Halogenated Meroditerpenoids from a South Pacific Collection of the

Article Cite This: J. Nat. Prod. 2018, 81, 2446−2454

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Halogenated Meroditerpenoids from a South Pacific Collection of the Red Alga Callophycus serratus Victoria H. Woolner,†,§ Rose M. A. Gordon,‡,§ John H. Miller,‡,§ Matthias Lein,† Peter T. Northcote,*,†,§,⊥ and Robert A. Keyzers*,†,§ †

School of Chemical and Physical Sciences, ‡School of Biological Sciences, and §The Center for Biodiscovery, Victoria University of Wellington, P.O. Box 600, Wellington 6012, New Zealand

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S Supporting Information *

ABSTRACT: A detailed examination of the red alga Callophycus serratus collected in Tonga led to the isolation of six new halogenated meroditerpenoids: callophycol C (1), callophycoic acid I (2), iodocallophycols E (3) and F (4), iodocallophycoic acid B (5), and callophycoic acid J (6). Of these, compounds 3−5 are new iodinated additions to the growing family of Callophycus meroditerpenoids. The relative configurations of compounds 1−6 were deduced by analyses of 1D NOE data and 1H−1H scalar coupling constants, and 3−6 are proposed to differ from the closely related compounds reported in the literature, iodocallophycoic acid A and iodocallophycols A−D. Iodocallophycol E (3) exhibited moderate cytotoxicity against the promyelocytic leukemia cell line HL-60 with an IC50 value of 6.0 μM.

R

their structure, bringing the total number of iodinated Callophycus metabolites reported to eight.

ed algae of the genus Callophycus have been found by the Kubanek group to be prolific producers of structurally diverse meroditerpenoids.1−8 The majority of those reported are halogenated, predominantly by bromine, but recently, a report featuring the first Callophycus-derived di-, tri-, and tetraiodinated meroditerpenoids was published.8 The ostensibly low number of iodine-containing marine natural products present in the literature has led to the perception that iodinated compounds are rarer than those that contain bromine and chlorine.9−12 However, when the relative halide concentrations in seawater are considered (0.5, 0.8 × 10−3, 0.4 × 10−6 mol L−1,13 giving an approximate ratio of 1 million:2000:1 for chloride, bromide, and iodide ions, respectively), the ratio of iodinated to brominated and chlorinated metabolites reported is significantly higher on a relative scale.8 Biohalogenation processes that incorporate halides into organic substrates and the higher reactivity of the iodide ion due to its enhanced oxidation potential are believed to be responsible.8 Specimens of C. serratus were collected from ′Eua, Tonga, and examined in this study. Purification was guided using HRESIMS in addition to 1H NMR spectroscopy, which resulted in the isolation of six new compounds, callophycol C (1), callophycoic acid I (2), iodocallophycols E (3) and F (4), iodocallophycoic acid B (5) and callophycoic acid J (6),14 in addition to the known macrolides bromophycolides A (7) and T (8). As the name implies, iodocallophycols E (3) and F (4) and iodocallophycoic acid B (5) incorporate iodine within © 2018 American Chemical Society and American Society of Pharmacognosy

■

RESULTS AND DISCUSSION Methanolic extracts of C. serratus (181.5 g, wet weight) were semipurified using polystyrene(divinylbenzene) (PSDVB), and the resulting fractions analyzed by HMBC NMR spectroscopy. In a search for structurally interesting, potentially new natural products featuring unusual functional/structural elements, our attention was drawn to a series of aromatic proton resonances and a pair of deshielded methyl singlets that were found through HMBC correlations to form a substituted dimethylallyl substructure. Further fractionation by silica and diol benchtop column chromatography, followed by purification using HILIC or C18 HPLC, yielded compounds 1−8. Callophycol C (1) was isolated as a white, amorphous solid. Negative-ion-mode HRESIMS data provided a deprotonated molecule at m/z 632.9787, consistent with the parent molecular formula C26H34Br3ClO, which required eight hydrogen deficiencies. The isotopic distribution pattern confirmed the presence of bromine and chlorine. The 13C NMR spectrum revealed 26 carbons, and a multiplicity-edited HSQC experiment accounted for 33 protons connected to 17 carbons. The remaining nine nonprotonated carbon centers comprised seven olefinic/aromatic carbons and two quaternary Received: June 14, 2018 Published: November 8, 2018 2446

DOI: 10.1021/acs.jnatprod.8b00487 J. Nat. Prod. 2018, 81, 2446−2454

Journal of Natural Products

Article

A substituted dimethyl allyl moiety was established based on the HMBC correlations from H3-18 and H3-26 to the other’s carbon and shared correlations to C-16 and C-17. Due to the near identical chemical shifts of the olefinic carbons, a bandselective HMBC (bandwidth δC 105−155) confirmed that the methyls were indeed correlating to two separate olefinic carbons (Supporting Information). COSY correlations observed between H2-14 and H2-15, in addition to HMBC correlations from H2-15 to C-16 and C-17, provided the connection between the gem-dimethyl and decalin ring motifs (Table 1). The bromines were assigned to C-2, C-4, and C-12 based on comparable carbon chemical shifts to callophycols A and B,7 which subsequently left chlorine to be positioned on the nonprotonated olefinic C-16 carbon to provide the planar structure of 1. In a 1D-NOE experiment, correlations were observed from H3-24 to H2-7, H-10a, H-11a, H-22b, and H3-25 and from H325 to H-11a, H-22b, and H3-24, implying these to be on the same face of the bicyclic ring system, while correlations observed from H-12 to H-10b, H-11b, H-15a, and H-23 indicated these resonances to be on the opposite face (Figure 1). These observations implied a trans-fused decalin system as is found in callophycols A and B.7 The relative configuration of 1 is therefore provided as 8R*,9S*,12R*,13R*,23S*. Callophycoic acid I (2) was isolated as a white, amorphous solid and produced a deprotonated molecule at m/z 501.1650, suitable for the molecular formula C27H35BrO4. Sequential COSY and HMBC correlations led to the elucidation of a monosubstituted p-hydroxybenzoic acid and a decalin ring system analogous to that in callophycol C (1). Compared with 1, the end of the isoprenoid chain presented a point of difference. In 2, the deshielded methyl H3-27 protons correlated to ketone carbonyl C-14, the nonprotonated olefin C-15, and methylidene C-16. From H2-16, HMBC correlations were observed to C-14, C-15, and C-27. These observations established the attachment of CH2-16 and CH3-27 to C-15 with the ketone carbonyl carbon adjacent to C-15 forming an α,β-unsaturated ketone. Chemical shift (Table 1) and NOE correlation data for 2 were analogous to those reported for callophycoic acids G and H,7 suggesting the relative configuration to be 6R*,7S*,10R*,13R*,23S*. Iodocallophycol E (3) was isolated as a white, amorphous solid. A molecular formula of C26H34Br3IO was indicated from the observation of an HRESIMS deprotonated molecule at m/ z 724.9131, for which eight double-bond equivalents are required. A characteristic 1:3:3:1 isotopic “quartet” supported the presence of three bromine atoms. The increased mass of the parent ion and, although extremely weak, the observation

carbons. The absence of one proton resonance in the HSQC experiment indicated this to be exchangeable. A 2,4,6-trisubstituted phenol, like that of previously reported related compounds,7 was established from the diagnostic 1 H−1H scalar coupling constants and HMBC correlations from the aromatic protons (H-315 and H-5) and those of the side chain (H2-7 and H-8) (Figure 1) to carbons within the aromatic ring. An HMBC correlation from H2-20 to C-19 established an exomethylene motif, and additional correlations to C-8 and C-21 established a connection to the aromatic substructure through the C-8/C-19 bond. Allylic COSY correlations observed between H2-20 and both H-8 and H221, and between H2-21, H2-22, and H-23, supported by HMBC correlations, led to further extensions of this segment. A cyclohexane ring was established through HMBC correlations observed from H3-24 to C-8, C-9, C-10, and C23. Successive COSY correlations between H2-10, H2-11, and H-12, in addition to HMBC correlations from quaternary methyl H3-25 to C-12, C-13, C-14, and C-23, gave rise to a decalin ring system.

Figure 1. Key COSY, HMBC, and NOE correlations observed for callophycol C (1) (600 MHz, CDCl3). 2447


Journal of Natural Products Table 1.

13

Article

C (150 MHz) and 1H (600 MHz) NMR Data for Compounds 1 and 2 in CDCl3 1

pos.

δC, type (1JCH in Hz)

1 2 3 4 5

149.5, 110.9, 131.2, 112.3, 132.2,

6 7 8

131.5, C 24.6, CH2 (128) 55.7, CH (126)

9

40.0, C

10

40.3, 126) 31.3, 129) 63.8, 42.0,

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1-OH

C C CH (172) C CH (165)

CH2 (129, CH2 (130, CH (148) C

37.8, CH2 (128) 29.3, CH2 (128, 126) 127.6, C

2

δH, mult. (J in Hz)

HMBC (1H to 13C)

δC,b type

7.39, d (2.3)

1,2,4,5

7.12, d (2.3)

1,2,3,4,7

169.5, C 121.4, C 132.3, CH 128.1, C 23.4, CH2

2.75, d (6.8) 2.16, m

1,2,5,6,8,9,19 6,7,9,13,19,20,21,23,24

55.8, CH 40.0, C 40.1, CH2 31.4, CH2

1.88, 1.36, 2.26, 2.19, 4.24,

dt (13.4, 3.5) td (13.3, 3.8) qd (13.1, 3.4) q (3.7) dd (12.5, 4.2)

9,11,12,23,24 8,9,11,12,22,23,24 9,12,13 9,12 10,11,13, 14,25

1.69, dd (9.3, 7.8) 2.45, dt (13.4, 8.6) 2.12, dt (13.5, 8.1)

12,13,15,16, 23,25 14,16 16,17

63.5, CH

1.81, s

16,17,26

4.81, 4.61, 2.39, 2.00, 1.81, 1.48, 1.45,

8,19,21 8,19,21 8,19,20, 22,23 19,20,22,23 19,21,23 23 8,9,12,13,21, 22,24,25

15.3, CH3 (126) 19.6, CH3 (128) 20.3, CH3 (127)

0.87, s 0.97, s 1.78, s

8,9,10,23 12,13,14,23 16,17,18

5.75, br s

1,2,6

br s br s ddd (13.2, 4.8, 3.3) m m qd (12.3, 4.2) d (11.2)

7.83, d (1.9) 2.81, dd (15.4, 10.2) 2.71, dd (15.4, 2.0) 2.24, m 1.92, 1.39, 2.26, 2.20, 4.20,

dt (13.4,3.5) td (13.4, 3.5) m m dd (12.6, 4.3)

41.8, C 34.6, CH2 30.8, CH2

1.76, m 2.80, dd (12.3, 4.8) 2.37, dd (12.2, 4.9)

201.9, C 144.2, C 125.2, CH2

127.4, C 22.0, CH3 (127) 146.6, C 108.8, CH2 (157, 155) 37.9, CH2 (129, 126) 25.2, CH2 (128, 127) 50.2, CH (121)

δH, mult. (J in Hz)

129.7, CH 115.1, CH 158.3, C −c 108.6, CH2 37.7, CH2 25.0, CH2 50.2, 15.2, 19.6, 17.7,

CH CH3 CH3 CH3

6.08, 5.83, 7.80, 6.76,

br s d (1.3) dd (8.3, 2.0) d (8.3)

4.83, 4.71, 2.36, 1.98, 1.70, 1.46, 1.44, 0.90, 1.00, 1.88, 5.45,

s s m m m m m s s s br s

a

Interchangeable. bData obtained from HSQC/HMBC spectra. cNot observed.

of an in-source fragment ion at m/z 126.9052 both signified the presence of iodine. All 26 carbon resonances appeared in the 13C NMR spectrum, 19 of which were protonated for a total of 33 out of the 34 protons. The seven nonprotonated carbons were attributed to five aromatic/olefinic centers and two quaternary carbons. The sole exchangeable proton was observed as a broad singlet in the 1H NMR spectrum. An analysis of the 1D and 2D NMR data for CH-2′, CH-5′, and CH-6′15 established an ortho-, para-substituted phenol ring like that observed in 2. In addition to COSY correlations between the aromatic protons, successive correlations were observed between H-2′, H2-1 (long-range), and H-2. HMBC correlations from H2-18 established a connection to C-10, C11, and C-12, supported by allylic COSY couplings of H2-18 with H-10 and H2-12. Further extensions to this substructure were achieved following further analyses that revealed successive COSY correlations between H-10, H2-9 and H2-8,

and H2-12, H2-13, and H-14, all of which were supported by HMBC data. Methyl singlets H3-19 and H3-20 displayed reciprocal and shared HMBC correlations to C-10, C-14, and C-15, establishing a gem-dimethyl moiety and a connection between C-10 and C-14 through C-15, giving rise to a cyclohexane ring. HMBC correlations from H3-17 to methines C-2 and C-6, quaternary carbon C-7, and methylene C-8 established the connectivity between C-2 and C-8 via C-7. Sequential COSY correlations between H-6, H2-5, and H2-4 established the connectivity between their respective carbons. Nonprotonated olefinic carbon C-3 was positioned between C2 and C-4 based on HMBC correlations observed from both H2-1 and H2-5 to C-3, thus establishing a second cyclohexane motif. While the chemical shift of C-3 (δC 146.7) was clearly indicative of a double bond, it had no obvious olefinic partner (i.e., >δH 4.0 and >δC 100). The only candidate yet to be assigned was CH-16 (δH 5.75; δC 75.7), whose proton 2448



Article

Table 2. 1H (600 MHz) NMR Data for Compounds 3−6

chemical shift was typical for an alkenyl proton, but the carbon chemical shift was not.16 However, its 192 Hz 1JCH value was consistent with an sp2-hybridized carbon,16 suggesting a terminal vinyl halide. Specifically, this combination of proton and carbon chemical shifts was consistent with those for the vinyl iodides of closely related meroditerpenoids.8 HMBC correlations from H-16 to C-2, C-3, and C-4, in addition to long-range allylic COSY coupling between H-16 and H-2 supported the connectivity between C-3/C-16. With all protons and carbons assigned within the structure, the placement of the halogens was determined based on chemical shift arguments. The iodine was already accounted for,8 while CH-1′, CH-6, and CH-14 were substituted by bromine due to their comparable proton and carbon chemical shifts to 1 (Tables 2 and 3). 1D NOE data coupled with 1H−1H scalar coupling constants were used to establish the relative configuration of 3. The relative configuration within each cyclohexane ring system and also between the ring systems both required consideration. The E-double bond configuration of Δ3,16 was established through correlations observed between H-16 and both H-1a and H-2. NOE correlations between H-2, H-6, and H2-9 indicated these protons to be on the same face of the cyclohexane ring. H-5b and H2-1 each displayed an NOE correlation to H3-17, suggesting these to be on the opposite face of the ring relative to H-2, H-6, and H2-9. On the second cyclohexane, NOE correlations from H-14 to H-10, H-12b, H13a, and H3-19 indicated these protons to be on the same face, while an NOE correlation from H3-20 to H-13b suggested these to be on the opposite face. These observations were found to be consistent with other reported iodinated meroditerpenoids,8 suggesting that the same relative configuration exists within each cyclohexane system of 3. This places the large, bulky groups, such as the aromatic ring, bromines, and the ethylene bridge (linking to the other cyclohexane ring), at equatorial positions. H-6 and H-14 both appear as a doublet of doublets with large (axial−axial) and small (axial− equatorial) coupling constants, providing evidence of both bromines occupying equatorial positions. With the relative configuration within each cyclohexane system established, the relative configuration between the cyclohexane systems was determined through an analysis of the 3 JHH coupling constants in relation to the Karplus curve and NOE correlation data. The defined multiplicities of H-8a (td, 12.9, 4.8 Hz), H-8b (ddd, 12.9, 12.1, 4.5 Hz), and H-10 (d, 10.7 Hz) suggested the molecule to be conformationally restricted and predominantly maintaining one conformation. The combination of large and small 3JHH coupling constants observed for each proton resonance of H2-8 suggested an antiperiplanar conformation across C-8/C-9 with approximate dihedral angles of 180° and 60° between the vicinally-coupled protons. The apparent “doublet” multiplicity of H-10 indicated ∼0 Hz coupling to one proton of H2-9 and a corresponding dihedral angle near 90°. This further signified a dihedral angle close to 150° with the other proton of H2-9, which was supported by the large coupling constant of 10.7 Hz. When visualized along the C-9/C-10 bond, four conformations of the possible two configurations at C-10 could account for these observations, and further analysis of the NOE data was required to narrow the possibilities to one (Figure 2). NOE correlations were observed from H-2 and H3-19 to H-9b and from H3-19 to H-2′, indicating that the gem-dimethyls were oriented toward the aromatic ring. An NOE correlation from

δH, mult. (J in Hz) 3a

4a

1a

3.03, dd (14.8, 11.8)

3.00, dd (14.8, 11.2)

3.02, m

1b

2.75, dd (14.8, 2.7) 2.64, dd (11.8, 2.3)

2.74, dd (14.8, 3.2) 2.66, dd (10.9, 2.7)

2.77, m

2.81, ddd (12.6, 4.8, 3.5) 2.18, dd (13.5, 4.1) 2.24, m

2.80, m

2.80, dt (12.7, 4.2) 2.08, td (12.7, 4.6) 2.25, ddd (12.7, 8.4, 4.3) 2.00, qd (12.1, 4.0) 4.49, dd (11.0, 4.5)

pos.

2 3 4a 4b 5a 5b 6 7 8a

2.12, ddd (12.8, 8.6, 4.2) 4.41, dd (12.8, 4.1)

9a

1.83, td (12.9, 4.8) 1.20, ddd (12.9, 12.1, 4.5) 1.63, m

9b

1.56, m

10

1.64, d (10.7)

2.11, m 2.22, ddd (11.8, 8.4, 4.4) 2.08, m 4.37, dd (9.5, 4.3)

5b

3.01, m

6a 2.98, dd (14.9, 10.2) 2.81, dd (14.9, 2.1) 2.67, d (10.2) 2.35,c d (7.8) -d 2.26, m 2.09, m 4.34, dd (6.6, 4.4)

1.60, td (12.9, 4.1) 1.21, td (13.1, 3.7) 1.40, tt (13.1, 3.3) 1.30, qd (12.2, 4.6) 1.70, dd (11.2, 3.2)

1.67, td (11.2, 3.7) 1.28, m

1.70, m

1.56, tt (12.6, 3.0) 1.35, qd (12.2, 4.3) 1.68, d (10.4)

1.40, m

2.80, m

12b

2.34, dd (8.6, 3.5) 2.02, m

2.87, d (19.5) 2.57, d (19.5)

13a

2.24, m

5.50, dt (9.9, 3.4)

2.86, dquin (19.8, 2.2) 2.54, ddd (19.8, 3.2, 1.6) 5.5, dt (9.8, 3.3)

13b 14

2.02, m 4.09, dd (10.4, 4.4)

5.34, d (9.9)

5.34, d (9.8, 3.3)

5.75, 1.06, 4.94, 4.78, 1.13, 0.89,

5.85, 1.00, 4.86, 4.69, 0.96, 0.95,

6.00, 0.94, 4.83, 4.70, 0.93, 0.95,

8b

11 12a

15 16 17 18a 18b 19 20 1′ 2′ 3′ 4′ 4′-OH 5′ 6′

br s s br s br s s s

2.56, d (19.7)

br s s br s br s s s

s s s s s s

1.29, m

1.30, m 1.67, m

5.50, dt (9.9, 3.4) 5.32, d (9.9)

4.87, 4.73, 0.97, 4.85, 4.71, 0.91, 0.87,

s s s s s s s

7.10, d (2.5)

7.12, d (2.3)

7.76, s

7.88, s

4.87, br s 6.59, d (8.5) 7.14, dd (8.5, 2.5)

6.60, d (8.4) 7.14, dd (8.4, 2.5)

6.78, d (8.3) 7.68, d (8.3)

6.78, d (8.3) 7.82, d (8.3)

a Acquired in CDCl3. bAcquired in CD3OD. cTentatively assigned from the COSY experiment. dNot observed.

H-8a to H-18b placed these two protons in close proximity to each other and indicated the methylidene moiety to be oriented on the opposite side of the ethylene bridge relative to the aromatic ring; the NOE correlation between H-1b and H8b supported this assignment. H-8b also showed an NOE correlation to H-10 (Figure 3). These observations eliminated 2449


Journal of Natural Products Table 3.

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Article

C (150 MHz) NMR Data for Compounds 3−6 δC, type (1JCH in Hz) a

pos. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 7′

a

3 27.0, 51.8, 146.7, 35.0, 33.4, 61.6, 44.3, 38.5, 19.6, 53.6, 145.3, 37.0, 36.0, 67.0, 41.9, 75.7, 19.7, 109.8, 28.7, 17.5, 113.1, 132.8, 129.5, 152.3, 117.0, 130.0,

CH2 CH C CH2 CH2 CH (150) C CH2 CH2 CH (129) C CH2 CH2 CH (150) C CH (192) CH3 (126) CH2 (156, 156) CH3 (126) CH3 (126) C CH (165) C C CH (158) CH (167)

5b

4 26.9, 51.6, 147.1, 36.1, 33.6, 62.0, 44.3, 37.5, 19.4, 53.3, 145.6, 31.8, 123.5, 136.5, 37.2, 75.4, 19.3, 110.3, 30.8, 25.9, 113.2, 133.0, 129.7, 152.3, 117.1, 130.0,

CH2 (124, 124) CH (124) C CH2 CH2 (135, 135) CH (150) C CH2 (125, 125) CH2 (125, 125) CH (127) C CH2 (124, 124) CH (165) CH (161) C CH (192) CH3 (126) CH2 (156, 156) CH3 (126) CH3 (126) C CH (162) C C CH (158) CH (167)

27.5, CH2 51.1, CH 148.8, C 38.4,c CH2 35.0, CH2 63.4, CH 45.5, C 38.4,c CH2 20.3, CH2 54.6, CH 147.1, C 32.7, CH2 124.3, CH 137.6, CH 38.0, C 75.3, CH (192)d 18.4, CH3 110.4, CH2 31.2, CH3 26.3, CH3 127.8,e C 132.9, CH 127.8,e C 160.1, C 115.5, CH 130.3, CH 171.8 C

6a 26.5, 47.4, 146.2, −f 35.4, 63.0, 44.3, 37.3, 19.1, 53.3, 145.9, 31.8, 123.6, 136.5, 37.1, 110.6, 17.8, 110.1, 30.8, 25.8, 121.8, 132.7, 128.0, 158.4, 115.4, 130.0, 170.1,

CH2 CH C CH2 CH C CH2 CH2 CH C CH2 CH CH C CH2 CH3 CH2 CH3 CH3 C CH C C CH CH C

a f

Acquired in CDCl3. bAcquired in CD3OD. cInterchangeable. dMeasured from the unsuppressed 1JCH in the HMBC experiment. eInterchangeable. Not observed.

Figure 2. Newman projections along C-7/C-8 and C-9/C-10 for the possible configurations and conformations of iodocallophycol E (3). Options B, C, F, and G were discounted as possibilities based on predicted H-10 coupling constants.

and 9.5 Hz coupling constant, while for H-10/H-9b, 78.4° and 0.1 Hz were determined (Supporting Information). These results suggest that the relative configuration linking the two cyclohexane rings of 3 is different from those of the iodinated meroditerpenoids reported in the literature,8 with the relative configuration of 3 tentatively assigned as shown. Based on the established 2S, 6S, 7S configurations for several related compounds, 1 −8 a relative configuration of 2S*,6S*,7S*,10S*,14R* is proposed for compound 3, with C-2, C-6, and C-7 reflecting those reported in the literature

options A, D, and E illustrated in Figure 2, indicating that option H depicts the configuration at C-10 and preferred conformation across the C-9/C-10 bond. DFT computational data revealed similarities between conformers A and H with their relative energies being the same within the margin of error for the calculation, however a 13C NMR-based DP4 analysis17 of these four conformers assigned H with an 88.7% probability and A, D, and E, each with less than 6%. Calculated dihedral angles and coupling constants support the experimental data, with H-10/H-9a giving a −166.1° dihedral angle 2450



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correlations between H-6′ and both H-2′ and H-5′ and HMBC correlations from H-2′ to C-1, C-4′, and C-6′, from H5′ to C-1′, C-3′, and C-4′, and from H-6′ to C-2 and C-4′. An HMBC correlation to the carboxyl carbon was lacking, and the carbon resonance was absent from the 13C NMR spectrum acquired using standard acquisition parameters. Initially we thought that the large longitudinal relaxation (T1) of carbonyl carbons may have been the problem and reran the 13C spectrum with a longer relaxation delay (Figure S31). To our surprise this seemed to exacerbate the situation, weakening some of the other carbon signals. This result suggested rapid signal loss due to short relaxation times. We obtained another 13 C NMR spectrum of 5 with a shortened relaxation delay and acquisition time and increased scans. With these acquisition parameters, signals for the carboxyl carbon C-7′ along with C2′ and C-6′ (very weak in the 13C NMR spectrum acquired from standard parameters) were observed, albeit with somewhat broadened peaks. NOE correlations were analogous to those observed for 3 and 4, leading to the assignment of an Edouble bond configuration of the vinyl iodide and a 2S*,6S*,7S*,10S* relative configuration for iodocallophycoic acid B (5). A molecular formula of C27H35BrO3 was determined for callophycoic acid J (6) from a deprotonated molecule observed at m/z 485.1684 in negative-ion-mode HRESIMS. The molecular formula required 10 double-bond equivalents and differed from 5 by the replacement of iodine by additional hydrogen. All COSY and HMBC correlations determined the carbon framework to be analogous to 5, and a complete methylidene motif was found within 6 at the position where the vinyl iodide was observed in 5. The carboxyl carbon resonance was absent from the 13C NMR spectrum but was observed by altering the acquisition parameters in the same fashion as that used for 5 (Figure S31). Based on NOE correlation data, the relative configuration of 6 is proposed to be analogous to compounds 3−5. Iodocallophycols E (3) and F (4) and bromophycolides A (7) and T (8) were tested against the promyelocytic leukemia cell line HL-60, which showed moderate cytotoxicity for compounds 3, 7, and 8 with IC50 values of 5.1, 6.2, and 6.0 μM, respectively, while 4 was inactive. This investigation of C. serratus yielded six new meroditerpenoids, bringing the total number of Callophycus isolates to 52. The purification process was guided by a combination of MS and NMR spectroscopy and was quite successful from a dereplication perspective when considering the significant number of previously reported C. serratus metabolites. Of those that are halogenated (48), each contains bromine, close to 17% contain iodine, and only 4% contain chlorine. This is consistent with the disproportionately high number of reported iodinated marine natural products relative to the concentration of the halides in seawater, which consequently arises because of the high oxidation potential of iodide relative to bromide and chloride. Perhaps the number of iodinated marine natural products is considerably greater than the literature accounts for, but they are inadvertently overlooked because of the ease by which brominated and chlorinated molecules can be detected and identified from mass spectrometric data. The fact that both chloro- and bromo-peroxidase enzymes are known to catalyze the oxidation of iodide ions19 suggests that the number could be greater. The organism under study was that of a single species, namely, C. serratus, and interestingly, this study is the first to

Figure 3. 3D representation of iodocallophycol E (3) showing key NOE correlations establishing the relative configurations between the cyclohexane ring systems.

and C-1018 and C-14 being the opposite configuration.8 Establishing the relative configuration between chiral entities connected by short, rotatable methylene chains is challenging. The difference in relative configuration between the two ring sections of the new callophycol derivatives when compared to those previously reported may reflect a slight difference in biosynthetic machinery catalyzing the cyclization process. Iodocallophycol F (4) was isolated as a white, amorphous solid. Negative-ion-mode HRESIMS data provided a molecular formula of C26H33Br2IO, indicating a difference of one additional double-bond equivalent and the loss of one hydrogen and bromine from iodocallophycol E (3). A detailed analysis of the NMR data revealed close similarities to 3 and established the p-bromophenol and bromo(iodomethylene)cyclohexane moieties to be conserved in 4, and the difference was found in the presence of a disubstituted olefin and the loss of HBr. The gem-dimethyls H3-19 and H3-20 displayed reciprocal and shared HMBC correlations to methine C-10, quaternary C-15, and olefinic carbon C-14, establishing C-15 as the attachment point. Sequential COSY correlations were observed between H-14, H-13, and H2-12. HMBC correlations were observed from methylidene protons H2-18 to C-10, the nonprotonated olefinic carbons C-11 and C-12 establishing a methylene−cyclohexene motif. As with 3, an ethylene bridge was found to connect the two six-membered ring systems. NOE data were comparable to those found in 3, leading to an identical E-configuration of the vinyl iodide and the tentative relative configuration of 2S*,6S*,7S*,10S* for 4. Iodocallophycoic acid B (5), isolated as a white, amorphous solid, dissolved readily in CDCl3; however, broad signals were observed in the NMR spectra. When the sample was dissolved in CD3OD, all the peaks resolved to provide a spectrum like that of previously isolated callophycols. Negative-ion-mode HRESIMS produced a deprotonated molecule at m/z 611.0668 consistent with the parent molecular formula C27H34BrIO3, for which 10 double-bond equivalents were requiredone more than 4from the addition of a carboxyl group. An analysis of the NMR data of 5 established the presence of the methylene−cyclohexene moiety connected to the bromo(iodomethylene)cyclohexane via the ethylene bridge, analogous to the structure of 4. The structural change from 4 was found in the aromatic ring where a carboxylic acid replaced the bromine at C-1′, as evidenced by COSY 2451



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A second batch of the alga (79.0 g) was extracted in MeOH (400 mL), cyclic loaded onto PSDVB, and eluted with (1) 30% Me2CO/ H2O, (2) 75% Me2CO/H2O, and (3) 100% Me2CO (D1−D3) in a similar fashion to that described above. The 100% Me2CO fraction (D3) was further purified on a DIOL (15 mL) column from 45 mL volumes of n-hexane, 5%, 10%, 20%, and 50% CH2Cl2/n-hexane, CH2Cl2, EtOAc, and MeOH to generate 47 fractions, which were combined, following a TLC analysis (mobile phase: 80% CH2Cl2/nhexane, fractions 1−30; 5% MeOH/CH2Cl2, fractions 28−47), into 14 samples (E1−E14). Samples E5−E7 were recombined due to their similar 1H NMR spectra, and the resulting sample was purified by HPLC (C18) using 80% MeCN/H2O to afford iodocallophycoic acid B (5) (tR = 16.6 min). E9 was fractionated using C18 HPLC, eluting isocratically with 90% MeCN/H2O, which afforded pure bromophycolide T (8) (0.1 mg, tR = 4.8 min) and additional mass of 3 (tR = 22.5 min) and 4 (tR = 21.6 min). All samples identified as 3 were combined (1.3 mg), as were those of 4 (1.8 mg). The 75% Me2CO/ H2O fraction (D2) was further fractionated by flash column chromatography on DIOL, eluting with n-hexane, 30%, 50%, and 70% CH2Cl2/n-hexane, CH2Cl2, 10%, 20%, and 50% EtOAc/CH2Cl2, EtOAc, and MeOH, which resulted in 50 fractions that were combined to give 11 samples (F1−F11). Additional purification of F5 was performed on C18 HPLC, eluting at 80% MeCN/H2O isocratically for 12.5 min, then ramping to 100% MeCN between 12.5 and 13.0 min ,and eluting for 12 min, providing fractions G1− G3. Bromophycolide A (7) was purified from fraction G1 using C18 HPLC eluting with 75% MeCN/H2O (1.2 mg, tR = 14.5 min). Fractions B2 (first batch extraction) and G7 were subjected to HPLC C18, eluting with 80% MeCN/H2O, to afford iodocallophycoic acid B (5) (tR = 16.6 min), callophycoic acid J (6) (tR = 11.6 min), and callophycoic acid I (0.6 mg) (2) (tR = 7.0 min). Fractions that contained 5 were combined (0.8 mg), as were those that contained 6 (1.0 mg). Callophycol C (1): white, amorphous solid; [α]20 D +60 (c 0.006, MeOH); UV (95% MeCN/H2O) λmax 225, 288 nm; IR νmax 3357, 2925, 1524, 1421, 1258 cm−1; 1H and 13C NMR data, Table 1; HRESI-MSMS (40 eV) m/z (% relative intensity) 638.9696 (0.4), 636.9715 (0.8), 634.9743 (0.7), 632.9812 [M − H]− (0.2), 559.0454 (9.5), 557.0483 (50.8), 555.0503 (76.2), 553.0520 (29.0), 265.8586 (4.6), 263.8604 (10.6), 261.8617 (5.7), 80.9171 (96.3), 78.9193 (100); HRESIMS m/z 632.9787 [M − H]− (calcd for C26H33Br3ClO, 632.9776). Callophycoic Acid I (2): white, amorphous solid; [α]20 D +19 (c 0.06, MeOH); UV (80% MeCN/H2O) λmax 258 nm; IR νmax 3330, 3068, 2925, 1679, 1275 cm−1; 1H and 13C NMR data, Table 1; HRESIMSMS (30 eV) m/z (% relative intensity) 505.1576 (0.8), 503.1650 (3.5), 501.1658 [M − H]− (2.2), 423,2413 (9.7), 422.2405 (37.0), 421.2378 (87.1), 377.2477 (8.4), 80.9173 (100), 78.9194 (85.9); HRESIMS m/z 501.1650 [M − H]− (calcd for C27H34BrO4, 501.1646). Iodocallophycol E (3): white, amorphous solid; [α]20 D −27.1 (c 0.17, CHCl3); UV (90% MeCN/H2O) λmax 225, 288 nm; IR νmax 3378, 2953, 1267 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESI-MSMS (5 eV) m/z (% relative intensity) 730.9107 (30.8), 728.9128 (100), 726.9143 (93.2), 724.9153 [M−H]− (20.4), 126.9054 (93.6), 80.91711 (7.3), 78.9192 (7.9); HRESIMS m/z 724.9131 [M − H]− (calcd for C26H33Br3IO, 724.9132). Iodocallophycol F (4): white, amorphous solid; [α]20 D −49.2 (c 0.12, CHCl3); UV (90% MeCN/H2O) λmax 225, 288 nm; IR νmax 3378, 2956, 1265 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESI-MSMS (5 eV) m/z (% relative intensity) 648.9877 (28.4), 646.9896 (53.3), 644.9900 [M − H]− (17.5), 126.9058 (100), 80.9173 (3.6), 78.9195 (3.4); HRESIMS m/z 644.9880 [M − H]− (calcd for C26H32Br2IO, 644.9870). Iodocallophycoic acid B (5): white, amorphous solid; [α]20 D −28 (c 0.08,CHCl3); UV (80% MeCN/H2O) λmax 258 nm; IR νmax 3320, 3068, 2925, 1683, 1273 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESI-MSMS (5 eV) m/z (% relative intensity) 615.0654 (2.0), 613.0625 (30.3), 611.0630 [M − H]− (17.7), 126.9053 (100),

report the isolation of macrolide meroditerpenoids alongside non-macrolide meroditerpenoids. This suggests that in previous studies environmental, rather than genetic, differences were the probable reason behind finding one compound class exclusive of the other.7

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a Rudolph Research analytical Autopol IV polarimeter. IR spectra were recorded on a Bruker Alpha Platinum ATR FT-IR spectrometer. A 600 MHz Varian Direct Drive spectrometer equipped with a triple-resonance HCN cryogenic probe operating at 25 K was used to record all NMR spectra (600 MHz for 1H nuclei and 150 MHz for 13C). The residual solvent peak was used as an internal reference for 1H (δH 7.26, CDCl3; 3.31, CD3OD) and 13C (δC 77.16, CDCl3; 49.00, CD3OD) chemical shifts.20 Samples were quantified using an internal CH3NO2 standard and acquisition parameters described by West.21 1H, 13C, 1D NOE, COSY, HSQC, HMBC, and band-selective HMBC (compound 1 only) NMR experiments were used to elucidate the structures of compounds isolated in this study. High-resolution (ESI) mass spectrometric data were obtained with an Agilent 6530 Accurate-Mass Q-TOF LC-MS equipped with a 1260 Infinity binary pump. HPLC purifications were carried out using a Rainin Dynamax SD-200 solvent delivery system with 25 mL pump heads (analytical and semipreparative chromatography) and a Varian Prostar 335 diode array detector. Bench-top column chromatography was performed using Pure Science silica gel 60 (40−63 μm), YMC DIOL (2,3-dihydroxypropoxy-propyl-derivatized silica) (12 nm, S-50 μm), or Mitsubishi Diaion HP20 poly(styrene-divinylbenzene). HPLC was carried out using Phenomenex Luna hydrophilic interaction liquid chromatography (HILIC) (analytical: 4.6 × 250 mm, 5 μm) or Phenomenex Prodigy octadecyl-derivatized silica gel (C18) (analytical: 4.6 × 250 mm, 5 μm) at a flow rate of 1 mL/min. All solvents used for column chromatography were of HPLC grade, and H2O was glass distilled. Solvent mixtures are reported as % v/v unless otherwise stated. TLC analyses were carried out using Macherey-Nagel Polygram Sil G/UV254 plates. Developed plates were visualized under UV light (λ = 254 nm) before analyzing by dipping in 5% H2SO4/MeOH followed by 0.1% vanillin/EtOH and heating. Algal Material. Specimens of Callophycus serratus were collected by scuba from the Deep Resort Beach, ′Eua, Tonga, in June 2016 and stored at −20 °C until extraction. The alga was identified by Dr. G. Zuccarello at the School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand, where a voucher specimen (PTN4_34F) is also stored. Extraction and Isolation. The alga (102.5 g wet weight) was extracted in MeOH (400 mL) twice overnight. The second, then first extracts were cyclic loaded onto PSDVB (80 mL) and eluted with (1) 30% Me2CO/H2O, (2) 75% Me2CO/H2O, and (3) 100% Me2CO (fractions A1−A3). Fraction A2 was loaded onto DIOL (15 mL) that was pre-equilibrated in n-hexane overnight and batch-eluted with 45 mL portions of CH2Cl2, 10%, 20%, and 50% EtOAc/CH2Cl2, EtOAc, and MeOH (B1−B6). Fraction B3 was further purified using C18 HPLC, eluting isocratically with 85% MeOH/H2O to afford callophycoic acid J (6) (tR = 12.2 min). Fraction A3 was dry-loaded onto silica gel (20 mL), and flash column chromatography was performed eluting with 60 mL portions of n-hexane, 5%, 10%, 20%, and 50% CH2Cl2/n-hexane, CH2Cl2, 25% and 50% EtOAc/CH2Cl2, and 20% MeOH/EtOAc, yielding 84 fractions that were combined on the basis of TLC (mobile phase: 80% CH2Cl2/n-hexane, fractions 1− 49; 5% MeOH/CH2Cl2, fractions 48−68; 10% MeOH/CH2Cl2, fractions 62−84) to provide 22 fractions (C1−C22). Fraction C3 was found to contain pure callophycol C (1, 5.2 mg). Both C12 and C13 were separately subjected to further purification on a HILIC HPLC column, eluting with 10% EtOAc/n-hexane. This resulted in the purification of iodocallophycol F (4) from fraction C12 (tR = 24.4 min) and both iodocallophycols E (3) and F (4) (tR = 16.6 min) from fraction C13. 2452



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80.9173 (13.2), 78.9172 (14.3); HRESIMS m/z 611.0668 [M − H]− (calcd for C27H33BrIO3, 611.0663). Callophycoic acid J (6): white, amorphous solid; [α]20 D +46 (c 0.1, CHCl3); UV (80% MeCN/H2O) λmax 258 nm; IR νmax 3291, 3068, 2952, 1683, 1274 cm−1; 1H and 13C NMR data, Tables 2 and 3; HRESI-MSMS (30 eV) m/z (% relative intensity) 487.1723 (1.4), 485.1722 [M − H]− (1.0), 405.2445 (49.9), 150.0356 (24.8), 80.9171 (100), 78.9190 (78.9); HRESIMS m/z 485.1684 [M − H]− (calcd for C27H34BrO3, 485.1697). Computational Details. Computations used the hybrid functional of Perdew, Burke, and Ernzerhof (PBE0)22−26 in conjunction with Grimme’s empirical dispersion correction (D3)27,28 for structural optimizations. Alrichs’ triple-ζ basis set29 (def2-TZVP) with a very fine grid for the numerical integration and very tight SCF convergence criteria in order to achieve greater numerical accuracy was used throughout. All examined structures were identified as minima on the potential energy hypersurface by the absence of imaginary frequencies. Single-point NMR calculations were carried out with the previously obtained structures with the same level of theory (PBE0-D3) and basis set (def2-TZVP). In order to improve the accuracy of the calculated chemical shifts and coupling constants, the basis set for all carbon and hydrogen atoms was decontracted. All calculations were performed using the respective quantum chemical methods as implemented in Gaussian 09 (revision D.01).30 Bioassays. Samples were submitted to the School of Biological Sciences, where they were tested for biological activity. A standard 48 h MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) cell proliferation assay was used to evaluate cytotoxic activity against the HL-60 promyelocytic leukemia cell line (n = 3 independent experiments with triplicate wells per experiment) as previously described.31 Cells were treated with compound at various concentrations, and a dose−response curve was generated relative to a control of untreated HL-60 cells. Peloruside A (HL-60: IC50 10 ± 4 nM) was used as a positive control.

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Fish and Dive Tonga and Mr. M. Page and D. Crossett for assistance with sample collection.

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(1) Teasdale, M. E.; Shearer, T. L.; Engel, S.; Alexander, T. S.; Fairchild, C. R.; Prudhomme, J.; Torres, M.; Le Roch, K.; Aalbersberg, W.; Hay, M. E.; Kubanek, J. J. Org. Chem. 2012, 77, 8000−8006. (2) Stout, E. P.; Prudhomme, J.; Roch, K. L.; Fairchild, C. R.; Franzblau, S. G.; Aalbersberg, W.; Hay, M. E.; Kubanek, J. Bioorg. Med. Chem. Lett. 2010, 20, 5662−5665. (3) Lin, A. S.; Stout, E. P.; Prudhomme, J.; Le Roch, K.; Fairchild, C. R.; Franzblau, S. G.; Aalbersberg, W.; Hay, M. E.; Kubanek, J. J. Nat. Prod. 2010, 73, 275−8. (4) Lane, A. L.; Stout, E. P.; Lin, A.-S.; Prudhomme, J.; Le Roch, K.; Fairchild, C. R.; Franzblau, S. G.; Hay, M. E.; Aalbersberg, W.; Kubanek, J. J. Org. Chem. 2009, 74, 2736−2742. (5) Kubanek, J.; Prusak, A. C.; Snell, T. W.; Giese, R. A.; Fairchild, C. R.; Aalbersberg, W.; Hay, M. E. J. Nat. Prod. 2006, 69, 731−735. (6) Kubanek, J.; Prusak, A. C.; Snell, T. W.; Giese, R. A.; Hardcastle, K. I.; Fairchild, C. R.; Aalbersberg, W.; Raventos-Suarez, C.; Hay, M. E. Org. Lett. 2005, 7, 5261−5264. (7) Lane, A. L.; Stout, E. P.; Hay, M. E.; Prusak, A. C.; Hardcastle, K.; Fairchild, C. R.; Franzblau, S. G.; Le Roch, K.; Prudhomme, J.; Aalbersberg, W.; Kubanek, J. J. Org. Chem. 2007, 72, 7343−7351. (8) Lavoie, S.; Brumley, D.; Alexander, T. S.; Jasmin, C.; Carranza, F. A.; Nelson, K.; Quave, C. L.; Kubanek, J. J. Org. Chem. 2017, 82, 4160−4169. (9) Wang, L.; Zhou, X.; Fredimoses, M.; Liao, S.; Liu, Y. RSC Adv. 2014, 4, 57350−57376. (10) Campagnuolo, C.; Fattorusso, E.; Taglialatela-Scafati, O. Eur. J. Org. Chem. 2003, 2003, 284−287. (11) Sun, J.-F.; Huang, H.; Chai, X.-Y.; Yang, X.-W.; Meng, L.; Huang, C.-G.; Zhou, X.-F.; Yang, B.; Hu, J.; Chen, X.-Q.; Lei, H.; Wang, L.; Liu, Y. Tetrahedron 2011, 67, 1245−1250. (12) Woolner, V. H.; Jones, C. M.; Field, J. J.; Fadzilah, N. H.; Munkacsi, A. B.; Miller, J. H.; Keyzers, R. A.; Northcote, P. T. J. Nat. Prod. 2016, 79, 463−469. (13) Gribble, G. W. Prog. Chem. Org. Nat. Prod. 1996, 68, 1−423. (14) The compound naming of 1−6 is adopted from nomenclature established for closely related compounds. (15) The locant numbering system used for compounds 1−6 follows that used for compounds in the literature comprising the same carbon skeleton. (16) Silverstein, R. M.; Webster, F. X.; Kiemle, D. J. Spectrometric Identification of Organic Compounds, 7th ed.; John Wiley & Sons, Inc.: Hoboken, NJ, 2005. (17) Smith, S. G.; Goodman, J. M. J. Am. Chem. Soc. 2010, 132, 12946−12959. (18) While the nomenclature describing the relative configuration of C-10 in 3−6 is the same as iodocallophycoic acid A and iodocallophycols A−D, the configuration is in fact the opposite as a result of the cyclohexane ring lacking an additional vinyllic iodide. (19) Carter-Franklin, J. N.; Butler, A. J. Am. Chem. Soc. 2004, 126, 15060−15066. (20) Fulmer, G. R.; J, M. A.; Sherden, N. H.; Gottlieb, H. E.; Nudelman, A.; Stoltz, B. M.; Bercaw, J. E.; Goldberg, K. I. Organometallics 2010, 29, 2176−2179. (21) West, L. M. Ph.D. Thesis, Victoria University of Wellington, Wellington, 2001. (22) Perdew, J. P. In Electronic Structures of Solids; Ziesche, P., Eschrig, H., Eds.; Akademie Verlag: Berlin, 1991; p 11. (23) Perdew, J. P.; Chevary, J. A.; Vosko, S. H.; Jackson, K. A.; Pederson, M. R.; Singh, D. J.; Fiolhais, C. Phys. Rev. B: Condens. Matter Mater. Phys. 1992, 46, 6671−6687. (24) Perdew, J. P.; Burke, K.; Ernzerhof, M. Phys. Rev. Lett. 1996, 77, 3865−3868. (25) Perdew, J. P.; Burke, K.; Wang, Y. Phys. Rev. B: Condens. Matter Mater. Phys. 1996, 54, 16533−16539.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00487. 1 H, 13C, COSY, HSQC, and HMBC NMR data of 1−6 and computational Cartesian coordinates (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Ph: +64-4-463-0065. *E-mail: [email protected]. Ph: +64-4-463-5117. ORCID

John H. Miller: 0000-0001-6383-1037 Matthias Lein: 0000-0002-5164-8638 Peter T. Northcote: 0000-0002-2086-9972 Robert A. Keyzers: 0000-0002-7658-7421 Present Address ⊥

Ferrier Research Institute, Victoria University of Wellington, PO Box 33436, Petone, New Zealand.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding for the collection was kindly provided by a VUW University Research Fund grant. Funding from a Victoria Doctoral Scholarship (V.H.W.) is greatly appreciated. We gratefully acknowledge the Tongan Ministry of Fisheries for allowing collection in their territorial waters and Whale Swim, 2453



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(26) Burke, K.; Perdew, J. P.; Wang, Y. Electronic Density Functional Theory: Recent Progress and New Directions; Plenum Press: New York, 1998. (27) Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. J. Chem. Phys. 2010, 132, 154104. (28) Grimme, S.; Ehrlich, S.; Goerigk, L. J. Comput. Chem. 2011, 32, 1456−1465. (29) Weigend, F.; Ahlrichs, R. Phys. Chem. Chem. Phys. 2005, 7, 3297−3305. (30) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09 Revision D.01; Gaussian, Inc.: Wallingford, CT, 2009. (31) Hood, K. A.; West, L. M.; Northcote, P. T.; Berridge, M. V.; Miller, J. H. Apoptosis 2001, 6, 207−219.

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Halogenated Meroditerpenoids from a South Pacific Collection of the

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