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).
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.
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
Table T3: NMR data of compound 3 (CDCl3, 400MHz)
δC DEPT δH mult. (J in Hz) HMBC COSY 1H-1H NOESY 1H-1H
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
Table T4: NMR data of compound 4 (CDCl3, 400MHz)
δC DEPT δH mult. (J in Hz) HMBC COSY 1H-1H NOESY 1H-1H
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
Table T5: NMR data of compound 5 (CDCl3, 400MHz)
δC DEPT δH mult. (J in Hz) HMBC COSY 1H-1H NOESY 1H-1H
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
Table T6: NMR data of compound 6 (CDCl3, 400MHz)
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
log[compound 1] (µM)
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
log[compound 2] (µM)
Sett
led c
yprids (
%)
-2 -1 0 1 20
20
40
60
80
100
replicate #1
replicate #2
log[compound 2] (µM)
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).
A. amphitrite B. perforatus
-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
log[compound 1] (µM)
Mort
alit
y (%
)
0.0 0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 1] (µM)
Mort
alit
y (%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 6] (µM)
Mort
alit
y (%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 1] (µM)
Mort
alit
y (%
)
TBTO
CuPT
Compound6
Compound1
Figure S17: Toxicity of compounds towards barnacle stage I/II nauplii (Bars represent the means ± SD).
A. amphitrite B. perforatus
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
log[compound 1] (µM)
Mort
ality
(%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 1] (µM)
Mort
ality
(%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 6] (µM)
Mort
ality
(%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 6] (µM)
Mort
ality
(%
)
0.0 0.5 1.0 1.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 2] (µM)
Mort
ality
(%
)
Compound 2 Not determined
Figure S18: Toxicity of compounds towards barnacle stage VI nauplii (Bars represent the means ± SD).
A. amphitrite B. perforatus
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
log[compound 1] (µM)
Mort
ality
(%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 1] (µM)
Mort
ality
(%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 6] (µM)
Mort
ality
(%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
log[compound 6] (µM)
Mort
ality
(%
)
0.5 1.0 1.5 2.0 2.50
20
40
60
80
100
replicate #1
replicate #2
repliacte #3
log[compound 2] (µM)
Mort
ality
(%
)
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