Supplementary Materials for - Springer Static Content Server10.1007/s10811-015-0668... · Supplementary Materials for ... Their chemotaxonomic significance. Phytochemistry 40: 827-831

Supplementary Materials for

Settlement inhibition of marine biofilm bacteria and barnacle larvae by

compounds isolated from the Mediterranean brown alga Taonia atomaria

Ahlem Othmani1, Robert Bunet2, Jean-Luc Bonnefont2, Jean-François Briand1 and Gérald

Culioli1,*

1 Université de Toulon, MAPIEM EA 4323, 83957 La Garde Cedex, France.

2 Institut Océanographique Paul Ricard, Ile des Embiez, 83140 Six-Fours-les-Plages, France.

* Corresponding author. Tel.: (+33) 4 94 14 29 35. E-mail: [email protected] (G. Culioli).

mailto:[email protected]

I - Structural characterization of compounds 1-8

Compound 1 was purified as an optically active greenish oil. The presence of a peak at m/z

203.17943 [M-H2O+H]+ in its ESI-HRMS spectrum, in addition to NMR data, allowed to the

determination of its molecular formula as C15H24O. Its IR absorptions indicated the presence of

a hydroxyl group (3348 cm-1) and conjugated double bonds (1608 and 1648 cm-1). The 13C

NMR spectrum of 1 showed more precisely that this compound contained 15 carbon atoms

including two quaternary (both sp2), five methines (two sp2 and three sp3, comprising one

oxymethine), six methylenes (two sp2 and four sp3, one of which being a hydroxyl-bearing

carbon) and two methyl carbons. The 1H NMR spectrum of 1 presented six olefinic protons

[H 6.00, d (J = 16.0 Hz), H-5 ; 5.43 dd (J = 16.0 and 10.5 Hz), H-6; 5.27 s and 5.00 s, Ha,b-14;

4.92 s and 4.84 s, Ha,b-15], one oxymethine proton [H 3.77 dd (J = 12.0 and 4.0 Hz), H-9] and

two doublet methyls [H 0.82 d (J = 6.5 Hz), H3-12; 0.90 d (J = 6.5 Hz), H3-13]. The high value

of the 3J-coupling between H-5 and H-6 (16.0 Hz) indicated an E 1,2-disubstituted double bond,

while other NMR signals disclosed two additional 1,1-disubstituted double bonds. Moreover,

1H-1H COSY correlations between the two methyl signals (H3-12 and H3-13) and the same

methine proton signal (H-11) are typical of an isopropyl moiety while specific 1H and 13C

signals reveal the occurrence of an oxymethine group (H-9). 1H-1H COSY and HMBC spectra

granted the knowledge to connect all these structural features and then allowed the assignment

of compound 1 as germacra-4(15),5,10(14)-trien-9-ol (Fig. S1). To our knowledge, this

sesquiterpenoid which stereochemistry at C-7 and C-9 remained to be determined had never

been described before.

Figure S1: Key NMR correlations (HMBC and 1H-1H COSY) for compound 1

For compound 2, the comparison of its experimental NMR data with those coming from

literature allowed the determination of its planar structure as that of a spiroaxane sesquiterpene

previously isolated from samples of T. atomaria collected in the northern Adriatic Sea (De Rosa

et al., 1994), as well as of the sponge Eurypon sp. from New Zealand (Barrow et al., 1988). The

analysis of its 1H-1H NOESY spectrum was used to define the relative stereochemistry at C-5

on the basis of the nOe between H-1/H-7 and Hb-9. The relative configuration at C-6 was

obtained through the small value of 3JH6,H7 that fixed the hydroxyl in axial position, the

isopropyl group bonded at C-7 being in equatorial position. Finally, after a comparison of its

spectral dataset with those of the literature, the absolute configuration of compound 2 was

determined to be identical to that of (-)-gleenol obtained by synthesis (Blay et al., 2005;

Nakazaki et al., 2007).

Examination of the experimental spectroscopic data of compound 3 led to the determination of

its chemical structure as those of a cadinane sesquiterpene. Comparison of its NMR data with

literature allowed its identification as -cadinol methyl ether (Dupré et al., 1991), a compound

described for the first time as a metabolite of T. atomaria.

Experimental NMR data of compound 4 were in agreement with a calamenane sesquiterpenoid

structure. More particularly, its spectroscopic data were identical in all aspects to those reported

in literature for (-)-trans-calamenene, a molecule obtained by synthesis (Nakashima et al.,

2002) and already isolated from T. atomaria [from an algal sample misidentified as D. fasciola

(Amico et al., 1979)].

The NMR data of compound 5 allowed the characterization of its chemical structure as those

of an acetylated sesquiterpene, (1S, 5E, 7S) 1-acetoxygermacra-4(15),5,10(14)-triene,

previously described from T. atomaria [erroneously identified as D. fasciola (Fattorusso et al.,

1978)]. The stereochemistry at C-1 and C-7 of this particular compound was determined on the

basis of a previous work dealing with the structural analysis of its enantiomer by electronic

circular dichroism (Nagashima et al., 1990).

The full NMR data set of compound 6 was found to be identical to those of 4-peroxymuurol-5-

ene, a sesquiterpene previously isolated from the soft coral Sarcophyton erhenbergi (Shaker et

al., 2010) and a terrestrial plant (Nagashima and Asakawa, 2001). However, the value of the

specific optical rotation of 6 was opposite to that previously described, thus compound 6 should

be its enantiomer: it might be considered as original but for this statement, its stereochemistry

at C-1 and C-4 remained to be determined.

The analysis of the spectral data of compound 7 and the comparison with the literature allowed

its identification as (5Z, 8Z, 11Z, 14Z, 17Z)-eicosa-5,8,11,14,17-pentaenoic acid. This

polyunsaturated fatty acid is commonly found in various marine organisms: in particular, it has

been previously isolated from the brown alga Zonaria tournefortii, where it was considered as

a biosynthetic precursor of a series of acylphloroglucinols (El Hattab et al., 2009).

Comparison of NMR data of 8 with those from literature led to the identification of this

compound as sn-3-O-(geranylgeranyl)glycerol. This molecule has been already isolated from

T. atomaria [erroneously identified as D. fasciola (Amico et al., 1977)] but also from other

brown algae of the Dictyotaceae family: Dictyota spp. (Othmani et al., 2014) and T. lacheana

(Tringali et al., 1995).

References

Amico V, Oriente G, Piattelli M, Tringali C, Fattorusso E, Magno S, Mayol L (1977) (-)-(R)-1-O-

Geranylgeranylglycerol from the brown alga Dilophus fasciola. Cell Mol Life Sci 33: 989-990

Amico V, Oriente G, Piattelli M, Tringali C, Fattorusso E, Magno S, Mayol L (1979) Sesquiterpenes

based on the cadalane skeleton from the brown alga Dilophus fasciola. Experientia 35: 450-451

Barrow CJ, Blunt JW, Munro MHG (1988) Sesquiterpenes from a New Zealand sponge of the genus

Eurypon. Aust J Chem 41: 1755-1761

Blay G, Collado AM, García B, Pedro JR (2005) Silicon guided rearrangement of epoxydecalines to

spirocyclic compounds. Synthesis of gleenol and axenol from carvone. Tetrahedron 61: 10853-

10860

De Rosa S, De Giulio A, Iodice C, Zavodink N (1994) Sesquiterpenes from the brown alga Taonia

atomaria. Phytochemistry 37: 1327-1330

Dupré S, Grenz M, Jakupovic J, Bohlmann F, Niemeyer HM (1991) Eremophilane, germacrane and

shikimic acid derivatives from chilean Senecio species. Phytochemistry 30: 1211-1220

El Hattab M, Bouzidi N, Ortalo-Magné A, Daghbouche Y, Richou M, Chitour SE, de Reviers B, Piovetti

L (2009) Eicosapentaenoic acid: Possible precursor of the phloroglucinol derivatives isolated

from the brown alga Zonaria tournefortii (J.V. Lamouroux) Montagne. Biochem Syst Ecol 37:

55-58

Fattorusso E, Magno S, Mayol L, Amico V, Oriente G, Piattelli M, Tringali C (1978) Isolation of

(2S,8R)-germacra-1(11),5(12),E6-trien-2-ol acetate from the brown alga Dilophus fasciola.

Tetrahedron Lett 19: 4149-4152

Nagashima F, Asakawa Y (2001) Sesqui- and diterpenoids from two Japanese and three European

liverworts. Phytochemistry 56: 347-352

Nagashima F, Toyota M, Asakawa Y (1990) Terpenoids from some Japanese liverworts.

Phytochemistry 29: 2169-2174

Nakashima K, Imoto M, Sono M, Tori M, Nagashima F, Asakawa Y (2002) Total synthesis of (-)-

(7S,10R)-calamenene and (-)-(7S,10R)-2-hydroxycalamenene by use of a ring-closing

metathesis reaction. A comparison of the cis- and trans-isomers. Molecules 7: 517-527

Nakazaki A, Era T, Kobayashi S (2007) Total synthesis of (±)-gleenol and (±)-axenol via a

functionalized spiro[4.5]decane. Chem Pharm Bull 55: 1606-1609

Othmani A, Bouzidi N, Viano Y, Alliche Z, Seridi H, Blache Y, El Hattab M, Briand J-F, Culioli G

(2014) Anti-microfouling properties of compounds isolated from several Mediterranean

Dictyota spp. J Appl Phycol 26: 1573-1584

Shaker KH, Müller M, Ghani MA, Dahse H-M, Seifert K (2010) Terpenes from the soft corals

Litophyton arboreum and Sarcophyton ehrenbergi. Chem Biodivers 7: 2007-2015

Tringali C, Piattelli M, Spatafora C (1995) Sesquiterpenes and geranylgeranylglycerol from the brown

algae Taonia lacheana and Taonia atomaria f. ciliata : Their chemotaxonomic significance.

Phytochemistry 40: 827-831

II - Spectral data for compounds 1-8

Compound 1: greenish oil; ][25

D -134° (c 0.10, MeOH); HRESIMS m/z 203.17943 [M-

H2O+H]+ (calcd for C15H23, 203.17943) (See Figure S2); IR: 3348, 2953, 2926, 2870, 2852,

1648, 1608, 1460, 1384, 1367, 1038, 1011, 971, 888 cm-1; 1H and 13C spectra, and a table with

the NMR data of 1 are provided in Figures S3-S4 and Table T1.

Compound 2: greenish oil; ][25

D -22° (c 0.10, MeOH); (+)-ESI-MS: m/z 205 [M-H2O+H]+; IR:

3462, 3039, 2952, 2923, 2871, 2850, 1652, 1608, 1460, 1444, 1379, 1132, 1047, 928 cm-1; 1H

and 13C spectra, and a table with the NMR data of 2 are given in Figures S5-S6 and Table T2.

Compound 3: pale yellow oil; 1H and 13C spectra and a table with the NMR data of 3 are given

in Figures S7-S8 and Table T3.

Compound 4: pale yellow oil; (+)-ESI-MS: m/z 203 [M+H]+; 1H and 13C spectra, and a table

with the NMR data of 4 are given in Figures S9-S10 and Table T4.

Compound 5: yellow oil; ][25

D -104° (c 0.05, MeOH); (+)-ESI-MS: m/z 203 [M-AcOH+H]+;

IR: 3068, 2950, 2933, 2871, 1735, 1644, 1608, 1456, 1441, 1373, 1236, 1016, 976, 953, 867 cm-

1; 1H and 13C spectra, and a table with the NMR data of 5 are given in Figures S11-S12 and

Table T5.

Compound 6: orange oil; ][25

D -59° (c 0.05, MeOH); (+)-ESI-MS: m/z 259 [M+Na]+,

237 [M+H]+, and 219 [M-H2O+H]+; 1H and 13C spectra, and a table with the full NMR data of

6 are given in Figures S13-S14 and Table T6.

Figure S2: UPLC-HRMS-DAD analysis of compound 1

Figure S3: 1H NMR spectrum of compound 1 (CDCl3, 400MHz)

Figure S4: 13C NMR spectrum of compound 1 (CDCl3, 100MHz)











Table T1 NMR data of compound 1 (CDCl3, 400 MHz)

n.o: not observed

δC DEPT δH mult. (J in Hz) HMBC 1H-1H COSY 1H-1H NOESY

1 34.7 CH2 a: 1.64 m a: C-10, C14

b: C-10

a: H b-1

b: Ha-1, Ha,b-2, Ha,b-14

a: H b-1, H-9, Ha-14

b: Ha-1, Ha,b-2, Ha-14

b: 2.62m

2 36.3 CH2 a: 1.65 m a: C-3, C10 a: Ha,b-3, H-1, Hb-2 a: Hb-2, Ha-3, Hb-1

b: 2.05 m b: C-10 b: Ha,b-3, H-1, Ha-2 b: Ha-2, Hb-1

3 30.1 CH2 a: 2.19 ddd (13.0, 5.5, 3.0) a: C-5 a: Hb-3, Ha,b-2 a: Hb-3, Hb-15

b: 2.43 td (13.0, 5.0) b: C-2, C-15, C-5, C-4 b: Ha-3, Ha,b-2 b: Ha-3, H-6

4 146.9 C - - - -

5 129.7 CH 6.00 d (16.0) C-3, C-4, C-7, C-15 H-6 H-7

6 138.1 CH 5.43 dd (16.0, 10.5) C-4, C-7 H-5, H-7, Ha,b-15 Hb-3

7 52.6 CH 1.80 m n.o. H-6, Ha,b-8, H-11 H-5, H-11, H3-12

8 36.4 CH2 a: 1,68 m a: C-9, C10 a: H-7, Hb-8, H-9 a: Hb-8, H-9

b: 2.05 m b: C-6, C-10 b: H-7, Ha-8, H-9 b: Ha-8, H-9, Hb-14,H3-12

9 76.2 CH 3.77 dd (12.0, 4.0) C-10, C-14 Ha,b-8, Ha-14 Ha-1, Ha,b-8

10 153.6 C - - - -

11 32.0 CH 1.49 oct (6.5) C-6, C-7, C-8, C-12, C13 H3-12, H3-13, H-7 H-7,H3-12,H3-13

12 20.6 CH3 0.82 d (6.5) C-7, C-11, C-13 H-11, H3-13 H-7, Ha,b-8, H-11

13 20.9 CH3 0.90 d (6.5) C-7, C-11, C-12 H-11, H3-12 H-11

14 110.7 CH2 a: 5.00 s a: C-1, C-9, C-10 a: Hb-1, H-9 a: Ha,b-1

b: 5.27 s b: C-1, C-9 b: Hb-1 b: Hb-8

15 113.1 CH2 a: 4.84 s a: C-3, C-5 a: H-6 a: n.o

b: 4.92 s b: C-3, C-5 b: H-6 b: Ha-3

Table T2: NMR data of compound 2 (CDCl3, 400MHz)

δC DEPT δH mult. (J in Hz) HMBC COSY 1H-1H NOESY 1H-1H

1 125.7 CH 5.17 br sext. (1,5) C-2, C-3, C-4, C-5, C-15 H2-3, H3-15 H-6, H-7, Hb-9, H3-15

2 142.9 C - - - -

3 36.5 CH2 2.21 m C-1, C-2, C-4, C-5 H-1, Ha,b-4 H-6, H3-14, H3-15

4 34.1 CH2 a: 1.88 ddd (13.5, 9.5, 7.0)

b: 1.81 ddd (13.5, 8.0, 5.5)

a: C-1, C-2, C-3, C-5, C-10, C-6

b: C-1, C-2, C-3, C-5, C-10, C-6

a: H2-3, Hb-4

b: H2-3, Ha-4

a: H-6, H3-15

b: H3-14, H3-15

5 59.0 C - - - -

6 76.6 CH 3.53 br d (1.5) C-1, C-5, C-7, C-8, C-10, C-11 H-7 H-1, H2-3, Ha-4, H-7, Hb-9, H-

11, H3-12, H3-13

7 45.5 CH 1.14 m C-5, C-8 H-6, Ha,b-8, H-11 H-1, H-6, Ha,b-8, H3-12, H3-13

8 24.5 CH2 a: 1.65 m

b: 1.28 qd (13.0, 4.0)

a: C-6, C-7, C-9, C-10, C-11

b: C-6, C-7, C-9, C-10, C-11

a: H-7, Hb-8, Ha,b-9

b: H-7, Ha-8, Ha-9

a: H-7, Hb-8, H3-12, H3-13

b: H-7, Ha-8

9 31.9 CH2 a: 1.45 dq (13.0, 3.5)

b: 1.08 qd (13.0, 4.0)

a: C-5, C-7, C-8, C-10, C-14

b: C-5, C-7, C-8, C-10, C-14

a: Ha,b-8, Hb-9, H-10

b: Ha-8, Ha-9, H-10

a: Hb-9, H3-14

b: H-1, H-6, Ha-9, H3-14

10 34.2 CH 1.69 m C-1, C-5, C-6, C-8, C-9, C-14 Ha,b-9, H3-14 H3-14

11 29.4 CH 1.54 hept.d (7.0, 2.5) C-6, C-7, C-8, C-12, C-13 H-7, H3-12, H3-13 H-6, H3-12, H3-13

12 20.9 CH3 0.91 d (7.0) C-7, C-11, C-13 H-11 H-6, H-7, Ha-8, H-11

13 21.4 CH3 0.92 d (7.0) C-7, C-11, C-12 H-11 H-6, H-7, Ha-8, H-11

14 16.4 CH3 0.74 d (7.0) C-5, C-9, C-10 H-10 H2-3, Hb-4, Ha,b-9, H-10

15 17.1 CH3 1.73 d (1.0) C-1, C-2, C-3 H-1 H-1, H2-3, Ha,b-4



1

46.7

CH

1.35 ddd (12.0, 10.5, 1.5)

C-3, C-6, C-9, C-10, C-14

Hb-2, H-6

Ha-2, H2-3

2 22.7 CH2 a: 1.95 m

b: 1.17 m

a: C-1, C-3, C-4, C-6

b: C-3, C-6, C-10

a: Hb-2

b: H-1, Ha-2, H2-3

a: H-1, Hb-2

b: Ha-2, H2-3

3 31.0 CH2 1.95 m C-1, C-2, C-4, C-5 Hb-2, H-5 H-1, Hb-2

4 135.2 C - - - -

5 122.6 CH 5.51brs C-1, C-3, C-6, C-7, C-15 H2-3, H-6, H3-15 H-6, H-11, H3-12,

H3-15

6 39.7 CH 1.77 m C-1, C-7, C-8 H-1, H-5, H-7 H-5, Hb-8

7 46.8 CH 0.99 m C-8 H-6, Ha-8 Ha-8

8 39.7 CH2 a: 1.62 m

b: 1.09 m

a: C-6, C-7, C-9, C-10

b: C-11

a: H-7, Hb-8, Ha,b-9

b: Ha-8, Ha,b-9

a: H-7, Hb-8

b: H-6, Ha-8

9 35.6 CH2 a: 1.80 m

b: 1.45 td (12.5, 4.0)

a: C-1, C-7, C-8, C-10, C-14

b: C-1, C-7, C-8, C-10, C-14

a: Ha,b-8, Hb-9

b: Ha,b-8, Ha-9

a: Hb-9, H3-16

b: Ha-9

10 76.3 C - - - -

11 26.1 CH 2.15 hept. d (7.0, 3.0) C-8, C-12, C-13 H3-12, H3-13 H-5

12 15.3 CH3 0.77 d (7.0) C-7, C-11, C-13 H-11 H-5

13 21.7 CH3 0.92 d (7.0) C-7, C-11, C-12 H-11 n.o.

14 17.6 CH3 1.06 s C-1, C-9, C-10 n.o. n.o.

15 24.0 CH3 1.67 s C-3, C-4, C-5 H-5 H-6

16 48.3 CH3 3.20 s C-10 n.o. Ha-9

n.o.: not observed



1 140.2 C - - - -

2 126.9 CH 7.12 d (8.0) C-4, C-6, C-10 H-3 H3-14

3 126.3 CH 6.95 br d (8.0) C-1, C-5, C-15 H-2, H3-15 H3-15

4 134.6 C - - - -

5 128.9 CH 7.02 br s C-1, C-3, C-7, C-15 H3-15 H-7, H-11, H3-15

6 140.1 C - - - -

7 43.9 CH 2.69 br q (6.5) C-1, C-8, C-11, C-12, C-13 Ha,b-8, H-11 H-5, Ha,b-8, Hb-9, H-11

8 21.6 CH2 a: 1.83 m

b: 1.60 m n.o.

a: Hb-8, H-7

b: Ha-8, H-7

a: H-7, Hb-8, H3-12

b: H-7, Ha-8

9 31.0 CH2 a: 1.95 m

b: 1.34 m n.o.

a: Hb-9, H-10

b: Ha-9, H-10

a: Hb-9, H-10, H3-14

b: H-7, Ha-9, H-10

10 32.6 CH 2.76 sext (7.0) n.o. Ha,b-9, H3-14 Ha,b-9, H3-14

11 32.0 CH 2.24 oct. (7.0) C-7, C-12, C-13 H-7, H3-12, H3-13 H-5, H-7, H3-13, H3-12

12 17.5 CH3 0.71 d (7.0) C-7, C-11, C-13 H-11 Ha-8, H-11, H3-13

13 21.4 CH3 1.00 d (7.0) C-7, C-11, C-12 H-11 H-11, H3-12

14 22.5 CH3 1.26 d (7.0) C-1, C-9, C-10 H-10 H-2, H-10, Ha-9

15 21.3 CH3 2.30 s C-3, C-4, C-5 H-3, H-5 H-3, H-5

n.o.: not observed



1 77.6 CH 5.05 dd (13.0, 4.0) C-3, C-9 Ha,b-2 Ha-2

2 33.0 CH2 a: 2.10 m

b: 1.63 m

a: C-4

b: n.o.

a: H-1, Hb-2

b: H-1, Ha-2

a: H-1

b: n.o.

3 29.8 CH2 a: 2.48 m

b: 2.20 dq (13.5, 6.0, 3.0)

a: C-2, C-4, C-15

b: C-15

a: Hb-3

b: Ha-3

a: Hb-3

b: Ha-3

4 146.1 C - - - -

5 129.6 CH 6.10 d (16.0) n.o. H-6 n.o.

6 138.4 CH 5.44 dd (16.0, 10.5) C-4 H-5, H-7 H-7

7 52.6 CH 1.82 m n.o. H-6, Hb-8 n.o.

8 36.1 CH2 a: 2.03 m

b: 1.60 m

a: C-10

b: n.o.

a: Hb-8, Ha-9

b: H-7, Ha-8

a: Hb-8

b: Ha-8

9 34.5 CH2 a: 2.48 m

b: 1.70 m

a: n.o.

b: C-10

a: Ha-8, Hb-9, Ha,b-14

b: Ha-9

a: Hb-9

b: Ha-9

10 149.3 C - - - -

11 32.0 CH 1.50 t (14.0, 7.0) C-12, C-13 H3-12, H3-13 H3-12, H3-13

12 20.7 CH3 0.82 d (7.0) C-7, C-11, C-13 H-11 H-11

13 20.9 CH3 0.90 d (7.0) C-7, C-11, C-12 H-11 H-11

14 114.1 CH2 a: 5.36 s

b: 5.14 s

a: C-1, C-9

b: C-1, C-9

a: Ha-9

b: Ha-9

a: n.o.

b :n.o.

15 113.4 CH2 a: 4.93 s

b: 4.89 s

a: C-3, C-5

b: C-3, C-5

a: n.o.

b: n.o.

a: n.o.

b: n.o.

16 170.6 C - - - -

17 21.6 CH3 1.97 s C-16 n.o. n.o.

n.o.: not observed


δC (ppm) DEPT δH mult. (J en Hz) HMBC COSY 1H-1H NOESY 1H-1H

1

77.8

C

-

-

-

-

2 27.3 CH2 a: 1.27 ddd (13.0, 12.0, 4.0) a: C-1, C-3, C-4, C-10 a: Hb-2, Ha,b-3 a: Hb-2, H3-14

b: 2.35 ddd (13.0, 9.5, 4.0) b: C-1, C-3, C-4, C-6, C-10 b: Ha-2, Hb-3 b: Ha-2, Hb-3

3 30.2 CH2 a: 1.53 m a: C-1, C-2, C-4, C-5, C-15 a: Ha-2, Hb-3 a: Hb-3

b: 2.01 ddd (13.0, 9.5, 4.0) b: C-1, C-2, C-4, C-5, C-15 b: Ha,b-2, Ha-3 b: Hb-2, Ha-3, H3-15

4 75.1 C - - - -

5 130.0 CH 6.09 d (2.0) C-1, C-3, C-4, C-7, C-15 H-7 H-11, H3-15

6 147.5 C - - - -

7 43.0 CH 2.51 dddd (8.5, 7.5, 6.0, 2.0) C-1, C-5, C-6, C-8, C-11, C-12,C-13 H-5, Ha,b-8, H-11 n.o.

8 23.0 CH2 a: 1.45 m a: C-7, C-10, C-11 a: H-7, Hb-8 n.o.

b: 1.77 m b: C-6, C-7, -9, C-10, C-11 b: H-7, Ha-8, H2-9 n.o.

9 28.2 CH2 1.51 m C-1, C-7, C-8, C-10, C-14 Hb-8, H-10 H3-14

10 37.3 CH 1.72 m C-1, C-2, C-8, C-9, C-14 H2-9, H3-14 H3-14

11 32.5 CH 1.90 oct (7.0) C-6, C-7, C-8, C-12, C-13 H-7, H3-12, H3-13 H-5

12 18.3 CH3 0.82 d (6.5) C-7, C-11, C-13 H-11 n.o.

13 20.8 CH3 0.89 d (7.0) C-7, C-11, C-12 H-11 n.o.

14 14.6 CH3 1.03 d (7.0 ) C-1, C-9, C-10 H-10 Ha-2, H2-9, H-10

15 21.9 CH3 1.36 s C-3, C-4, C-5 n.o. H-5

n.o.: not observed

III - Bioassays

Figure S15: Settlement inhibition of barnacle cypris larvae by AF agents (Bars represent the

means ± SD).

-2.0 -1.5 -1.0 -0.5 0.0 0.50

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[TBTO] (µM)

Sett

led c

yprids (

%)

Settlement inhibition of barnacle cyprids by AF agents

-2.0 -1.5 -1.0 -0.5 0.0 0.50

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[TBTO] µM

Sett

led c

yprids (

%)

-2.0 -1.5 -1.0 -0.5 0.00

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[CuPT] (µM)

Sett

led c

yprids (

%)

-2.0 -1.5 -1.0 -0.5 0.00

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[CuPT] (µM)

Sett

led c

yprids (

%)

0.0 0.5 1.0 1.5 2.00

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[compound 1] (µM)

Se

ttle

d c

ypri

ds

(%)

0.0 0.5 1.0 1.5 2.00

20

40

60

80

100

replicate #1

replicate #2


Se

ttle

d c

ypri

ds

(%)

0.0 0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2

replicate #3


Sett

led c

yprids (

%)

-2 -1 0 1 20

20

40

60

80

100

replicate #1

replicate #2


Se

ttle

d c

ypri

ds

(%)

A. amphitrite B. perforatus

TBTO

CuPT

Compound2

Compound1

Figure S16: Toxicity of compounds towards barnacle cyprids (Bars represent the means ± SD).


-2.0 -1.5 -1.0 -0.5 0.0 0.50

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[TBTO] (µM)

Mort

alit

y (%

)

Toxicity of compounds towards barnacle cyprids

-2.0 -1.5 -1.0 -0.5 0.0 0.50

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[TBTO] (µM)

Mort

alit

y (%

)

-2.0 -1.5 -1.0 -0.5 0.00

20

40

60

80

100replicate #1

replicate #2

replicate #3

log[CuPT] (µM)

Mort

alit

y (%

)

-2.0 -1.5 -1.0 -0.5 0.00

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[CuPT] (µM)

Mort

alit

y (%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

alit

y (%

)

0.0 0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

alit

y (%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

alit

y (%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

alit

y (%

)

TBTO

CuPT

Compound6

Compound1

Figure S17: Toxicity of compounds towards barnacle stage I/II nauplii (Bars represent the means ± SD).


Toxicity of compounds towards barnacle stage I/II nauplii

TBTO

CuPT

Compound 6

Compound 1

-2.0 -1.5 -1.0 -0.5 0.00

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[TBTO] (µM)

Mort

ality

(%

)

-2.0 -1.5 -1.0 -0.5 0.0 0.50

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[TBTO] (µM)

Mort

ality

(%

)

-2.0 -1.5 -1.0 -0.50

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[CuPT] (µM)

Mort

ality

(%

)

-2.0 -1.5 -1.0 -0.50

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[CuPT] (µM)

Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

0.0 0.5 1.0 1.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

Compound 2 Not determined

Figure S18: Toxicity of compounds towards barnacle stage VI nauplii (Bars represent the means ± SD).


Toxicity of compounds towards barnacle stage VI nauplii

TBTO

CuPT

Compound 6

Compound 1

Compound 2 Not determined

-2.0 -1.5 -1.0 -0.5 0.00

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[TBTO] (µM)

Mort

ality

(%

)

-2.0 -1.5 -1.0 -0.5 0.0 0.50

20

40

60

80

100

replicate #3

replicate #1

replicate #2

log[TBTO] (µM)

Mort

ality

(%

)

-2.0 -1.5 -1.0 -0.5 0.00

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[CuPT] (µM)

Mort

ality

(%

)

-2.0 -1.5 -1.0 -0.5 0.00

20

40

60

80

100

replicate #1

replicate #2

replicate #3

log[CuPT] (µM)

Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2


Mort

ality

(%

)

0.5 1.0 1.5 2.0 2.50

20

40

60

80

100

replicate #1

replicate #2

repliacte #3


Mort

ality

(%

)

Figure S19: Adhesion response (EC50) for TBTO and compounds 1, 2, and 8 against the five bacterial

strains and the two species of barnacle larvae.

0

20

40

60

80

100

TBTO Compound 8 Compound 10 Compound 2

EC50 (µM)

Documents

Supplementary Materials for - Springer Static Content Server10.1007/s10811-015-0668... · Supplementary Materials for ... Their chemotaxonomic significance. Phytochemistry 40: 827-831