrolysis, then O, then M), C (conversion to related analogue or co-metabolite and comparison of optical properties), C2 (comparison of NMR and specific rotation properties). See cited reference for complete details. cNMR data acquired in CDCl3 unless otherwise noted. dn.r., not reported. eNatural product is N-acetylated. fNatural product is O-acetylated. gNatural product is N,O-diacetylated. hNatural product is an N-formyl-O-acetyl derivative. Optical and NMR data shown in table are for the natural product. iThe structure 45 has Δ4 unsaturation, which appears to have some influence on the NH 1H NMR chemical shift value of the corresponding diacetate derivative (see text). A similar effect on NH chemical shift was reported in a synthetic study involving a Z-alkene-containing analogue of a 2-AA (Table 2, entry 37).30 jAC originally proposed as 2S,3S based upon degradative studies.10 AC corrected to 2R,3S by total synthesis.28 kAC originally proposed as 2S,3R based upon degradative studies.10 AC corrected to 2R,3R by total synthesis.28

chemical shift (δH 5.59) was anomalous compared to other erythro diastereomers.2 The presence of Δ4 unsaturation appears to be the influencing factor, with a similar NH chemical shift (δH 5.51−5.60) being reported for a model compound prepared as part of a synthetic study (Table 2, entry 37).30 The data suggest that NH δH and J values are not suitable for establishing the relative configuration of 2-AAs that bear Δ4 unsaturation, but that the C-1 methyl δC is still reliable. The [α]D/δC C-1 methyl AC predictive relationship is also apparent for mono N-acetyl 2-AA derivatives with examples reported as natural products in their own right (37, 38, 39, 43, 44)8,9 (structures in Figure 5) or as synthetic or semisynthetic intermediates (51−57) (Figure 6).4,6,10,12,13 As summarized in

diastereomers [i.e., (2S,3R) and (2R,3S)] and δC ∼18 for threo diastereomers [i.e., (2S,3S) and (2R,3R)]. These observations have been put to use by several authors, using diacetyl 2-AA derivative [α]D and 13C NMR data to support AC determined for specific natural products by other more classical routes.5,12 To determine whether Mori et al.’s observations hold true for a larger data set of examples, we have compiled data reported for diacetyl derivatives of 2-AAs being either natural products, semisynthetic derivatives, or synthesized material (Table 2). In all cases, where the original authors have prepared and characterized such derivatives, these relationships hold and [α]D (obtained in either MeOH or CHCl3) and 13C NMR data (CDCl3) are entirely predictive of the determined 2-AA absolute configuration. Comparison with other 13C chemical shifts, specifically for methyl resonances of NAc and OAc groups and methines C-2 and C-3, showed no discernible relationship with AC (Table S1). In light of the previous report from Nicholas and Molinski25 of the relationship between benzamide NH 1H NMR chemical shift and coupling constant being predictive of the relative configuration for N,O-dibenozyl derivatives of 2-AAs, we also examined 1H NMR data reported for diacetyl derivatives of 2AAs of defined AC. While no relationship was discernible for NAc, OAc, or C-1 methyl or C-2 and C-3 methine chemical shifts (Table S2), the acetamide NH shift and coupling constant do appear to align with AC, with erythro diastereomers [δH 5.90−5.73 (x̅ = 5.800 ± 0.014; n = 14), J = 8.0−7.2 Hz (x̅ = 7.71 ± 0.09; n = 12)] being statistically different (δH p-value ≈ 0, J p-value ≈ 0) from the threo diastereomers [δH 5.53−5.50 (x̅ = 5.521 ± 0.004; n = 12), J = 9.3−7.8 Hz (x̅ = 8.97 ± 0.14; n = 10)] (Table 2). We conclude that analysis of acetamide NH chemical shift (CDCl3) and its coupling constant can also be used to determine the relative configuration of 2-AAs. In the current context, the N,Odiacetyl derivative of distaminolyne B (50) exhibited [α]D −19.8 (MeOH) [and [α]D −19.1 (CHCl3)] (levorotatory equating to 2S configuration) and C-1 Me δC 18.6 and NH δH 5.54 (J = 9.3 Hz) (both equating to threo relative configuration), establishing an AC of (2S,3S), which agrees with that determined by the dibenzoate ECCD method. An important point to note arising from the summarized data in Table 2 concerns the diacetate derivative of erythro(2R*,3S*)-crucigasterin 225 (45), where the reported NH

Figure 6. Structures of semisynthetic mono N-Ac derivatives of 2AAs.

Table 3, levorotation again indicates 2S AC, although the smaller magnitude versus diacetate derivatives makes this data somewhat less reliable. The magnitude of Me-1 δC also correctly identifies relative configuration (threo ∼18 ppm, erythro ∼14−15 ppm). Of note for these monoacetates, however, is that the acetamide NH (δH and J) chemical shift and coupling constant values are not predictive of relative configuration. The AC of several examples of 2-AAs remain unresolved. Jimenez and Crews reported three examples, xestoaminols A− C (32−34), from a Fijian Xestospongia sp.11 Erythro relative configuration was assigned to xestoaminol A via analysis of an E



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means it is not possible to say whether the two natural products (14 and 16) are identical or are in fact enantiomers. In summary, our investigation of the chemistry of a New Zealand collection of the ascidian P. cereum has afforded a new acetylenic 2-AA lipid. Absolute configuration was assigned by the established dibenzoate exciton coupled circular dichroism method. An alternative workflow made use of the N,O-diacetyl derivative, whereby analysis of specific rotation, C-1 methyl δC, and amide NH δH and J values established an AC that was in agreement with that determined by the ECCD method. Similar analysis of 2-AA diacetate derivatives also correctly predicted the AC of all previously known, AC-defined, marine 2-AAs. These results highlight a robust, high-yielding, and methodology- and equipment-friendly approach to the determination of AC for 2-AA natural products.

Table 3. Summary of Spectroscopic Data Reported for Mono N-Acetyl 2-AA Derivatives cmpd

AC

[α]D

51 51 52

2S,3S 2S,3S 2S,3R

53

2S,3R

53 54 55 37

2S,3R 2S,3R 2S,3R 2R,3S

38 39 43

2R,3S 2R,3S 2R,3S

44

2R,3S

56 57

2R,3S 2R,3R

−6.3 (0.16, CHCl3) n.r.b −14.6 (0.005, CH2Cl2) −20.1 (0.017, CH2Cl2) n.r. n.r. n.r. +11.4 (0.0022, MeOH) +8 (0.0013, MeOH) +3 (0.0012, MeOH) +9.05 (0.0005, MeOH) +6.6 (0.0014, MeOH) +42.5 (1.13, CHCl3) +43.9 (0.41, CHCl3)

Me-1 (δC)a

NH [δH (J in Hz)a

ref

18.5 n.r. 14.8

5.68 (8.5) 5.86 (s) n.r.

13 6 12

15.1c

n.r.

12

n.r. n.r. n.r. 14.1

5.75 5.79 5.79 5.80

(6.8) (6.9) (6.8) (7.1)

4 4 4 8

14.26 14.25 14.0

5.76 (7.0) 5.76 (7.0) 5.74 (m)

8 8 9

14.4

5.77 (7.0)

9

14.1 18.4

n.r. n.r.

10 10

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EXPERIMENTAL SECTION

General Experimental Procedures. General experimental details have been reported elsewhere.35 Specific rotations were recorded on an Autopol IV polarimeter using a 1 dm cell (concentration units of g/100 mL), while ECD spectra were recorded on an Applied PhotoPhysics Chirascan spectrophotometer (concentration units of M). NMR referencing used proto-deutero solvent signals or tetramethylsilane (TMS) (CD3OD: δH 3.31, δC 49.00; CDCl3: δH TMS, δC 77.16). Animal Material. Specimens of the ascidian Pseudodistoma cereum were collected on November 24, 2002, at a depth of 15 m from Princes Island (34.1786° S, 172.0418° E), Three Kings island group, Northland, New Zealand, and kept frozen until used. A voucher specimen is held at NIWA, Wellington, New Zealand, as MNP7042. A color in situ photograph is included in the Supporting Information. Isolation and Purification. The freeze-dried ascidian specimens (22.42 g dry wt) were extracted repeatedly with MeOH (4 × 200 mL), filtered, and concentrated in vacuo. The combined green solid (4.25 g) was subjected to initial C18 reversed-phase column chromatography using a step gradient of acidified (0.05% TFA) H2O to MeOH. Distaminolyne A (1, 8.5 mg, 0.04% dry wt) and distaminolyne B (2, 16 mg, 0.07% dry wt) eluted with 70% MeOH/ H2O + 0.05% TFA as pale yellow gums. Distaminolyne A trifluoroacetate salt, 1: [α]20D +1.5 (c 0.275, MeOH); 1H NMR (CD3OD, 500 MHz) spectrum was identical to the published data;20 HRESIMS m/z 262.2157 [M + H]+ (calcd for C17H28NO, 262.2165). Distaminolyne B trifluoroacetate salt, 2: [α]22D −4.4 (c 0.75, MeOH); UV (MeOH) λmax (log ε) 204 (3.80), 215 (3.64), 240 (3.39), 252 (3.33), 268 (3.32), 282 (3.24) nm; IR νmax (ATR) 2934, 2859, 2229, 1670, 1200, 1182, 1135, 800, 722 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 276.2317 [M + H]+ (calcd for C18H30NO, 276.2322). N,O-Dibenzoyl Distaminolyne B 3: A solution of EDC·HCl (15 mg, 0.08 mmol), benzoic acid (7 mg, 0.06 mmol), and DMAP (11 mg, 0.10 mmol) was stirred in CH2Cl2 (1 mL) under N2 at 0 °C for 10 min, after which distaminolyne B (5 mg, 0.02 mmol) in CH2Cl2 (0.5 mL) was added. The solution was allowed to come to room temperature (rt) and stirred under N2 for 20 h. CH2Cl2 (10 mL) was added, and the mixture was washed in turn with 10% HCl (10 mL), H2O (10 mL), aqueous saturated NaHCO3 (10 mL), and H2O (10 mL) before being dried under vacuum. The crude product was passed through a DIOL Sep-Pak, with the product N,O-dibenzoyl distaminolyne B eluting with 100% CH2Cl2 as a colorless oil (8.4 mg, 96%): ECD (c 1.04 × 10−4 M, MeOH), 222 (Δε −3.5), 232 (Δε 0), 239 nm (Δε +1.9) and Figure 1; 1H NMR (CDCl3, 500 MHz) δ 8.04 (2H, m, H-2′ and H-6′), 7.73 (2H, m, H-2″ and H-6″), 7.57 (1H, m, H-4′), 7.50−7.39 (5H, m, H-3′, H-5′, H-3″, H-4″, H-5″), 6.39 (1H, br d, J = 9.4 Hz, NH-2), 5.84 (1H, ddt, J = 16.8, 10.2, 6.5 Hz, H-17), 5.22 (1H, m, H-3), 5.10−5.02 (2H, m, H2-18), 4.54 (1H, m, H-2), 2.33 (2H, m, H2-15), 2.27 (2H, m, H2-16), 2.20 (2H, m, H210), 1.77 (2H, m, H2-4), 1.48 (2H, m, H2-9), 1.43 (2H, m, H2-5),

a

CDCl3 solvent. bn.r., not reported. cPyridine-d5 NMR solvent.

oxazolidinone derivative, while AC was ascribed to all three natural products on biogenetic grounds by reference to related 2-AAs isolated by Gulavita and Scheuer from a Papua New Guinean Xestospongia sp.10,28 Ichihashi and Mori determined the AC of xestoaminol C as being erythro (2S,3R) by total synthesis.29 Of use in the current discussion was that Jimenez and Crews prepared and characterized the diacetate derivative of xestoaminol A, reporting [α]D +15 (i.e., 2R) and C-1 (δC 13.9) (i.e., erythro), leading us to propose the natural product to possess an AC of (2R,3S).33 What is interesting about this proposal is that the same marine organism specimen appears to be capable of biosynthesizing 2-AAs belonging to different stereochemical series (viz., 2R,3S-xestoaminol A and 2S,3Rxestoaminol C).34 Not enough spectroscopic data were reported for xestoaminol B to make any predictions as to its AC. A further example of a 2-AA with unresolved AC is that of one of two unnamed compounds 13 and 14 isolated from a South African collection of Pseudodistoma sp.3 Relative configuration (threo) was assigned to both metabolites by analysis of oxazolidinone derivatives, while the corresponding diacetate derivatives exhibited noticeably different specific rotations (13 [α]D −20, 14 [α]D +14). AC of 2S,3S was assigned to the former metabolite when it was reisolated by Ciavatta et al. and characterized by Mosher’s analysis.5 Given the preceding, it is tempting to ascribe a 2R,3R configuration to 14 (based upon the dextrorotatory [α]D); however with no NH chemical shift nor 13C NMR data reported in the original isolation paper, there is no additional support for this proposal. A counterpoint is that the authors took both oxazolidinone derivatives of 13 and 14 and hydrogenated them and obtained products that exhibited identical specific rotation. Pseudoaminol B 166 (Figure 3), also from a Pseudodistoma sp., shares the same planar structure as 14 with an AC of (2S,3S) assigned to the former using chemical modification and ECCD analysis of the dibenzoylated derivative. With the original report of 14 only describing the diacetate derivative and the pseudoaminol series study characterizing the natural product and dibenzoylated derivative, the lack of commonality of described data F



Article

ORCID

1.36−1.27 (9H, H2-6 to H2-8, including 1.29 (3H, d, J = 6.67 Hz, H31); 13C NMR (CDCl3, 125 MHz, * = interchangeable) δ 167.1 (2C, −OCOBz, −NHCOBz), 136.6 (C-17), 134.7 (C-1″), 133.4 (C-4′), 131.6 (C-4″), 130.0 (C-1′), 129.8 (C-2′ and C-6′), 128.8, 128.7 (C3′, C-5′, C-3″, C-5″), 127.0 (C-2″ and C-6″), 116.1 (C-18), 77.9 (C11), 77.4 (C-3, obsc.), 76.7 (C-14), 65.9 (C-12*), 65.3 (C-13*), 48.5 (C-2), 32.6 (C-16), 31.8 (C-4), 29.4, 29.0, 28.8 (C-6, C-7, C-8), 28.4 (C-9), 25.4 (C-5), 19.3 (C-10*), 19.2 (C-15*), 18.7 (C-1); HRESIMS m/z 484.2830 [M + H]+ (calcd for C32H38NO3 484.2846). Bis-(S)-MPA Derivative of Distaminolyne B, 6. A solution of EDC· HCl (3 mg, 16 μmol), S-(+)-α-methoxyphenylacetic acid (2.4 mg, 14 μmol), and DMAP (3.7 mg, 30 μmol) was stirred in CH2Cl2 (0.5 mL) under N2 at 0 °C for 10 min, after which distaminolyne B (1.5 mg, 6 μmol) in CH2Cl2 (0.5 mL) was added. The solution was allowed to come to rt and stirred under N2 for 20 h. CH2Cl2 (10 mL) was added, and the mixture was washed in turn with 10% HCl (10 mL), H2O (10 mL), saturated NaHCO3 (10 mL), and H2O (10 mL) before being dried under vacuum to yield 2.9 mg (93%) of crude product. The product was analyzed without further purification. 1H NMR (CDCl3, 400 MHz) diagnostic CαH and OMe resonances of bis-MPA: δ 4.84 (1H, s), 4.47 (1H, s), 3.46 (3H, s), 3.37 (3H, s); HRESIMS m/z 594.3203 [M + Na]+ (calcd for C36H45NNaO5 594.3190). N,O-Diacetyl Distaminolyne B, 50. A solution of premixed pyridine (0.5 mL) and acetic anhydride (0.5 mL) was added to distaminolyne B (6.3 mg, 23 μmol) and stirred at rt under N2 for 24 h. The mixture was dried under high vacuum, then taken up in CH2Cl2 (10 mL) and washed in turn with 10% HCl (10 mL), H2O (10 mL), saturated NaHCO3 (10 (mL), and finally H2O (10 mL) before being dried under vacuum to yield diacetyl distaminolyne B (6.4 mg, 77%) as a yellow oil. No further purification was carried out. [α]23D −19.8 (c 0.48, MeOH); [α]19D −19.1 (c 0.35, CHCl3); 1H NMR (CDCl3, 500 MHz) δ 5.84 (1H, ddt, J = 17.1, 10.4, 6.7 Hz, H17), 5.54 (1H, br d, J = 9.3 Hz, NH-2), 5.08 (1H, ddt, J = 16.9, 1.7, 1.3 Hz, H-18a), 5.04 (1H, m, H-18b), 4.86 (1H, m, H-3), 4.21 (1H, m, H-2), 2.34 (2H, m, H2-15), 2.28 (2H, m, H2-16), 2.23 (2H, t, J = 6.7 Hz, H2-10), 2.09 (3H, s, OAc), 1.99 (3H, s, NHAc), 1.52 (2H, m, H2-4), 1.50 (2H, m, H2-9), 1.36 (2H, m, H2-8), 1.31−1.24 (6H, m, H2-5 to H2-7), 1.10 (3H, d, J = 7.3 Hz, H3-1); 13C NMR (CDCl3, 125 MHz, * = interchangable) δ 171.1 (−OCOCH 3 ), 169.7 (−NHCOCH3), 136.6 (C-17), 116.1 (C-18), 77.9 (C-11), 76.7 (C14), 76.6 (C-3), 65.8 (C-13*), 65.3 (C-12*), 47.3 (C-2), 32.6 (C16), 31.7 (C-4), 29.3, 29.0, 28.8, 28.4 (C-6 to C-9), 25.3 (C-5), 23.6 (−NHCOCH3), 21.1 (−OCOCH3), 19.3 (C-10*), 19.2 (C-15*), 18.6 (C-1); HRESIMS m/z 382.2354 [M + Na]+ (calcd for C22H33NNaO3 382.2353).

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Robert A. Keyzers: 0000-0002-7658-7421 Brent R. Copp: 0000-0001-5492-5269 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge general funding from the University of Auckland and the New Zealand Foundation for Research Science and Technology, contract CO1X0205, for collection of the ascidian material. We thank Dr. M. Schmitz for assistance with NMR data acquisition, Mr. Tony Chen for MS data, and Dr. T. Molinski (UCSD) and Dr. L. Pilkington (UoA) for helpful discussions.

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(1) Pruett, S. T.; Bushnev, A.; Hagedorn, K.; Adiga, M.; Haynes, C. A.; Sullards, M. C.; Liotta, D. C.; Merrill, A. H., Jr. J. Lipid Res. 2008, 49, 1621−1639. (2) Jares-Erijman, E. A.; Bapat, C. P.; Lithgow-Bertelloni, A.; Rinehart, K. L.; Sakai, R. J. Org. Chem. 1993, 58, 5732−5737. (3) Hooper, G. J.; Davies-Coleman, M. T.; Coetzee, P. S. Nat. Prod. Lett. 1995, 6, 31−35. (4) Garrido, L.; Zubia, E.; Ortega, M. J.; Naranjo, S.; Salva, J. Tetrahedron 2001, 57, 4579−4588. (5) Ciavatta, M. L.; Manzo, E.; Nuzzo, G.; Villani, G.; Varcamonti, M.; Gavagnin, M. Tetrahedron 2010, 66, 7533−7538. (6) Won, T. H.; You, M.; Lee, S. H.; Rho, B. J.; Oh, D. C.; Oh, K. B.; Shin, J. Mar. Drugs 2014, 12, 3754−3769. (7) Kossuga, M. H.; MacMillan, J. B.; Rogers, E. W.; Molinski, T. F.; Nascimento, G. G. F.; Rocha, R. M.; Berlinck, R. G. S. J. Nat. Prod. 2004, 67, 1879−1881. (8) Aiello, A.; Fattorusso, E.; Giordano, A.; Menna, M.; Navarrete, C.; Munoz, E. Bioorg. Med. Chem. 2007, 15, 2920−2926. (9) Aiello, A.; Fattorusso, E.; Giordano, A.; Menna, M.; Navarrete, C.; Munoz, E. Tetrahedron 2009, 65, 4384−4388. (10) Gulavita, N. K.; Scheuer, P. J. J. Org. Chem. 1989, 54, 366−369. (11) Jimenez, C.; Crews, P. J. Nat. Prod. 1990, 53, 978−982. (12) Clark, R. J.; Garson, M. J.; Hooper, J. N. A. J. Nat. Prod. 2001, 64, 1568−1571. (13) Dasyam, N.; Munkacsi, A. B.; Fadzilah, N. H.; Senanayake, D. S.; O’Toole, R. F.; Keyzers, R. A. J. Nat. Prod. 2014, 77, 1519−1523. (14) Rinehart, K. L.; Fregeau, N. L.; Warwick, R. A.; Garcia Gravalos, D.; Avila, J.; Faircloth, G. T. PCT Int. Appl. WO 99/52521, 1999. (15) Martinkova, M.; Gonda, J.; Jackova, D. Tetrahedron: Asymmetry 2016, 27, 1187−1212. (16) Cuadros, R.; Montejo de Garcini, E.; Wandosell, F.; Faircloth, G.; Fernandez-Sousa, J. M.; Avila, J. Cancer Lett. 2000, 152, 23−29. (17) Fabisikova, M.; Martinkova, M.; Hirkova, S.; Gonda, J.; Pilatova, M. B.; Gonciova, G. Carbohydr. Res. 2016, 435, 26−36. (18) Du, L.; Zhu, X.; Gerber, R.; Huffman, J.; Lou, L.; Jorgenson, J.; Yu, F.; Zaleta-Rivera, K.; Wang, Q. J. Ind. Microbiol. Biotechnol. 2008, 35, 455−464. (19) Pearce, A. N.; Copp, B. R.; Molinski, T. F. Molecules 2019, 24, 90. (20) Wang, J.; Pearce, A. N.; Chan, S. T. S.; Taylor, R. B.; Page, M. J.; Valentin, A.; Bourguet-Kondracki, M.-L.; Dalton, J. P.; Wiles, S.; Copp, B. R. J. Nat. Prod. 2016, 79, 607−610. (21) Sun, D.-Y.; Han, G.-Y.; Gong, J.-X.; Nay, B.; Li, X.-W.; Guo, Y.W. Org. Lett. 2017, 19, 714−717. (22) Dumpala, M.; Theegala, S.; Palakodety, R. K. Tetrahedron Lett. 2017, 58, 1273−1275. (23) Molinski, T. F.; Salib, M. N.; Pearce, A. N.; Copp, B. R. J. Nat. Prod. 2019, 82, 1183−1189. (24) Searle, P. A.; Molinski, T. F. J. Org. Chem. 1993, 58, 7578− 7580.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00504.

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REFERENCES

Color in situ photo of Pseudodistoma cereum, NMR spectra (1H, 13C, COSY, HSQC, HMBC) of 2 and N,Odiacetate 50, 1H NMR spectrum of N,O-dibenzoyl derivative 3, and tables summarizing 1H and 13C NMR data reported for diacetylated derivatives of 2-AA marine natural products (PDF) Raw NMR data (FIDs) for 1 (ZIP) Raw NMR data (FIDs) for 2 (ZIP) Raw NMR data (FIDs) for 50 (ZIP)

AUTHOR INFORMATION

Corresponding Author

*Tel (B. R. Copp): +64 9 373 7599, ext 88284. E-mail: b. [email protected]. G



Article

(25) Nicholas, G. M.; Molinski, T. F. J. Am. Chem. Soc. 2000, 122, 4011−4019. (26) We thank Prof. T. Molinski for electronic copies of ECD data for 4 and 5. (27) Leiro, V.; Freire, F.; Quinoa, E.; Riguera, R. Chem. Commun. 2005, 5554−5556. (28) Mori, K.; Matsuda, H. Liebigs Ann. Chem. 1992, 1992, 131− 137. (29) Ichihashi, M.; Mori, K. Biosci., Biotechnol., Biochem. 2003, 67, 329−333. (30) Abraham, E.; Davies, S. G.; Millican, N. L.; Nicholson, R. L.; Roberts, P. M.; Smith, A. D. Org. Biomol. Chem. 2008, 6, 1655−1664. (31) Archer, S. G.; Csatayova, K.; Davies, S. G.; Fletcher, A. M.; Roberts, P. M.; Thomson, J. E. Tetrahedron Lett. 2016, 57, 1270− 1272. (32) These are examples that have AC secured by robust chemical and physical methods. (33) The authors reported an acetamide NH chemical shift of δH 12.13 and no coupling constant. We consider the anomolous chemical shift to be a misprint and have ignored it. (34) Ichihashi and Mori commented that, at the genus level, Xestospongia sp. sponges were capable of biosynthesizing 2-AAs of variable relative configuration.29 This capability now appears to be possible at the individual specimen level. (35) Khalil, I. M.; Barker, D.; Copp, B. R. J. Nat. Prod. 2012, 75, 2256−2260.

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