Supporting Information

Heterofibrins: inhibitors of lipid droplet formation from a deep-water southern Australian marine sponge, Spongia (Heterofibria) sp.

Angela A. Salim, James Rae, Frank Fontaine, Melissa Conte, Zeinab Khalil, Sally Martin,

Robert G. Parton, Robert J. Capon

Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland, 4072, Australia

Table of Contents

Experimental Details………………………………………………………………...…. 3

Table S1. NMR (600 MHz, CDCl3) data for heterofibrin A1 (1)…………………...... 5

Table S2. NMR (600 MHz, CDCl3) data for heterofibrin A2 (2)…………………...... 6

Table S3. NMR (600 MHz, CDCl3) data for heterofibrin A3 (3)……………………... 7

Table S4. NMR (600 MHz, CDCl3) data for heterofibrin B1 (4)…………………....... 8

Table S5. NMR (600 MHz, CDCl3) data for heterofibrin B2 (5)……………………... 9

Table S6. NMR (600 MHz, CDCl3) data for heterofibrin B3 (6)………………...…… 10

Figure S1. NMR (600 MHz, CDCl3) spectrum for heterofibrin A1 (1)…………………. 11

Figure S2. NMR (600 MHz, CDCl3) spectrum for heterofibrin A2 (2)…………….….. 12

Figure S3. NMR (600 MHz, CDCl3) spectrum for heterofibrin A3 (3)……………….. 13

Figure S3a NMR (600 MHz, CDCl3) spectrum for 13-methylmyristic acid………… 14

Figure S4. NMR (600 MHz, CDCl3) spectrum for heterofibrin B1 (4)………………... 15

Figure S5. NMR (600 MHz, CDCl3) spectrum for heterofibrin B2 (5)………………... 16

Figure S6. NMR (600 MHz, CDCl3) spectrum for heterofibrin B3 (6)……………….. 17

Figure S6a NMR (600 MHz, CDCl3) spectrum for palmitic acid…………………….. 18

Figure S7. NMR (600 MHz, CDCl3) spectrum for 2’S-lactyl linoleoate ester (7a)…… 19

Figure S8. NMR (600 MHz, CDCl3) spectrum for 2’R-lactyl linoleoate ester (7b)…… 20

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Figure S9. NMR (600 MHz, CDCl3) spectrum for 2’S,2”S-dilactyl linoleoate ester (8a)

……………………………………………………………………..…… 21

Figure S10. NMR (600 MHz, CDCl3) spectrum for 2’R,2”R-dilactyl linoleoate ester

(8b)……………………………………………………………………… 22

Figure S11. NMR (600 MHz, CDCl3) spectrum for 2’S,2”R-dilactyl linoleoate ester

(8c)……………………………………………………………………… 23

Figure S12. NMR (600 MHz, CDCl3) spectrum for 2’R,2”S-dilactyl linoleoate ester

(8d)……………………………………………………………………… 24

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Experimental Details

Taxonomy. A description of the specimen is as follows: Growth form spreading,

lobate; colour in life and in EtOH beige; texture soft and spongy but tough to tear; oscules

discrete and sunken in hollows; surface translucent, conulose, with a skin-like covering

over surface of the hollows; spicules none; ectosome a fine tract of foreign spicules aligned

tangentially to the surface; choanosome exhibits a regular, polygonal mesh of

interconnecting, homogeneous, secondary fibres clear of laminations, pith and detritus with

each intersection formed by three fibres. Sub-primary fibres display two distinct size-

classes. Primary fibres are reduced in size and number, ascend to the surface in support of

conules and are cored with a single tract of spicular detritus. The specimen was identified

as Class: Demospongiae, Order: Dictyoceratida, Family: Spongiidae, Genus: Spongia

(Heterofibria). A voucher sample was deposited with Museum Victoria (Reg No.

MVF166231).

(2′R)-lactyl linoleoate (7b). 1H NMR (600 MHz, CDCl3) δ 5.38 (2H, m, H-10 and H-

12), 5.33 (2H, m, H-9 and H-13), 5.10 (1H, q, J = 7.0, H-2′), 2.77 (2H, dd, J = 6.9, 6.9 Hz,

H2-11), 2.40 (1H, m, H-2a), 2.35 (1H, m, H-2b), 2.04 (4H, dt, J = 7.1, 7.1 Hz, H2-8 and H2-

14), 1.65 (2H, tt, J = 7.5, 7.5 Hz, H2-3), 1.52 (3H, d, J = 7.0 Hz, H3-3′), 1.38-1.24 (14 H,

m), 0.89 (3H, t, J = 6.9 Hz, H3-18); 13C NMR (150 MHz, CDCl3) δ 176.3 (C-1′), 173.5 (C-

1), 130.4 (C-13), 130.2 (C-9), 128.2 (C-10), 128.1 (C-12), 68.4 (C-2′), 34.1 (C-2), 31.7 (C-

16), 29.8-29.2 (methylenes), 27.4 (2C, C-8 and C-14), 25.8 (C-11), 24.9 (C-3), 22.8 (C-

17), 17.1 (C-3′), 14.3 (C-18).

(2′R,2′ ′R)-Dilactyl linoleoate (8b). NMR (600 MHz, CDCl3) δ 5.38 (2H, m, H-10

and H-12), 5.33 (2H, m, H-9 and H-13), 5.20 (1H, q, J = 7.0 Hz, H-2′′), 5.11 (1H, q, J =

7.0 Hz, H-2′), 2.77 (2H, t, J = 6.9 Hz, H2-11), 2.40 (1H, m, H-2a), 2.35 (1H, m, H-2b),

2.04 (4H, dt, J = 7.1, 7.1 Hz, H2-8 and H2-14), 1.65 (2H, tt, J = 7.5, 7.5 Hz, H2-3), 1.57

(3H, d, J = 7.0 Hz, H3-3′′), 1.55 (3H, d, J = 7.0 Hz, H3-3′), 1.38-1.24 (14 H, m), 0.89 (3H,

t, J = 6.9 Hz, H3-18); 13C NMR (150 MHz, CDCl3) δ 173.7 (C-1′′), 173.6 (C-1), 170.6 (C-

1′), 130.4 (C-13), 130.3 (C-9), 128.2 (C-10), 128.1 (C-12), 68.7 (C-2′′), 68.4 (C-2′), 34.1

(C-2), 31.7 (C-16), 29.8-29.2 (methylenes), 27.4 (2C, C-8 and C-14), 25.8 (C-11), 24.9 (C-

3), 22.8 (C-17), 16.9 (2C, C-3′ and C-3′′), 14.3 (C-18).

(2′R,2′ ′S)-Dilactyl linoleoate (8d). NMR (600 MHz, CDCl3) δ 5.38 (2H, m, H-10

and H-12), 5.32 (2H, m, H-9 and H-13), 5.16 (1H, m, H-2′′), 5.14 (1H, m, H-2′), 2.77 (2H,

t, J = 6.9 Hz, H2-11), 2.40 (1H, m, H-2a), 2.35 (1H, m, H-2b), 2.04 (4H, dt, J = 7.1, 7.1 Hz,

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H2-8 and H2-14), 1.64 (2H, tt, J = 7.5, 7.5 Hz, H2-3), 1.53 (3H, d, J = 7.0 Hz, H3-3′′), 1.52

(3H, d, J = 7.0 Hz, H3-3′), 1.38-1.24 (14 H, m), 0.89 (3H, t, J = 6.9 Hz, H3-18); 13C NMR

(150 MHz, CDCl3) δ 175.0 (C-1′′), 173.5 C-1), 170.4 (C-1′), 130.4 (C-13), 130.3 (C-9),

128.2 (C-10), 128.1 (C-12), 69.0 (C-2′′), 68.6 (C-2′), 34.1 (C-2), 31.7 (C-16), 29.8-29.2

(methylenes), 27.4 (2C, C-8 and C-14), 25.8 (C-11), 24.9 (C-3), 22.8 (C-17), 17.0 (C-3′ or

C-3′′), 16.9 (C-3′ or C-3′′), 14.3 (C-18).

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Table 1. NMR (600 MHz, CDCl3) data for heterofibrin A1 (1)

δC δH (m, J Hz) HMBC (1H to 13C) COSY

1 177.8 - - -

2 32.6 2.51, t (7.3) C-1, C-3, C-4 H-3

3 23.4 1.87, tt (7.3, 6.8) C-1, C-2, C-4, C-5 H-2, H-4

4 19.1 2.42, t (6.8) C-2, C-3, C-5, C-6, C-7, C-8 H-3

5 81.9 - - -

6 66.5a - - -

7 72.7a - - -

8 74.8 - - -

9 108.6 5.47, br d (15.9) C-6, C-7, C-10, C-11 H-10, H-11

10 148.9 6.29, dt (15.9, 7.2) C-8, C-9, C-11, C-12 H-9, H-11

11 33.5 2.11, dtd (7.2, 7.2, 1.3) C-9, C-10, C-12 H-9, H-10, H-12

12 28.7 1.38, tt (7.2, 7.2) C-10, C-11, C-13 H-11, H-13

13 29.6 b 1.26, m overlap H-12

14 29.4 b 1.26, m overlap overlap

15 29.3 b 1.26, m overlap overlap

16 32.1 1.25, m C-17 overlap

17 22.8 1.29, m C-16, C-18 H-18

18 14.3 0.88, t (7.0) C-16, C-17 H-17

a,b assignments are interchangeable

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Table S2. NMR (600 MHz, CDCl3) data for heterofibrin A2 (2)

δC δH (m, J Hz) HMBC (1H to 13C) COSY

1 172.6 - - - 2a 2b

32.8 2.56, m, 1H 2.51, m, 1H

C-1, C-3, C-4 H-3

3 23.6 1.90, tt (7.3, 6.9), 2H C-1, C-2, C-4, C-5 H-2, H-4 4 19.1 2.42, t (6.9), 2H C-2, C-3, C-5, C-6, C-7, C-8 H-3 5 82.1 - - - 6 66.4a - - - 7 72.8a - - - 8 74.7 - - - 9 108.6 5.47, br d (15.9), 1H C-6, C-7, C-10, C-11 H-10, H-11

10 148.9 6.29, dt (15.9, 7.2), 1H C-8, C-11, C-12 H-9, H-11 11 33.5 2.11, dtd (7.2, 7.0, 1.3), 2H C-8, C-9, C-10, C-12 H-10, H-12 12 28.7 1.38, tt (7.0, 7.0), 2H C-11, C-13 H-11 13 29.6b 1.26, m overlap H-12 14 29.4b 1.26, m overlap overlap 15 29.3b 1.26, m overlap overlap 16 32.1 1.25, m C-17 overlap 17 22.9 1.29, m C-16, C-18 H-18 18 14.3 0.88, t (7.0), 3H C-16, C-17 H-17 1′ 173.6 - - - 2′ 68.7 5.14, q (7.1), 1H C-1, C-1′, C-3′ H-3′ 3′ 17.0 1.54, d (7.1), 3H C-1′, C-2′ H-2′

a,b Carbon assignments are interchangeable

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Table S3. NMR (600 MHz, CDCl3) data for heterofibrin A3 (3)

a,b Carbon assignments are interchangeable

δC δH (m, J Hz) HMBC (1H to 13C) COSY

1 172.2 - - - 2a 2b

32.8 2.56, m, 1H 2.51, m, 1H

C-1, C-3, C-4

H-3

3 23.6 1.89, tt (7.3, 6.8), 3H C-1, C-2, C-4, C-5 H-2, H-4 4 19.1 2.42, t (6.8), 2H C-2, C-3, C-5, C-6, C-7, C-8 H-3 5 82.1 - - - 6 66.4a - - - 7 72.8a - - - 8 74.7 - - - 9 108.7 5.47, br d (15.9) C-6, C-7, C-10, C-11 H-10, H-11

10 148.9 6.29, dt (15.9, 7.2) C-8, C-11, C-12 H-9, H-11 11 33.5 2.11, dt (7.2, 7.0), 2H C-8, C-9, C-10, C-12 H-10, H-12 12 28.7 1.38, tt (7.0, 7.0), 2H C-10, C-11, C-13 H-11 13 29.6b 1.26, m overlap H-12 14 29.4b 1.26, m overlap overlap 15 29.3b 1.26, m overlap overlap 16 32.1 1.25, m C-17 overlap 17 22.9 1.29, m C-16, C-18 H-18 18 14.3 0.88, t (7.0), 3H C-16, C-17 H-17 1′ 170.5 - - - 2′ 68.3 5.12, q (7.1), 1H C-1, C-1′, C-3′ H-3′ 3′ 16.9 1.56, d (7.1), 3H C-1′, C-2′ H-2′ 1′′ 173.6 - - - 2′′ 68.6 5.21, q (7.1), 1H C-1′, C-1′′, C-3′′ H-3′′ 3′′ 16.9 1.57, d (7.1), 3H C-1′′, C-2′′ H-2′′

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Table S4. NMR (600 MHz, CDCl3) data for heterofibrin B1 (4)

δC δH (m, J Hz) HMBC (1H to 13C) COSY

1 175.2 - - - 2 32.1 2.51, t (7.1), 2H C-1, C-3, C-4 H-3 3 23.5 1.87, tt (7.1, 6.8), 2H C-1, C-2, C-4, C-5 H-2, H-4 4 19.1 2.42, t (6.8), 2H C-2, C-3, C-5, C-6, C-7, C-8 H-3 5 82.0 - - - 6 66.5a - - - 7 72.7a - - - 8 74.8 - - - 9 108.6 5.48, br d (15.8), 1H C-6, C-7, C-10, C-11 H-10, H-11

10 148.9 6.29, dt (15.8, 7.2), 1H C-8, C-11, C-12 H-9, H-11 11 33.5 2.11, dt (7.2, 7.2), 2H C-8, C-9, C-10, C-12 H-10, H-12 12 28.8 1.38, tt (7.2, 7.2), 2H C-10, C-11, C-13 H-11 13 29.9b 1.25, m overlap H-12 14 29.3b 1.25, m overlap overlap 15 27.5 1.25, m overlap overlap 16 39.2 1.15, dt (6.7, 6.7), 2H C-15, C-17, C-18, C-19 H-15 17 28.2 1.51, tq (6.7, 6.7), 1H C-15, C-16, C-18, C-19 H-18, H-19

18 19 22.9 0.86, d (6.7), 6H C-16, C-17 H-17 a,b Carbon assignments are interchangeable

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Table S5. NMR assignment for heterofibrin B2 (5)

δC δH (m, J Hz) HMBC (1H to 13C) COSY

1 172.6 - - - 2a 2b

32.8 2.56, m, 1H 2.51, m, 1H

C-1, C-3, C-4 H-3

3 23.6 1.90, tt (7.1, 6.9), 2H C-1, C-2, C-4, C-5 H-2, H-4 4 19.1 2.42, t (6.9), 2H C-2, C-3, C-5, C-6, C-7, C-8 H-3 5 82.1 - - - 6 66.4a - - - 7 72.8a - - - 8 74.7 - - - 9 108.7 5.47, br d (15.9), 1H C-6, C-7, C-10, C-11 H-10, H-11

10 148.9 6.29, dt (15.9, 7.1), 1H C-8, C-11, C-12 H-9, H-11 11 33.5 2.11, dt (7.0, 7.1), 2H C-8, C-9, C-10, C-12 H-10, H-12 12 28.8 1.38, tt (7.0, 7.0), 2H C-10, C-11, C-13 H-11 13 29.3b 1.25, m overlap H-12 14 29.9b 1.25, m overlap overlap 15 27.5 1.25, m overlap overlap 16 39.2 1.15, dt (6.7, 6.7), 2H C-15, C-17, C-18, C-19 H-15 17 28.2 1.51, tq (6.7, 6.7), 2H C-15, C-16, C-18, C-19 H-18, H-19

18 19 22.9 0.86, d (6.7), 6H C-16, C-17 H-17 1′ 174.0 - - - 2′ 68.3 5.13, q (7.1), 1H C-1, C-1′, C-3′ H-3′′ 3′ 17.0 1.54, d (7.1), 3H C-1′, C-2′ H-2′′

a,b Carbon assignments are interchangeable

(E)

O

O(S)1' 2'

3'

HO

O

19

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Table S6. NMR assignment for heterofibrin B3 (6)

δC δH (m, J Hz) HMBC (1H to 13C) COSY

1 172.7 - - - 2a 2b

32.8 2.56, m, 1H 2.51, m, 1H

C-1, C-3, C-4 H-3

3 23.6 1.89, tt (7.1, 7.0), 2H C-1, C-2, C-4, C-5 H-2, H-4 4 19.1 2.42, t (7.0), 2H C-2, C-3, C-5, C-6, C-7, C-8 H-3 5 82.1 - - - 6 66.4a - - - 7 72.8a - - - 8 74.7 - - - 9 108.7 5.47, br d (15.9), 1H C-6, C-7, C-10, C-11 H-10, H-11

10 148.9 6.29, dt (15.9, 7.1), 1H C-8, C-11, C-12 H-9, H-11 11 33.5 2.11, dt (7.1, 7.1), 2H C-8, C-9, C-10, C-12 H-10, H-12 12 28.8 1.38, tt (7.1, 7.1), 2H C-10, C-11, C-13 H-11 13 29.3b 1.25, m overlap H-12 14 29.9b 1.25, m overlap overlap 15 27.5 1.25, m overlap overlap 16 39.2 1.15, dt (6.7, 6.7), 2H C-15, C-17, C-18, C-19 H-15 17 28.2 1.51, tq (6.7, 6.7), 2H C-15, C-16, C-18, C-19 H-18, H-19

18 19 22.9 0.86, d (6.7), 6H C-16, C-17 H-17 1′ 170.5 - - - 2′ 68.6 5.12, q (7.1), 1H C-1, C-1′, C-3′ H-3′′ 3′ 16.9 1.56, d (7.1), 3H C-1′, C-2′ H-2′′ 1′′ 173.9 - - - 2′′ 68.8 5.21, q (7.1), 1H C-1′, C-1′′, C-3′′ H-3′′ 3′′ 16.9 1.57, d (7.1), 3H C-1′′, C-2′′ H-2′′

a,b Carbon assignments are interchangeable

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Figure S1. 1H-NMR (600 MHz, CDCl3) and UV-vis (inset) spectra of heterofibrin A1 (1)

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Figure S2. 1H-NMR (600 MHz, CDCl3) and UV-vis (inset) spectra of heterofibrin A2 (2)

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Figure S3. 1H-NMR (600 MHz, CDCl3) and UV-vis (inset) spectra of heterofibrin A3 (3)(× denotes peak from 13-methylmyristic acid)

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Figure S3a. 1H-NMR (600 MHz, CDCl3) spectrum of 13-methylmyristic acid (authentic sample)

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Figure S4. 1H-NMR (600 MHz, CDCl3) and UV-vis (inset) spectra of heterofibrin B1 (4).

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FigureS5.1H‐NMR(600MHz,CDCl3)andUV‐vis(inset)spectraofheterofibrinB2(5).

(E)

O

O(S)1' 2'

3'

HO

O

19

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FigureS6.1H‐NMR(600MHz,CDCl3)andUV‐vis(inset)spectraofheterofibrinB3(6).

(× denotes peaks from palmitic acid)

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Figure S6a. 1H-NMR (600 MHz, CDCl3) spectrum of palmitic acid (authentic sample)

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FigureS7.1H‐NMR(600MHz,CDCl3)spectrumof2’S‐lactyllinoleoateester

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FigureS8.1H‐NMR(600MHz,CDCl3)spectrumof2’R‐lactyllinoleoateester

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FigureS9.1H‐NMR(600MHz,CDCl3)spectrumof2’S,2”S‐dilactyllinoleoateester

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FigureS10.1H‐NMR(600MHz,CDCl3)spectrumof2’R,2”R‐dilactyllinoleoateester

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FigureS11.1H‐NMR(600MHz,CDCl3)spectrumof2’S,2”R‐dilactyllinoleoateester

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FigureS12.1H‐NMR(600MHz,CDCl3)spectrumof2’R,2”S‐dilactyllinoleoateester

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