Isolation and Stereospecific Synthesis of Janolusimide B from a New

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Isolation and Stereospecific Synthesis of Janolusimide B from a New Zealand Collection of the Bryozoan Bugula flabellata Jiayi Wang,† Michèle R. Prinsep,‡ Dennis P. Gordon,§ Michael J. Page,⊥ and Brent R. Copp*,† †

School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand Chemistry, School of Science, Faculty of Science and Engineering, University of Waikato, Private Bag 3105, Hamilton 3240, New Zealand § National Institute of Water & Atmospheric Research (NIWA) Ltd, Private Bag 14901, Wellington 6021, New Zealand ⊥ NIWA, PO Box 893, Nelson 7010, New Zealand ‡

S Supporting Information *

ABSTRACT: NMR-directed screening of New Zealand marine organisms has led to the isolation of the modified tripeptide janolusimide B from the common invasive bryozoan Bugula f labellata. The structure was established by NMR and MS analysis, degradative hydrolysis and derivatization, and stereoselective fragment synthesis. The bryozoan natural product is an N-methyl analogue of janolusimide, previously reported from the Mediterranean nudibranch Janolus cristatus, a species known to prey upon bryozoa. ryozoans (moss animals) are sessile filter-feeding organisms that are present in many benthic marine habitats. Although not that commonly studied, these organisms represent a rich source of bioactive secondary metabolites.1 While the most notable of these metabolites are the macrocyclic polyketides, the bryostatins,2 a growing number of alkaloids including the tambjamines,3 amathaspiramides,4 securamines,5 euthyroideones,6 and pterocellins7,8 have also been reported from bryozoans. Nudibranchs are major bryozoan colony predators, constituting a total of 29% of all predator species.9 While many nudibranch species are known to prey on bryozoa, only a limited number of predator−prey relationships have been defined. From a chemical ecological perspective, the most well studied relationship involves nudibranchs of the genus Tambja, e.g., Tambja abdere and T. eliora, which prey upon the bryozoan Sessibugula translucens10 and T. ceutae and T. stegosauriformis,3,11 that include Bugula dentata in their diet. In these cases, the bryozoan-derived 2,2′bipyrrolic alkaloids the tambjamines are also present in the predator nudibranchs, presumably sequestered as part of a chemical defense strategy.10 Bugula f labellata is a cosmopolitan species, reported worldwide as an invasive fouling organism.12 Specimens of the bryozoan were collected by scuba from mussel farm ropes at Stewart Island, New Zealand, and identified by one of us (D.P.G.) as Bugula f labellata. Extraction of the freeze-dried sample with MeOH, followed by fractionation by repeated C18 reversed-phase flash column chromatography using acidified solvents, led to the purification of janolusimide B (1) as an optically active yellow oil. A molecular formula of C20H36N3O5 for 1, requiring 5 degrees of unsaturation, was established by (+)-HRESIMS. NMR spectra for 1 were acquired in CD3OD, as well as CDCl3,

B

© XXXX American Chemical Society and American Society of Pharmacognosy

although the natural product was less soluble in the latter solvent. 1H NMR data accounted for 35 protons, composed of three methyl singlets [δH 2.90 (6H, br s), 1.24, and 1.20], five methyl doublets (δH 1.68, 1.24, 1.16, 1.11, and 0.83), six methines (δH 4.61, 4.27, 4.04, 3.94, 3.77, and 2.50), and two exchangeable resonances [δH 11.73 (br s) and 8.52 (d, J = 8.1 Hz)], the latter being observed in spectra acquired in CDCl3. Analysis of 13C and multiplicity-edited HSQC NMR spectra identified 20 carbons, comprising nine methyls, six methines, an alkyl quaternary carbon (δC 50.3), and three amide or amidelike (δC 179.0, 178.7, and 169.2) and ketone (δC 212.3) resonances (Table 1). Final confirmation of the molecular formula was obtained from 1H−15N HMBC data, which indirectly identified the presence of three 15N resonances at δN 170.3, 122.0, and 42.4. COSY data identified the presence of three 1H spin-systems, while a combination with correlations observed in the 1H−13C and 1H−15N HMBC spectra allowed for the construction of 1. The first spin-system, a methine quartet (δH 3.94, H-3) and methyl doublet (δH 1.68, H3-4), was identified as N,Ndimethylalanine based upon observation of 1H−13C HMBC Special Issue: Special Issue in Honor of William Fenical Received: September 25, 2014

A

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Table 1. 1H, 13C, and 15N NMR Data (CD3OD) for Janolusimide B (1)a position 1 1′ N-2 3 4 5 N-6 7 8 9 10 11 12 N-13 14 15 16 17 18 19 20 21 22

δC/δN, type 43.3, 40.7, 42.4, 65.8, 15.2, 169.2, 122.0, 47.2, 18.4, 77.5, 45.1, 13.9, 178.7, 170.3, 179.0, 50.3, 23.2, 19.8, 212.3, 70.4, 30.4, 17.4, 19.4,

CH3 CH3 NH CH CH3 C NH CH CH3 CH CH CH3 C N C C CH3 CH3 C CH CH CH3 CH3

δH, mult (J in Hz) 2.90, br s 2.90, br s 11.73, br sc 3.94, q (6.8) 1.68, d (6.8) 8.52, 4.27, 1.24, 3.77, 4.04, 1.11,

d (8.1)c ddd (8.6, 7.0, 6.7) d (7.0) dd (10.1, 1.2) m d (6.9)

HMBCb 1′, 2, 3 1, 2, 3 1, 1′, 5 2, 3, 5

5, 6, 6, 9, 9,

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

1.24, sd 1.20, sd

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

4.61, 2.50, 0.83, 1.16,

12, 13, 19, 19,

d (3.5) m d (6.9) d (7.5)

observed, CHCH(CH3)2, was composed of a methine doublet (δH 4.61, H-19) coupling to a methine multiplet (δH 2.50, H20), which in turn coupled to two methyl doublets at δH 0.83 (H3-21) and δH 1.16 (H3-22). This 1H spin-system was extended by the observation of 1H−13C HMBC correlations from methine H-19 to two carbonyl resonances at δC 179.0 (C14) and δC 212.3 (C-18). These same two carbonyl resonances also correlated to a gem-dimethyl subunit [δC 50.3 (C-15); δH 1.24 (H3-16), δC 23.2 (C-16); δH 1.20 (H3-17), δC 19.8 (C17)] requiring formation of a ring, the last remaining degree of unsaturation. Finally, an interfragment 1H−13C HMBC correlation from methine H-19 (δH 4.61) to C-12 (δ 178.7) resolved the connectivity of the molecule and established the planar structure of 1. Database substructure searching identified 1 to be an Nmethyl analogue of janolusimide (2), previously isolated from a Mediterranean collection of the nudibranch Janolus cristatus.13 Due to the presence of two possible N-methylation sites, to avoid ambiguity, we have named the new metabolite janolusimide B. The absolute configuration of 2 has been previously defined by a combination of hydrolysis and stereoselective synthesis.14,15 Comparison of 1H and 13C NMR chemical shifts (CDCl3) observed for 1 and reported for 2 (Figure S1) shows a high degree of similarity, differing only for those resonances associated with the alanine moiety. While similarity of specific rotation data between 1 ([α]D −13) and 2 ([α]D −10.3) suggested that both compounds shared the identical absolute configuration, we were mindful of the dangers of such assumptions and so undertook further studies to unequivocally establish the absolute configuration of 1. In a similar fashion to those studies undertaken with 2, alkaline hydrolysis of janolusimide B afforded methyl ester 3 and pyrrolidinedione 4 (Scheme 1). The NMR and chiroptical data observed for 4 ([α]D −26) agreed with those previously reported for the (−)-S enantiomer ([α]D −30.5)15 defining the 19S configuration of 1. In a departure from the protocol used by Sodano et al. to define the configuration of janolusimide (2), the hydrolysis reaction of 1 was repeated, followed by exhaustive acetylation of the crude reaction product, to afford 5 ([α]D −24) as an inseparable 3:1 mixture with alcohol 3. Details of our stereospecific synthesis of 5 are shown in Scheme 2. Homoallylic alcohol 7 was prepared by reaction of Boc-Lalaninal with organoborane reagent (E)-crotyl-(−)-Ipc2B (see Supporting Information). NMR data observed for 2S,3S,4S-7 (notice numbering scheme of synthetic products differs from natural product) agreed with those reported for its enantiomer,16 and as expected, an opposite signed specific rotation was observed (7 [α]D −36; ent-7 [α]D +3316). Removal of the Boc protecting group (TFA/CH2Cl2) afforded amine 8, which was then coupled under standard conditions with Boc-N-Me-L-Ala to afford 9. Acetylation (to 10) followed by ruthenium tetraoxide-catalyzed oxidation afforded carboxylic acid 11. Finally, the reaction sequence of methyl ester

13, 18, 20, 20,

14, 18, 20, 21 19 22 21

a1 H (400 MHz); 13C (100 MHz); 15N (40.5 MHz), chemical shift indirectly determined from 1H−15N HMBC NMR data. bHMBC correlations, optimized for 8.3 Hz, reported from the proton resonance to the indicated carbon or nitrogen resonance(s). cResonance observed in spectrum obtained in CDCl3. dAssignments are interchangeable.

correlations from both H-3 and H3-4 to a carbonyl resonance at δC 169.2 (C-5), from equivalent methyl resonances at δH 2.90 (H3-1 and H3-1′) to C-3 (δC 65.8), and 1H−15N HMBC correlations from both δH 2.90 (H3-1 and H3-1′) and δH 1.68 (H-4) to a nitrogen signal at δN 42.4 (N-2). An exchangeable proton resonance at δH 11.73 in the 1H NMR spectrum of 1 acquired in CDCl3 was observed to couple (COSY) to H3-1 and H3-1′, establishing the amino acid was present in the form of a trifluoroacetate salt. The second spin-system extended from a methyl doublet (δH 1.24, H3-8) through three sequential methine resonances [δH 4.27 (H-7) to δH 3.77 (H-9) to δH 4.04 (H-10)], before terminating at another methyl doublet (δH 1.11, H3-11). HSQC data identified C-9 (δC 77.5) to likely be an oxymethine (Table 1). HMBC data established correlations from H-9 (δH 3.77) and methyl H3-11 (δH 1.11) to a carbonyl resonance at δC 178.7 (C-12), while 1H−15N HMBC data indicated correlations from H-9 and methyl H3-8 (δH 1.24) to a nitrogen signal at δN 122.0 (N-6). An 1H−13C HMBC correlation between methine H-7 and C-5 linked this fragment to the N,N-dimethyl alanine residue. The third 1H spin-system Scheme 1. Hydrolysis of Janolusimide B (1)

B

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NH-2), 8.52 (1H, d, J = 8.1 Hz, NH-6), 4.56 (1H, d, J = 3.7 Hz, H16), 4.26−4.18 (1H, m, H-7), 4.18−4.11 (1H, q, J = 5.7 Hz, H-3), 4.03−3.95 (1H, m, H-10), 3.80 (1H, dd, J = 7.5, 2.4 Hz, H-9), 2.95 (6H, s, 2H3-1), 2.57−2.46 (1H, m, H-19), 1.73 (3H, d, J = 5.7 Hz, H34), 1.33 (3H, d, J = 6.8 Hz, H3-8), 1.27 (3H, s, H3-18), 1.24 (3H, s, H3-17), 1.18 (3H, d, J = 7.0 Hz, H3-21), 1.16 (3H, d, J = 6.7 Hz, H311), 0.88 (3H, d, J = 6.9 Hz, H3-20); 13C NMR (CDCl3, 75 MHz) δ 210.9 (C-15), 177.6 (C-13), 177.3 (C-12), 167.0 (C-5), 77.7 (C-9), 69.4 (C-16), 63.2 (C-3), 49.4 (C-14), 47.1 (C-7), 44.4 (C-10), 41.8 (C-1a), 39.2 (C-1b), 29.7 (C-19), 23.2 (C-17), 19.4 (C-18), 19.2 (C21), 17.9 (C-8), 17.1 (C-20), 14.6 (C-4), 14.0 (C-11); (+)-HRESIMS m/z 398.2652 [M + H]+ (calcd for C20H36N3O5, 398.2649). Hydrolysis of Janolusimide B. A solution of janolusimide B (4.0 mg, 0.010 mmol) in methanolic NaHCO3 solution (1.5 mL, MeOH/ H2O, 3:1, 1% saturated aqueous NaHCO3) was stirred at room temperature (rt) for 3 h. H2O (5 mL) was added, and the resultant solution extracted with CH2Cl2 (2 × 5 mL). The combined organic layers were washed with brine (3 mL), dried (MgSO4), and concentrated in vacuo. The crude residue was purified by silica gel column chromatography (EtOAc to THF/EtOAc/MeOH, 3:6:1) to yield methyl ester 3 as a yellow oil (1.0 mg, 38%) and pyrrolidinedione 4 as a brown oil (0.7 mg, 41%). Methyl 4-(2-(dimethylamino)propanamido)-3-hydroxy-2-methylpentanoate 3: [α]20D −40 (c 0.020, MeOH); 1H NMR (CDCl3, 400 MHz) δ 7.38 (1H, d, J = 10.0 Hz, NH-7), 4.21−4.11 (1H, m, H4), 3.72 (3H, s, H3-12), 3.67 (1H, dd, J = 8.7, 2.5 Hz, H-3), 3.01 (1H, q, J = 7.2 Hz, H-9), 2.53 (1H, dq, J = 8.7, 7.2 Hz, H-2), 2.24 (6H, s, 2H3-11), 1.24 (3H, d, J = 6.4 Hz, H3-5), 1.22 (3H, d, J = 7.2 Hz, H310), 1.20 (3H, d, J = 7.2 Hz, H3-6); (+)-HRESIMS m/z 261.1813 [M + H]+ (calcd for C12H25N2O4, 261.1809). (5S)-Isopropyl-3,3-dimethylpyrrolidine-2,4-dione (4): [α]20D −26 (c 0.085, CHCl3) (lit.15 −38.3 (c 0.70, CHCl3)); 1H NMR (CDCl3, 300 MHz) δ 5.91 (1H, br s, NH-1), 3.90 (1H, d, J = 4.3 Hz, H-5), 2.25−2.12 (1H, m, H-8), 1.25 (3H, s, H3-6), 1.21 (3H, s, H3-7), 1.03 (3H, d, J = 7.0 Hz, H3-9), 0.90 (3H, d, J = 6.6 Hz, H3-10); (+)-HRESIMS m/z 192.0993 [M + Na]+ (calcd for C9H15NNaO2, 192.0995). The spectroscopic data were found to be in agreement with literature.13 Semisynthetic (2R,3S,4S)-Methyl 3-acetoxy-4-((S)-2(dimethylamino)propanamido)-2-methylpentanoate (5). A solution of janolusimide B (4.6 mg, 0.012 mmol) in methanolic NaHCO3 solution (1.6 mL, MeOH/H2O, 3:1, 1% saturated aqueous NaHCO3) was stirred at rt for 3 h. H2O (5 mL) was added, and the resultant solution extracted with CH2Cl2 (2 × 5 mL). The combined organic layers were washed with brine (3 mL), dried (MgSO4), and concentrated in vacuo. To this yellow crude product in pyridine (1 mL) was added acetic anhydride (1.5 μL, 0.016 mmol). The reaction was left to stir under N2 for 60 h, then concentrated in vacuo to give a yellow oil, which was subjected to silica gel column chromatography (EtOAc/MeOH, 4:1) to give an inseparable mixture of alcohol 3 and acetate 5 (0.7 mg, 17% yield) in a 1:3 ratio: [α]20D −24 (c 0.11, MeOH); 1H NMR (CD3OD, 500 MHz) δ 5.08 (1H, dd, J = 9.0, 3.7 Hz, H-3), 4.39−4.33 (1H, m, H-4), 3.65 (3H, s, H3-14), 3.02 (1H, q, J = 7.0 Hz, H-9), 2.73−2.66 (1H, m, H-2), 2.29 (6H, s, 2H3-11), 2.06 (3H, s, H3-13), 1.23 (3H, d, J = 7.0 Hz, H3-10), 1.19 (3H, d, J = 7.3 Hz, H3-6), 1.12 (3H, d, J = 6.7 Hz, H3-5); (+)-HRESIMS m/z 303.1924 [M + H]+ (calcd for C14H27N2O5, 303.1914). Synthetic (2R,3S,4S)-Methyl 3-acetoxy-4-((S)-2-(dimethylamino)propanamido)-2-methylpentanoate (5). To a solution of methylamine 13 (5.3 mg, 0.018 mmol) in H2O (1 mL) were added formaldehyde (38% w/w, 4.4 μL, 0.055 mmol) and palladium on activated carbon (10% w/w, 2.0 mg, 1.8 μmol). The reaction mixture was stirred under H2 for 4.5 h and filtered, and the solute concentrated in vacuo. The resultant red oil was then dissolved in saturated aqueous Na2CO3 (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic extracts were dried (MgSO4) and concentrated in vacuo to give a red gum, which was subjected to silica gel column chromatography (EtOAc/MeOH, 9:1) to yield 5 as a colorless oil (2.1 mg, 38% yield): Rf 0.20 (EtOAc/MeOH, 4:1); [α]19D −37 (c 0.087, MeOH); IR νmax (ATR) 3357, 2979, 2950, 1743, 1663, 1508, 1226,

Scheme 2. Stereoselective Synthesis of 2R,3S,4S,9S-5

formation (12, 68% yield), amine deprotection to give 13 (quantitative yield), and reductive methylation (CH2O, Pd/C, H2; 38% yield) afforded 2R,3S,4S,9S-5. 1H NMR and specific rotation data ([α]D −37) observed for this synthetic fragment agreed with those observed for the degradative fragment of janolusimide B (5, as an inseparable mixture with alcohol 3 at a ratio of 3:1, [α]D −24), defining the absolute configuration of the natural product to be as shown. In summary, our investigation of the chemistry of a New Zealand collection of the cosmopolitan fouling bryozoan B. f labellata has led to the isolation and structural characterization of a modified tripeptide, janolusimide B. The natural product is an N-methyl analogue of a metabolite previously reported from a Mediterranean collection of the nudibranch J. cristatus, an organism known to prey upon bryozoans. The determination that both natural products are of the same absolute configuration suggests that the janolusimides represent only the second example of a chemical ecological linkage between a bryozoan and a nudibranch. Rigorous confirmation of this linkage will require identification of a predator of the New Zealand populations of B. f labellata and/or identification of the prey of the Mediterranean J. cristatus.

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EXPERIMENTAL SECTION

General Experimental Procedures. The majority of the experimental details have been reported elsewhere.17 Optical rotations were recorded on a PerkinElmer 341 polarimeter using a 0.1 dm cell, while 15N chemical shifts were referenced using the Ξi unified scale18 as implemented in Bruker library function xiref. Animal Material. The bryozoan was collected on August 28, 2007, by scuba at a depth of 10 m from mussel farm ropes in Big Glory Bay, Stewart Island, Southland, New Zealand, and kept frozen until used. A voucher specimen of the peach-colored bryozoan Bugula f labellata (Thompson, in Gray, 1848) (phylum: Bryozoa, class: Gymnolaemata, order: Cheilostomata, family: Bugulidae) is held at the NIWA, Private Bag 14901, Wellington, New Zealand, as NIWA 76320 (MNP9185). A color in situ photograph is included in the Supporting Information. Isolation and Purification. Freeze-dried specimens (15.15 g) were extracted with MeOH (5 × 200 mL) to afford a yellow extract (6.01 g), which was subjected to repeated C18 reversed-phase flash column chromatography [H2O (0.05% TFA) to MeOH (0.05% TFA)] with the compound of interest eluting at 15% MeOH/H2O (TFA), to yield janolusimide B (1) as a yellow oil (30 mg, 0.002% dry weight), being the trifluoroacetate salt. Janolusimide B (1): [α]20D −13 (c 0.83, CHCl3); UV (MeOH) λmax (log ε) 207 (9.22), 218 (8.95), 268 (7.69), 326 (7.03) nm; IR νmax (ATR) 3315, 2926, 2782, 2739, 1721, 1647, 1531, 1451, 1359, 1219, 1085, 1019 cm−1; 1H NMR (CDCl3, 500 MHz) δ 11.73 (1H, br s, C

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1199, 1162, 1021 cm−1; 1H NMR (CD3OD, 500 MHz) δ 5.08 (1H, dd, J = 9.0, 3.3 Hz, H-3), 4.39−4.33 (1H, m, H-4), 3.65 (3H, s, H314), 3.02 (1H, q, J = 6.9 Hz, H-9), 2.69 (1H, dq, J = 9.0, 7.0 Hz, H-2), 2.29 (6H, s, 2H3-11), 2.05 (3H, s, H3-13), 1.23 (3H, d, J = 6.9 Hz, H310), 1.19 (3H, d, J = 7.0 Hz, H3-6), 1.12 (3H, d, J = 7.3 Hz, H3-5); 13C NMR (CD3OD, 75 MHz) δ 175.7 (C-1), 175.6 (C-8), 171.8 (C-12), 78.2 (C-3), 65.4 (C-9), 52.4 (C-14), 45.7 (C-4), 43.1 (C-2), 42.4 (C11), 20.7 (C-13), 18.6 (C-5), 14.6 (C-10), 14.1 (C-6); (+)-HRESIMS m/z 303.1916 [M + H]+ (calcd for C14H27N2O5, 303.1914).

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(15) Giordano, A.; Della Monica, C.; Landi, F.; Spinella, A.; Sodano, G. Tetrahedron Lett. 2000, 41, 3979−3982. (16) Yakelis, N. A.; Roush, W. R. J. Org. Chem. 2003, 68, 3838−3843. (17) Khalil, I. M.; Barker, D.; Copp, B. R. J. Nat. Prod. 2012, 75, 2256−2260. (18) Harris, R. K.; Becker, E. D.; De Menezes, S. M. C.; Goodfellow, R.; Granger, P.; Hoffman, R. E.; Zilm, K. W. Pure Appl. Chem. 2008, 80, 59−84.

ASSOCIATED CONTENT

S Supporting Information *

Color in situ photo of Bugula flabellata, table of comparison of 1 H and 13C NMR data for 1 and 2 (CDCl3) and NMR chemical shift difference plots, copies of 1H, 13C, COSY, HSQC, 1H−13C HMBC, and NOESY NMR spectra for 1, 1H spectra of fragments 3−5, synthesis experimental details, and 1H and 13C spectra of synthetic intermediates 7−13. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +64 9 373 7599, ext 88284. Fax: +64 9 373 7422. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge funding from the University of Auckland and from the New Zealand Foundation for Research Science and Technology (contract CO1X0205) for organism collection. We thank Dr. D. Furkert for technical assistance, Dr. M. Schmitz for assistance with NMR data acquisition, and Dr. N. Lloyd for the HRMS data.

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DEDICATION Dedicated to Dr. William Fenical of Scripps Institution of Oceanography, University of California−San Diego, for his pioneering work on bioactive natural products.

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REFERENCES

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