Colony-wise Analysis of a Theonella swinhoei Marine Sponge with a

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

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Colony-wise Analysis of a Theonella swinhoei Marine Sponge with a Yellow Interior Permitted the Isolation of Theonellamide I Kazuya Fukuhara,† Kentaro Takada,*,†,§ Ryuichi Watanabe,‡ Toshiyuki Suzuki,‡ Shigeru Okada,† and Shigeki Matsunaga*,† †

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Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan ‡ National Research Institute of Fisheries Science, 2-12-4 Fukuura, Kanazawa, Yokohama 236-8648, Japan S Supporting Information *

ABSTRACT: There are several examples of marine organisms whose metabolic profiles differ among conspecifics inhabiting the same region. We have analyzed the metabolic profile of each colony of a Theonella swinhoei marine sponge with a yellow interior and noticed the patchy distribution of one metabolite. This compound was isolated and its structure was studied by a combination of spectrometric analyses and chemical degradation, showing it to be a congener in the theonellamide class of bicyclic peptides. Theonellamides had previously been isolated by us only from T. swinhoei with a white interior and not from those with a yellow interior.

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that the major constituents, such as onnamides, aurantosides, and orbiculamides,7 were distributed in common in all of the samples (Figures S1−S4), whereas the quantities of some metabolites varied among samples. The intensity of the peak that gave a pair of ions at m/z 1703 and 1705 in 1:1 ratio differed most significantly. Their contents were highest for two of the samples (YT2 and YT11), lower but detectable for five samples, and not detectable in the remaining eight samples. Then, we compared the LC-MS data of the extracts of small pieces dissected from two distant regions of a mass of the sponge YT11; the data were almost superimposable, suggesting an even distribution of secondary metabolites within the colony (Figure S3). We then set out to characterize this compound. The specimen YT-11 was extracted with EtOH, and the extract was subjected to a solvent partitioning scheme to give H2O, n-BuOH, and CHCl3 fractions. The n-BuOH fraction was subjected to ODS open column chromatography followed by two rounds of ODS-HPLC to afford theonellamide I (1).8 The molecular formula of 1 was determined to be C74H95BrN16O26 by HRESIMS. Initial analysis of the 1H NMR and HSQC data suggested that 1 was a peptide, as judged from the presence of considerable numbers of amideand α-protons. In order to facilitate the analysis of the signals buried under the H2O resonance, NMR spectra were measured at 30 and 50 °C in DMSO-d6−H2O (4:1) (Table 1 and Table

arine invertebrates such as sponges and tunicates are rich sources of biologically active secondary metabolites.1 Because identical or very closely related compounds have been isolated from marine organisms and terrestrial microorganisms, such natural products were speculated to be produced by symbiotic microorganisms.2 Recent studies using molecular biological techniques have revealed the biosynthetic gene clusters of such metabolites to reside in the genomes of the symbiotic bacteria, thereby demonstrating the involvement of symbiont(s) in their production.3 Patchy distribution of some marine natural products within the same species of organisms can be accounted for by the uneven distribution of the producing microorganisms, as exemplified by the studies on patellamide,4 patellazoles,5 and dysiherbaine.6 Even though the biosynthetic gene clusters for most of the secondary metabolites isolated from Theonella swinhoei with a yellow interior (TSY) have been located in the genome of “Candidatus Entotheonella factor”,3 we speculated that metabolic profiles of individual sponge specimens could be variable, reflecting the composition of the symbiotic bacteria. On the basis of this hypothesis, we have analyzed the constituents of individual specimens of TSY, which were collected in the same region at the same time. We have detected a new metabolite of the theonellamide class termed theonellamide I (1). In spite of the almost even distribution of other secondary metabolites that had been isolated from TSY,7 theonellamide I was present in only a few of the samples at higher concentrations. We examined 15 individuals of TSY (sponge ID: YT1− YT15), collected at Hachijo Island, Japan. The analysis showed © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 19, 2018

A

DOI: 10.1021/acs.jnatprod.8b00591 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

4-hydroxy-6-methyl-8-phenyloctanoic acid (H4-Apoa) residue by hydrogenation, which was liberated by acid hydrolysis.11 Authentic samples of histidinoalanine, BrPhe, OHAsp, and H4Apoa were prepared from the mixture of theonellamides A, D, and E (3−5).11 The Marfey’s analysis with detection by ESIMS revealed the presence of L-Phe, L-Ser, L-aThr, Lhistidino-D-alanine,14 L-Aad, (S)-Iser, L-BrPhe, (2S,3R)OHAsn, L-Asn, and (3S,4S)-Apoa (Figures S18−S26).15 The arabinose residue liberated by acid hydrolysis was converted to 3-phenylthiocarbamoylthiazoline-4(R)-carboxylate16 followed by LCMS analysis (Figure S27), demonstrating it to be in the L-form. Therefore, the structure of 1 was assigned as a demethyl derivative of theonegramide (2), which had been isolated from T. swinhoei collected at Negros Island, Philippines.17 However, there are discrepancies in the absolute configurations of the Ise and arabinose residues between the two compounds. The assignments of (R)-Ise and D-arabinose in theonegramide17 were made by GC-MS analysis of the acid hydrolysate with a chiral stationary phase, which is an established method for the configurational analysis of amino acids and monosaccharides. Because they used GC analysis with MS detection, the possibility of peak misassignments due to contaminants is improbable. The configurations of the Ise residue in theonellamides A and B and that of the Ara residue in theonellamide D are both identical with those in theonellamide I (1). In order to prove or disprove the assignments in theonegramide, stereochemical re-evaluation using an authentic sample is required. Theonellamide I (1) exhibited moderate cytotoxicity against HeLa cells with an IC50 value of 1.9 μM. This potency was similar to those of theonellamides A−E against P388 murine leukemia cells.11 Large populations of two types of T. swinhoei are found in the coastal areas of Hachijo Island. From the one with the yellow interior (TSY) we have isolated onnamides, polytheonamides, orbiculamides, cyclotheonamides, and aurantosides.7 Metagenomics and single-cell genomics studies have shown that all of these metabolites except the aurantosides are biosynthesized by the single symbiotic bacterium Ca. E. factor.3 The second phylotype with a white interior (TSW) is the source of the theonellamides11 and misakinolides,18 both of which have been shown to be produced by the symbiotic bacterium Ca. E. serta.19,20 From these studies the distribution of different secondary metabolites in the two phylotypes of sponges was accounted for by the presence of different symbionts with unique sets of biosynthetic gene clusters for secondary metabolites. These two types of sponges sometimes inhabit locations close to each other, but the compositions of secondary metabolites are distinct, suggesting the absence of horizontal transfer of the symbionts. Although the structure of theonellamide I (1) is closely related to those of theonellamides previously isolated from TSW, the same compound has not been isolated from TSW,11 indicating the presence of another theonellamide producer in select individuals of TSY. In this study, we have demonstrated the benefits of specimen-wise analysis of the sponge extract to find otherwise hidden metabolites.

S1).9 Interpretation of the COSY, TOCSY, NOESY, and HMBC data showed the presence of 3-amino-4-hydroxy-6methyl-8-phenyl-5,7-octadienoic acid (Apoa), Phe, Ser (two residues), Thr (or allo-Thr (aThr)), histidinoalanine (sHis (histidine portion) and sAla (alanine portion)), α-aminoadipic acid (Aad), isoserine (Iser), p-bromophenylalanine (BrPhe), βhydroxyasparagine (OHAsn), and Asn,10 suggesting that 1 was a member of the theonellamide class of peptides.11 One anomeric carbon signal (δC 88.2, δH 4.98) observed in the HSQC spectrum suggested glycosylation of the molecule. By considering the molecular formula, the remaining monosaccharide portion was assigned as a pentose. The 1H NMR signals for the C-5 methylene protons of the pentose were buried under the water signal, and the C-4 methine signal was partly obscured. However, a large coupling constant between H-2 and H-3 and a small coupling constant between H-3 and H-4 as inferred from the magnitudes of the COSY cross-peaks suggested the presence of an arabinopyranose residue. The amino acid sequence of 1 was determined on the basis of the NOESY data. The amide protons were well separated, permitting us to sequence the peptide on the basis of the NH/ α-H and NH/NH correlations between the neighboring residues. The glycosylation at the π-nitrogen in the imidazole ring of the histidinoalanine residue was demonstrated by the HMBC cross-peaks between the anomeric proton signal (δH 4.98) and carbon signals in the imidazole ring (δC 131.4 and 136.5).11 The (5E,7E)-configuration in the Apoa residue was elucidated on the basis of the 13C NMR chemical shift of the εmethyl signal (δC 12.8) and the 1H−1H coupling constant of 16.5 Hz between H-ζ and H-η. The absolute configuration of each amino acid residue in 1 was determined by a modification of the Marfey’s method using FDNP-Val as the chiral derivatization reagent.12,13 Because the Apoa residue decomposed during acid hydrolysis, it was converted to the 3-amino-

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EXPERIMENTAL SECTION

General Experimental Procedures. The optical rotation was measured on a Jasco DIP-1000 polarimeter. The UV spectrum was measured on a Shimadzu BioSpec-1600 spectrophotometer. NMR spectra were measured either on a JEOL alpha 600 or a Bruker B



Note

Table 1. 1H (600 MHz) and 13C NMR Data (150 MHz) of Theonellamide I (1) in DMSO-d6−H2O (4:1) at 50 °C amino acid

position

δC,a,b type

Apoa

CO α β γ δ ε ε-Me ζ η 1 2, 6 3, 5 4 NH CO α β NH CO α β NH CO α β CONH2 NH CO α β CONH2 NH CO α β 1 2, 6 3, 5 6 NH CO α β

172.5, C 36.9, CH2 52.4, CH 67.9, CH 132.4, CH 135.7, C 12.8, CH3 133.3, CH 127.7, CH 137.6, C 126.2, CH 128.9, CH 127.7, CH

Ser-1

sAla

Asn

OHAsn

BrPhe

Iser

172.5, C 56.4, CH 60.8, CH2 169.5, C 50.6, CH 49.8, CH2 171.3, C 51.5, CH 36.6, CH2 174.5, C 170.4, C 54.2, CH 72.0, CH 174.2, C 172.2, C 55.0, CH 36.6, CH2 137.6, C 131.2, CH 131.2, CH 120.0, CH

δH (J in Hz) 2.38, 4.10, 4.25, 5.20,

amino acid

m;c 2.11, dd (12.6, 3.3) m dd (8.8, 3.9) d (8.8)

Aad

1.66, s 6.63, d (16.5) 6.50, d (16.5) 7.40, 7.30, 7.19, 7.79,

sHis

d (7.7) t (7.7) m d (8.0)

3.72, m 3.63, m 7.86, m

Arabinose

5.05, m 4.89, brd (13.7), 4.20, m 8.22, d (10.1) 4.47, 2.56, 7.23, 7.80,

m m; 2.24, m m; 6.77, brs m

5.30, 4.03, 7.35, 8.33,

dd (7.4, 9.4) d (7.4) brs; 7.23, m m

aThr

Ser-2

Phe

4.28, dt (4.4, 8.5) 3.03, m; 2.66, m 7.04, d (8.3) 7.27, d (8.3)

NH CO α β γ δ COO− NH CO α β 2 4 5 NH 1 2 3 4 5 CO α β γ NH CO α β NH CO α β 1 2, 6 3, 5 4 NH

δH (J in Hz)

δC,a,b type 7.45, m 175.5, C 54.2, CH 31.7, CH2 21.9, CH2 35.4, CH2 174.3, C

3.93, 1.77, 1.40, 2.23,

m m; 1.53, m m m; 1.98, m

7.63, m 170.4, C 53.9, CH 26.0, CH2 136.5, CH 131.4, C 123.6, CH 88.2, CH 69.1, CH 72.6, CH NDd ND 172.3, C 58.0, CH 68.2, CH 21.0, CH3 169.5, C 56.2, CH 61.5, CH2 171.5, C 54.2, CH 38.6, CH2 137.0, C 129.2, CH 128.3, CH 126.8, CH

4.82, m 3.23, m; 2.99, m 8.90, s 7.24, 8.35, 4.98, 3.67, 3.43, ND ND

s m d (8.8) m m

4.22, 3.56, 0.90, 7.43,

m m d (6.1) m

4.48, m 3.66, m; 3.59, m 8.66, m 4.55, q (8.0) 2.82, dd (13.7, 8.0); 2.65, m 7.16, 7.22, 7.17, 8.02,

m m m brd (9.1)

a13 C chemical shifts of protonated carbons were determined by the HSQC spectrum. bChemical shifts of nonprotonated carbons were determined by the HMBC spectrum with either the 600 or 800 MHz spectrometer. cCoupling constant was not determined due to overlapped signals. dObscured by the H2O signal.

8.67, m 171.3, C 69.7, CH 43.0, CH2

position

4.11, m 3.90, m; 2.95, m

AVANCE 800 NMR spectrometer and referenced to the solvent peak: δH 2.49 and δC 39.5 for DMSO-d6. ESI mass spectra were recorded on a JEOL JMS-T100LC mass spectrometer. LC-MS experiments were performed on a Shimadzu LC-20AD solvent delivery system interfaced to a Bruker amaZon SL mass spectrometer. The result of the XTT assay was recorded with a Molecular Devices SPECTRA max M2. Analysis of the Chemical Profiles of Theonella swinhoei with a Yellow Interior. The specimens of TSY were collected at Hachijo Island in 1999 and kept frozen at −20 °C until processed. The small amounts (10−20 g) dissected from 15 colonies (YT-1−YT-15) were separately extracted with MeOH, and the extracts were dried in vacuo. Each extract was applied to a short column of ODS and eluted with MeOH. The MeOH eluates were analyzed by LC-MS on a COSMOSIL 2.5C18-MS-II column with gradient elution from H2O to MeCN in the presence of 0.5% acetic acid for 70 min. Extraction and Isolation. A specimen of T. swinhoei designated YT-11 (850 g, wet weight) was extracted with EtOH. The extract was

partitioned between H2O and CHCl3. The H2O layer was extracted with n-BuOH. The BuOH layer (2.0 g) was subjected to ODS column chromatography and eluted with a stepwise gradient of MeCN−H2O (1:4), MeCN−H2O (3:7), MeCN−H2O (2:3), and MeCN−H2O (1:1). The fraction eluted with MeCN−H2O (2:3) was subjected to ODS-HPLC on a COSMOSIL 5C18-MS-II column. Gradient elution was performed using solvent A (50 mM KH2PO4 aqueous solution) and solvent B (n-PrOH), from A−B (4:1) to A−B (1:1). The fraction that contained the compound exhibiting ions at m/z 1703 and 1705 in the ESIMS spectrum was further purified by ODS-HPLC on a COSMOSIL 5C18-AR-II column with gradient elution from MeCN− H2O (3:7) to MeCN−H2O (1:1) containing 0.5% acetic acid to afford theonellamide I (1, 1.2 mg). Additionally a one gram portion of the n-BuOH fraction (22 g) prepared from 22 kg wet weight of TSY colonies collected at Hachijo Island in 1999 was purified in the same manner to afford more theonellamide I (1, 2.2 mg). Theonellamide I (1): pale yellow powder; [α]D −19 [c 0.05, nPrOH−H2O (2:1)]; UV [n-PrOH−H2O (2:1)] λmax (log ε) 211 C



Note

(4.6) 278 (4.2); 1H and 13C NMR [DMSO-d6−H2O (4:1)], Table 1 and Table S1; HRESIMS m/z 1725.5701 (calcd for C74H9579BrN16O26Na, 1725.5684, Δ +1.7 mmu) . Marfey’s Analysis of Theonellamide I (1). Compound 1 (100 μg) was dissolved in 6 N HCl (200 μL) and heated at 110 °C for 3 h. The mixture of theonellamides A, D, and E11 was hydrolyzed in the same manner. The solvent was evaporated with a stream of N2 and redissolved in 0.6 M NaHCO3 (100 μL). To the solution was added 3% FDNP-L-Val in EtOH (80 μL), and the mixture was kept at 55 °C for 1 h.13 After neutralization with 3 N HCl (20 μL), the reaction mixture was analyzed by LC-MS on a COSMOCORE 2.6PBr column with gradient elution from MeCN−H2O (1:9) to MeCN−H2O (7:3) containing 0.5% acetic acid for 32 min. Standard amino acids were derivatized with either FDNP-L-Val or FDNP-D-Val and analyzed by LC-MS. Standard amino acids of L-BrPhe and (2S,3R)-β-OHAsp were obtained from the hydrolysate of the mixture of theonellamides A, D, and E.11 Retention times of the amino acids and LC-MS charts are shown in Table S2 and Figures S18−S24. Determination of the Absolute Configuration of the Histidinoalanine Residue. The mixture of theonellamide A, D, and E (500 μg) was dissolved in 6 N HCl (250 μL) and subjected to acid hydrolysis (110 °C, 16 h). A half-portion of the hydrolysate was derivatized with FDNP-L-Val and analyzed by LC-MS. In addition to 11 L-histidino-D-alanine, L-histidino-L-alanine was detected. The ratio of the LD-isomer and LL-isomer was 3:1. The rest of the hydrolysate was derivatized with FDNP-D-Val to prepare HPLC equivalents of Dhistidino-L-alanine and D-histidino-D-alanine.11 Retention times and LC-MS charts are shown in Table S4 and Figure S25. Determination of the Absolute Configuration of 3-Amino4-hydroxy-6-methyl-8-phenyl-5,7-octadienoic Acid Residue. To a solution of 1 (100 μg) in MeCN−H2O (1:1, 1 mL) was added 10% Pd/C (2 mg), and the mixture was stirred under an atmosphere of H2 for 18 h. The mixture was filtered through Celite, and the solvent was removed in vacuo to afford a diastereomeric mixture of tetrahydrotheonellamide I.18,19 The product was subjected to acid hydrolysis, derivatization with FDNP-L-Val, and LC-MS analysis as described above. The standard of a diastereomeric mixture of 3amino-6-methyl-8-phenyloctanoic acid was obtained from the mixture of theonellamides A, D, and E and derivatized with either FDNP-DVal or FDNP-L-Val and then analyzed by LC-MS in the same manner. Retention times and the LC-MS chart are shown in Table S5 and Figure S26. Analysis of the Absolute Configuration of the Arabinose Residue in Theonellamide I (1). Theonellamide I (1, 60 μg) was dissolved in 6 N HCl (200 μL) and heated at 110 °C for 3 h. The solvent was evaporated with a stream of N2, and the residue was dissolved in 10% HCl in MeOH (200 μL). The mixture was heated at 100 °C for 1 h. After evaporation of the solvent, to the product was added a solution of L-cysteine methyl ester hydrochloride in pyridine (2 mg/mL; 100 μL) and the solution was heated at 60 °C for 1 h. Then, a 5 μL portion of phenylisothiocyanate was added, and the solution was heated for 1 h at 60 °C. The solvent was evaporated, the product was dissolved in MeOH (100 μL), and the product was analyzed by LC-MS on a COSMOSIL 2.5C18-MS-II column with gradient elution from MeCN−H2O (1:9) to MeCN−H2O (3:17) containing 0.5% acetic acid for 60 min. L- and D-Arabinose were treated in the same manner. Retention times and the LC-MS chart are shown in Table S2 and Figure S27. XTT Assay against HeLa Cells. HeLa human cervical cancer cells were cultured in Dulbecco’s modified Eagle’s medium (Wako Chemical), supplemented with 100 U/mL of penicillin G (Wako Chemical), 100 μg/mL of streptomycin sulfate (Wako Chemical), and 10% fetal bovine serum (Gibco), at 37 °C under a 5% CO2 atmosphere. To each well of a 96-well microplate containing 200 μL of tumor cell suspension (1 × 104 cells/mL) was added a test solution after 24 h of preincubation, and the plate was incubated for 72 h. After the addition of 50 μL of 3′-[1-(phenylaminocarbonyl)-3,4tetrazolium]bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate (XTT) solution (1 mg/mL) containing 4% phenazine methosulfate solution (0.153 mg/mL) to each well, the plate was further incubated

for 4 h. The absorbance at 450 nm was measured with a microplate reader. Three replicates were examined to determine the IC50 value (Figure S28). A parallel analysis of adriamycin using MTT gave an IC50 value of 2 μM. The purity of 1 used for the cytotoxicity test was ca. 90%, as judged from the 1H NMR spectrum.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00591. Spectroscopic data (1H, COSY, HSQC, TOCSY, HMBC, and NOESY) and the results of the Marfey’s analysis of theonellamide I (1) (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Shigeki Matsunaga: 0000-0002-8360-2386 Present Address §

School of Marine Bioscience, Kitasato University, Sagamihara, Kanagawa 252-0373, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partly supported by JSPS KAKENHI Grant Numbers 25252037, 16H04980, 17J09477, and 17H06403 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank Professor J. Piel, ETA Zurich, for valuable discussion and R. Suo for experimental assistance.

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REFERENCES

(1) Blunt, J. W.; Carroll, A. R.; Copp, B. R.; Davis, R. A.; Keyzers, R. A.; Prinsep, M. R. Nat. Prod. Rep. 2018, 35, 8−53 and previous reviews in this series. . (2) Piel, J. Nat. Prod. Rep. 2009, 26, 338−362. (3) Wilson, M. C.; Vagstad, A. L.; Piel, J. Curr. Opin. Chem. Biol. 2016, 31, 8−14 and references therein . (4) Schmidt, E. W.; Nelson, J. T.; Rasko, D. A.; Sudek, S.; Eisen, J. A.; Haygood, M. G.; Ravel, J. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 7315−7320. (5) Kwan, J. C.; Donia, M. S.; Han, A. W.; Hirose, E.; Haygood, M. G.; Schmidt, E. W. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 20655− 20660. (6) Sakai, R.; Yoshida, K.; Kimura, A.; Koike, K.; Jimbo, M.; Koike, K.; Kobiyama, A.; Kamiya, H. ChemBioChem 2008, 9, 543−551. (7) Wilson, M. C.; Mori, T.; Ruckert, C.; Uria, A. R.; Helf, M. J.; Takada, K.; Gernert, C.; Steffens, U. A.; Heycke, N.; Schmitt, S.; Rinke, C.; Helfrich, E. J.; Brachmann, A. O.; Gurgui, C.; Wakimoto, T.; Kracht, M.; Crüsemann, M.; Hentschel, U.; Abe, I.; Matsunaga, S.; Kalinowski, J.; Takeyama, H.; Piel, J. Nature 2014, 506, 58−62. (8) Additional material was isolated from a fraction obtained in a larger scale extraction of TSY conducted in 1999. (9) Due to the peak broadening during preparative HPLC, the NMR spectrum was associated with inseparable impurities. Therefore, the purity of the compound was estimated to be ca. 90%. (10) There were two pairs of amide NH2 protons, which suggested Asn and OHAsn residues rather than Asp and OHAsp, satisfying the HRMS data. (11) Matsunaga, S.; Fusetani, N. J. Org. Chem. 1995, 60, 1177−1181. (12) Marfey, P. Carlsberg Res. Commun. 1984, 49, 591−596. D



Note

(13) Bhushan, R.; Kumar, R. Anal. Bioanal. Chem. 2009, 394, 1697− 1705. (14) The configurations of the histidinoalanine residue (L-histidinoD-alanine) are conserved among the known theonellamides. The alanine portion of this residue partially racemizes during acidic hydrolysis for 16 h to give L-histidino-L-alanine. In the Marfey’s analysis of the constituent amino acids, equivalents for D-histidino-Lalanine and D-histidino-D-alanine were prepared by derivatization of the standard 3:1 mixture of L-histidino-D-alanine and L-histidino-Lalanine with D-FDNP-Val instead of the conventional L-FDNPVal.12,13 Although the retention times of the DL- and LD-isomers are close, the liberation of the LL-isomer in the 16 h hydrolysate confirmed the presence of L-histidino-D-alanine in theonellamide I (1). (15) The relative configurations of OHAsp and Apoa residues were determined by comparing the coupling constants and chemical shifts with those reported for theonellamide A.11 (16) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899−901. (17) Bewley, C. A.; Faulkner, D. J. J. Org. Chem. 1994, 59, 4849− 4852. (18) Kato, Y.; Fusetani, N.; Matsunaga, S.; Hashimoto, K.; Sakai, R.; Higa, T.; Kashman, Y. Tetrahedron Lett. 1987, 28, 6225−6228. (19) Ueoka, R.; Uria, A. R.; Reiter, S.; Mori, T.; Karbaum, P.; Peters, E. E.; Helfrich, E. J. N.; Morinaka, B. I.; Gugger, M.; Takeyama, H.; Matsunaga, S.; Piel, J. Nat. Chem. Biol. 2015, 11, 705−712. (20) Mori, T.; Cahn, J. K. B.; Wilson, M. C.; Meoded, R. A.; Wiebach, V.; Martinez, A. F. C.; Helfrich, E. J. N.; Albersmeier, A.; Wibberg, D.; Dätwyler, S.; Keren, R.; Lavy, A.; Rückert, C.; Ilan, M.; Kalinowski, J.; Matsunaga, S.; Takeyama, H.; Piel, J. Proc. Natl. Acad. Sci. U. S. A. 2018, 115, 1718−1723.

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Colony-wise Analysis of a Theonella swinhoei Marine Sponge with a

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