Natural polyketide 6-pentyl-2H-pyrone-2-one and its synthetic analogues efficiently prevent marine biofouling


Contents of supporting information


Contents

Pages Figure S1 Structures of the compounds obtained from Trichoderma spp. 
Scheme 1-6 Synthesis schemes of α-pyrone-derivatives 6-7 Table S1 The barnacle inhibition results of synthesized compounds S1 -S6 

Table S2

The antibiofilm formation results of α-pyrone-derivatives toward three marine bacteria 

Table S3

The stock and final concentrations that tested compounds were prepared for bioassays in 96 well-plate. 

Table S4

The stock and final concentrations of compound S5 for bioassays in 96 well-plate. 

Figure S2

Antibacterial results of S5 (Repeated data) toward Grampositive L. hongkongensis and S. cohnii, observed for 24h (blue) and 48h (orange) incubation. 

Figure S3

Antibiofilm formation results of S5 (Repeated data) toward Gram-positive L. hongkongensis and S. cohnii, observed for 48h incubation. 

Table S5

The MIC 50 and BIC 50 values of S5 for 48h incubation Figure S4 1 H-NMR spectrum of compound 1 Figure S5 13 C-NMR spectrum of compound 1 Figure S6 LR-ESI-MS spectrum of compound 1 Figure S7 1 H-NMR spectrum of compound 2 Figure S8 13 C-NMR spectrum of compound 2 Figure S9 LR-ESI-MS spectrum of compound 2 Figure S10 1 H-NMR spectrum of compound 3 Figure S11 13 C-NMR spectrum of compound 3 Figure S12 LR-ESI-MS spectrum of compound 3 Figure S13 1 H-NMR spectrum of compound 4 Figure S14 13 C-NMR spectrum of compound 4 Figure S15 LR-ESI-MS spectrum of compound 4 Figure S16 1 H-NMR spectrum of compound 5 Figure S17 13 C-NMR spectrum of compound 5 Figure S18 LR-ESI-MS spectrum of compound 5 Figure S19 1 H-NMR spectrum of compound 6 Figure S20 13 C-NMR spectrum of compound 6 Figure S21 LR-ESI-MS spectrum of compound 6 Figure S22 1 H-NMR spectrum of compound 7 Figure S23 13 C-NMR spectrum of compound 7 Figure S24 LR-ESI-MS spectrum of compound 7 Figure S25 1 H-NMR spectrum of compound 8 Figure S26 13 C-NMR spectrum of compound 8 Figure S27 LR-ESI-MS spectrum of compound 8 Figure S28 1 H-NMR spectrum of compound 9 Figure S29 13 C-NMR spectrum of compound 9 Figure S30 LR-ESI-MS spectrum of compound 9 Figure S31 1 H-NMR spectrum of compound 10 Figure S32 13 C-NMR spectrum of compound 10 Figure S33 LR-ESI-MS spectrum of compound 10 Figure S34 1 H-NMR spectrum of compound S1 Figure S35 13 C-NMR spectrum of compound S1 Figure S36 HSQC spectrum of compound S1 Figure S37 ESI-MS spectrum of compound S1 Figure S38 1 H-NMR spectrum of compound S2 Figure S39 13 C-NMR spectrum of compound S2 Figure S40 HSQC spectrum of compound S2 Figure S41 ESI-MS spectrum of compound S2 Figure S42 1 H-NMR spectrum of compound S3 Figure S43 13 C-NMR spectrum of compound S3 Figure S44 HSQC spectrum of compound S3 Figure S45 ESI-MS spectrum of compound S3 Figure S46 1 H-NMR spectrum of compound S4 Figure S47 13 C-NMR spectrum of compound S4 Figure S48 HSQC spectrum of compound S4 Figure S49 ESI-MS spectrum of compound S4 Figure S50 1 H-NMR spectrum of compound S5 Figure S51 13 C-NMR spectrum of compound S5 Figure S52 HSQC spectrum of compound S5 Figure S53 ESI-MS spectrum of compound S5 Figure S54 1 H-NMR spectrum of compound S6 Figure S55 13 C-NMR spectrum of compound S6 Figure S56 HSQC spectrum of compound S6 Figure S57 ESI-MS spectrum of compound S6 Identification of T. atroviride and T. reesei by ITS sequence DNA Geno typing data of species identification of the marine bacterium 

45-47


Isolation of secondary metabolites from the EtOAc extracts of T. reesei (TR-13-01) and T. atroviride (TA-13-01)

By using the conventional process of natural product isolation, four polyketides, T. atroviride (Figure S1). The structures of the obtained compounds were determined using spectroscopy (NMR and MS) and the data of respective studies. The detailed information of isolation procedure is described as follows. 
namely
EtOAc layer of TR-13-01 was partitioned between methanol (MeOH) and inclusive of n-hexane to afford MeOH layer (2.88 g) and n-hexane (460.1 mg), respectively. 
MeOH layer (2.88 g) was subjected to sephadex LH20 column and several fractions were collected. Among them, Fr. 8 (171. prepared by the reaction between propiolic acid and 1-nonyne and S2 (6-benzyl-2Hpyran-2-one) was prepared by using propiolic acid and 3-phenyl-1-propyne. S3 (6benzyl-4-methyl-2H-pyran-2-one) was prepared through reaction of butynoic acid and 3-phenyl-1-propyne, while S4 (6-heptyl-4-methyl-2H-pyran-2-one) was obtained from reaction between butynoic acid and 1-Nonyne. A reaction of phenylpropiolic acid with 3-phenyl-1-propyne was carried out to obtain compound S5 (6-benzyl-4phenyl-2H-pyran-2-one), while S6 (6-heptyl-4-phenyl-2H-pyran-2-one) was prepared by using 1-Nonyne with phenylpropiolic acid. The structures of these compounds were confirmed based on spectroscopic and literature data comparison. The synthesis schemes of S1-S6 are shown below. 

[S1]

[S2] 
[S3] 
[S4] 
[S5] 
[S6] 

Marine biofouling bacteria used in this study

To evaluate the role of marine bacteria in biofouling, we collected sea water samples offshore from Kaohsiung harbor and bacteria were purified as single strain by fourway purification method. Later on, biofilm formation ability of the strains was confirmed by biofilm formation assay (O'Toole 2011). Among them, five bacterial strains apparently showed biofilm formation in the test. 16S rRNA sequencing analysis (Geno typing data of species identification) of those biofilm-forming strains were performed to identify as Gram-positive Loktanella hongkongensis, 

Staphylococcus cohnii, Staphylococcus saprophyticus and Gram-negative

Alteromonas macleodii and Photobacterium angustum, respectively. The brief introduction of marine bacteria is described here. 
Loktanella hongkongensis is a Gram-positive, non-motile and rod-shaped bacterium which was isolated from a marine biofilm in the subtropical seawater of Hong Kong. 

L. hongkongensis can induce larval settlement and metamorphosis of a ubiquitous

polychaete tubeworm hydroids elegans (Lau et al. 2004, Liang et al. 2015). 
Staphylococcus cohnii is a Gram-positive, coagulase-negative and consisting of clustered cocci. S. cohnii can be found in clinical isolates and, have shown high levels of antibiotic resistance (Szczuka et al. 2016). Staphylococcus saprophyticus is a 
Gram-positive bacterium, can form biofilm in marine environment. This bacteria was isolated from glass slides submerged in seawater from Dae-Ho Dike, Korea (Kwon et al. 2002). Photobacterium angustum is a Gram-negative bacteria and abundant in marine and freshwater environments was formerly, known as Vibrio angustum (Matallana-Surget et al. 2012). Alteromonas macleodii is a Gram-negative biofilm forming bacteria, also found in seawater (Vandecandelaere et al. 2008). Table S2. The antibiofilm formation results of α-pyrone-derivatives toward three marine bacteria 

Antibiofilm formation activity of the compound

Bacterium 1 S1 S2 S3 S4 S5 S6 Staphylococcus saprophyticus - - - - - - - Photobacterium angustum - - - - - - - Alteromonas macleodii - - - - - - -
Note: (-) sign refers to no growth inhibition activity. Copper pyrithione (CuPT) was served as positive control and can inhibit more than 60% of bacterial biofilm at 0.4 µg/mL concentration. 
Table S3. The stock and final concentrations that tested compounds were prepared for bioassays in 96 well-plate. The active compound, S5 was tested toward five marine bacteria in antibacterial and antibiofilm formation systems with newly prepared compound concentration as listed in table S4. The incubation time for the both tests were kept for 24 and 48 h, respectively. All the data (treated and untreated bacteria) was measured in triplicates. Hz, H-4), 5.97 (1H, dd, J = 6.3, 0.7 Hz), 2.48 (2H, t, J = 7.6 Hz), 1.68-1.60 (4H, m), 1.40-1.21 (2H, m), 0.96-0.8 (3H, m); 13 C-NMR (CDCl 3 , 100 MHz) δ C 166.9, 163.0, 143.8, 113.2, 102.7, 33.9, 31.2, 26.7, 22.4, 14.0 (Dombray et al. 2010 11.6 Hz), δ H 3.41 (1H, s), 2.99 (3H, s), 2.52 (1H, dd, J = 14.0, 10.4 Hz), 2.52 (1H, d, J = 10.4 Hz), 1.88 (3H, d, J = 6 Hz), 1.85 (1H, dd, J = 14.0, 7.2 Hz), 1.17 (3H, s), 1.16 (3H, s); 13 C-NMR (100 MHz, CD 3 OD) δ C 211.7, 202.3, 169.8, 142.6, 139.6, 132.3, 120.0, 111.1, 111.1, 92.8, 75.0, 69.8, 64.8, 62.9, 50.5, 50.6, 49.7, 38.7, 26.8, 18.8, 10.9 (Neumann et al. 2007). Hz), 2.45 (1H, dd, J = 11.6 Hz), 2.22 (1H, dd, J = 14.0, 9.6 Hz), 1.89 (3H, d), 1.67 (1H, dd, J = 13.6, 6.8 Hz), 1.14 (3H, s), 1.12 (3H, s); 13 C-NMR (100 MHz, CD 3 OD) δ C 212.5, 201.2, 168.3, 143.1, 140.1, 132.4, 120.1, 111.7, 111.5, 90.8, 75.2, 68.7, 65.3, 62.4, 50.7, 49.1, 49.1, 41.7, 26.8, 19.0, 11.2 (Neumann, Abdel-Lateff, Wright, Kehraus, Krick and König 2007). 200.5, 170.2, 143.1, 140.1, 132.5, 120.1, 111.8, 108.1, 90.8, 75.3, 68.7, 67.9, 65.2, 51.9, 50.7, 40.8, 26.9, 19.0, 11.3 192.7, 163.7, 158.8, 144.7, 141.3, 130.7, 130.7, 128.9, 122.0, 115.9, 110.5, 19.0, 15.7, 7.7 (Cooney and Lauren 1999) (Tarus et al. 2003). 135.8, 127.1, 82.0, 75.5, 69.0, 55.5, 45.5, 41.7, 41.4, 26.1, 24.8, 25.2, 23.3, 15.4, 13.7 (Kasitu et al. 1992). 

Trichodermanone B (3):


10(E)-Cyclonerotriol (7): Yellowish viscous oil; [α]

25 D +11.3 (c 0.27, CHCl 3 ); UV (MeOH) λ max 241, 284, 362 nm; IR (Neat) ν max 3390,2961,1730,1454,1378,1260,1104, 1026 cm -1 ; (+)ESI-MS m/z 279.1 [M+Na] + (calcd. for C 15 H 28 O 3 ). 1 H-NMR (400 MHz, CD 3 OD) δ H 5.28 (1H, t, J = 7.2 Hz), 4.08 (2H, s), 2.12 (2H, m), 1.83 (1H, m), 1.77 (3H, s), 1.64 (2H, m), 1.61 (2H, m), 1.60 (1H, m), 1.48 (2H, m), 1.23 (3H, s), 1.14 (3H, s), 1.02 (3H, d, J = 6.8 Hz); 13 C-NMR (100 MHz, CD 3 OD) δ C 135.7, 129.2, 82.2, 75.7, 61.5, 55.8, 45.7, 42.3, 41.6, 26.3, 25.3, 24.8, 23.4, 21.7, 15.5 (Kasitu, ApSimon, Blackwell, Fielder, Greenhalgh and Miller 1992). 1 C-NMR (100 MHz, CD 3 OD) δ C 170.9, 167. 1, 157.8, 132.3, 132.3, 127.8, 116.3, 116.3, 60.2, 58.1, 46.0, 37.8, 29.6, 22.9 (Wang et al. 2012). 175.3, 171.7, 60.4, 52.1, 46.4, 29.1, 23.6, 15.7 (Lenman et al. 1997). 1 H NMR (400 MHz, CDCl 3 ) δ H 5.69 (1H, d, J = 3.7 Hz), 5.46 (1H, d, J = 5.7 Hz), 5.44 (1H, dd, J = 10.5, J 3,4 = 9.5 Hz), 5.37 (1H, dd, J = 5.9 Hz, J = 5.7 Hz), 5.08 (1H, dd, J = 10.1 Hz, J = 9.5 Hz), 4.87 (1H, dd, J = 10.5 Hz, J = 3.7 Hz), 4.16-4.34 (8H, m), 2.17 (3H, s), 2.11 (3H, s), 2.11 (3H, s), 2.11 (3H, s), 2.10 (3H, s), 2.10 (3H, s), 2.04 (3H, s), 2.01 (3H, s); 13 C NMR (100 MHz, CDCl 3 ) δ C 170.9, 170.7, 170.3, 170.3, 170.1, 169.8, 169.7, 169.5, 104.2, 90.1, 79.3, 75.8, 75.1, 70.4, 69.8, 68.7, 68.4, 63.8, 63.0, 61.9, 20.7-20.6 (8C) (Uenishi and Ueda 2008). 

Cyclo(D-tyrosine-D-proline (8): White wax; [α]


(3S,8aS)-3-Methylhexahydropyrrolo


Chemical data of α-pyrone-derivatives 6-Heptyl-2H-pyran-2-one (S1): Yellowish oil; [α]

25 D +21.7(c 0.15, CHCl 3 ); λ max 228, 300 nm; IR (Neat) ν max 3085, 2928, 2862, 1738, 1627, 1557, 1459, 1365, 1208, 1170, 1087, 802, 724; (+) for C 12 H 18 O 2 ). 1 H-NMR (400 MHz, CDCl 3 ) δ H 7. dd,J = 9.3,6.5 Hz),6.17 (1H,d J = 9.3,0.8 Hz), 5.99 (1H, dd J = 6.6, 0.8 Hz), 2.50 (2H, t, J = 7.6 Hz), 1.71-1.63 (2H, m), 1.35-1.27 (8H, m), 0.91 (3H, t); 13 C-NMR (CDCl 3 , 100 MHz) δ C 167.0, 163. 1, 143.9, 113.2, 102.8, 34.0, 31.8, 29.1, 29.1, 27.0, 22.7, 14.2 (Wickel et al. 2013). 

6-Benzyl-2H-pyran-2-one (S2): Yellowish oil; [α]

25 D +12.7(c 0.25, CHCl 3 ); λ max 240, 295 nm; IR (Neat) ν max 3069, 3027, 1721, 1628, 1609, 1555, 1493, 1443, 1133, 1029, 703; (+)ESI-MS m/z 186.2 (calcd. for C 12 H 10 O 2 ). 1 H NMR (CDCl 3 , 400 MHz) δ H 7.24-7.35 (m, 6H), 6.13 (dd, J = 12 Hz), 5.86 (dd, J = 8.8 Hz), 3.78 (2H, s); 13 C NMR (CDCl 3 , 100 MHz) δ C 163.3, 162.8, 156.0, 135.0, 129.1, 128.7, 127.8, 110.8, 106.2, 39.8, 21.3. (Thibonnet et al. 2002) 

6-Benzyl-4-methyl-2H-pyran-2-one (S3): Yellowish oil; [α]

25 D +17.2 (c 0.01, CHCl 3 ); λ max 248, 296 nm; IR (Neat) ν max 3069, 3032, 1721, 1638, 1443, 1224, 1071; (+)ESI-MS m/z 200.2 (calcd. for C 13 H 12 O 2, ). 1 H NMR (400 MHz, CDCl 3 ) δ H 7.26-7.36 (5H, m), 5.95 (1H, s), 5.73 (1H, s), 3.77 (2H, s), 2.08 (3H, s); 13 C NMR (100 MHz, CDCl 3 ) δ C 163. 6, 163.1, 156.3, 135.3, 129.4, 129.0, 127.4, 111.0, 106.5, 40.0, 21.6. (Cui et al. 2014) 

6-Heptyl-4-methyl-2H-pyran-2-one (S4): Yellowish oil; [α]

25 D -1.4 (c 0.35, CHCl 3 ); λ max 249, 295 nm; ν max 3077, 3032, 1716, 1654, 1273, 1224, 939, 703; (+)ESI-MS m/z 208.0 (calcd. for C 13 H 20 O 2 ). 1 H NMR (400 MHz, CDCl 3 ) δ H 5.91 (1H, s), 5.81, 2.43 (2H, t, J = 7.6 Hz), 2.0 (3H, s), 1.65-1.59 (2H, m), 1.29-1.24 (8H, m), 0.85 (3H, t, J = 6.8 Hz); 13 C NMR ( 100MHz, CDCl 3 ) δ C 165. 1, 163.5, 156.4, 110.7, 105.8, 33.7, 31.7, 29.0, 29.0, 27.0, 22.7, 21.5, 14.2. (Sun and Fürstner 2013) 

6-Benzyl-4-phenyl-2H-pyran-2-one (S5): Yellowish oil [α]

25 D +22.0 (c 0.12, CHCl 3 ); λ max 254, 270, 316 nm; ν max 3735, 3069, 3037, 1750, 1665, 1495, 1428, 1255, 1137, 955, 755, 695; (+)ESI-MS m/z 262.3 (calcd. for C 18 H 14 O 2 ). 1 H NMR (400 MHz, CDCl 3 ) δ H 7. 44-7.52 (5H, m), 7.31-7.36 (5H, m), 6.36 (s), 6.21 (s), 3.87 (2H,s); 13 C NMR (CDCl 3 , 100 MHz) δ C 40.2, 103.4, 108.5, 126.6, 127.3, scalesiae, and therefore TA-13-01 was identified as T. atroviride. 



  trichodermanone A, trichodermanone B, trichodermanone C, and sorbicillinol, (2-5); two sesquiterpens, namely 10(Z)-cyclonerotriol (6) and 10(E)cyclonerotriol (7); one diketopiperazine, namely cyclo(D-tyrosine-D-proline (8); and one disaccharide, namely1,3,4,6-tetra-O-acetyl-b-D-fractofuranosyl)2,3,4,6-tetra-Oacetyl-a-D-glucopyranoside (10), were isolated from T. reesei. Similarly, one polyketide, namely 6-pentyl-2H-pyrone-2-one (1), and one diketopiperazine, namely (3S,8aS)-3-methylhexahydropyrrolo[1,2-a]pyrazine-1,4-dione (9), were isolated from 


Figure S3 .

 S3 Figure S3. Antibacterial results of S5 (Repeated data) toward Gram-positive L. hongkongensis and S. cohnii, observed for 24h (blue) and 48h (orange) incubation. 


Figure S4 .

 S4 Figure S4. Antibiofilm formation results of S5 (Repeated data) toward Gram-positive L. hongkongensis and S. cohnii, observed for 48h incubation. 




  hongkongensis and S. cohnii with the MIC 50 values at 12.5 and 25.6 µg/mL, respectively, and also exhibited inhibitory activities toward the biofilm formation of both marine bacteria with the BIC 50 values at 6.25 and 1.56 µg/mL, respectively. No significant activity of S5 was observed toward Gram-positive S. saprophyticus, and Gram-negative A. macleodii and P. angustum.Chemical data of compoundsfrom TR-13-01 and TA-13-01 (1-10) 6-Pentyl-2H-pyran-2-one (1): Colorless viscous oil; [α] 25 D +10.7 (c 1.21, CHCl 3 ); UV (MeOH) λ max 352 nm; IR (Neat) ν max 2931, 1731, 1634, 1597, 1462 cm -1 ; (+)ESI-MS m/z 167.0 [M+H] + , 333.2 [2M+H] + (calcd. for C 10 H 14 O 2 ).1 H-NMR (400 MHz, CDCl 3 ) δ H 7.25 (1H, dd, J = 9.2, 6.8 Hz),6.15 (1H, d, J = 9.2 




  1 H-NMR (400 MHz, CD 3 OD) 7.26 (1H, dd, J = 14.4, 10.4 Hz), 6.37 (1H, dd, J = 14.4, 10.4 Hz), 6.20 (1H, dd, J = 14.4, 7.2 Hz), 6.43 (1H, d, J = 13.2 Hz), 4.24 (1H, d, J = 11.6 Hz), 4.24 (1H, d, J = 11.6 Hz), 3.84 (1H, d, J = 11.6 Hz), 3.84 (1H, d, J = 




  Yellowish viscous oil; [α] 25 D +79.5 (c 0.63, MeOH); UV (MeOH) λ max 249, 361 nm; IR (Neat) ν max 3382, 2931, 1732, 1633, 1600, 1561, 1383 cm -1 ; (-)ESI-MS m/z 407.5 [M-H] -, 815.3 [2M-H] -(calcd. for C 20 H 26 O 8 ). 1 H-NMR (400 MHz, CD 3 OD) δ H 7.28 (1H, dd, J = 14.8, 11.6 Hz), 6.38 (1H, d, J = 13.2 Hz), 6.35 (1H, dd, J = 13.2, 11.2 Hz), 6.21 (1H, dq, J = 14.8, 6.4 Hz), 3.99 (2H, d, brm), 3.70 (1H, d, J = 11.6 Hz), 3.51 (1H, s), 3.24 (3H, s), 2.95 (1H, d, J = 11.6 




  λ max 249, 361 nm; IR (Neat) ν max3380, 2936, 1731, 1633, 1600, 1561, 1446, 1384 cm -1 ; (+)ESI-MS m/z 417.1 [M+Na] + (calcd. for C 19 H 24 O 8 ). 1 H-NMR (400 MHz, CD 3 OD) δ H 7.28 (1H, dd, J = 14.8, 11.2 Hz), 6.38 (1H, dd, J = 15.2, 11.2 Hz), 6.34 (1H, d, J = 14.8 Hz),6.22 (1H, dq, J = 14.8, 6.4 Hz), 4.20 (1H, d, J = 11.6 Hz), 3.89 (1H, d, J = 11.6 Hz), 3.36 (1H, s), 3.28 (2H, s), 2.62 (1H, dd, J = 9.6, 7.2 Hz), 2.13 (1H, dd, J = 14.4, 9.6 Hz),1.90 (3H, d, J = 6.4 Hz), 1.72 (1H, dd, J = 18.4, 11.6 Hz), 1.15 (3H, s), 1.13 (3H, s);13 C-NMR (100 MHz, CD 3 OD) δ C 212.2, 




  .3 (c 0.34, CHCl 3 ); UV (MeOH) λ max 242, 288, 361 nm; IR (Neat) ν max 3402, 2961, 1730, 1455, 1379, 1260, 1099, 1024 cm -1 ; (+)ESI-MS m/z 279.1 [M+Na] + , 535.3 [M+2Na] + (calcd. for C 15 H 28 O 3 ). 1 H-NMR (400 MHz, CD 3 OD) δ H 5.41 (1H, t, J = 7.2 Hz), 3.91 (2H, s), 2.11 (2H, m), 1.84 (1H, m), 1.67 (3H, d, J = 6.8 Hz), 1.59 (1H, m), 1.62 (2H, m), 1.54 (2H, m), 1.51 (2H, m), 1.23 (3H, s), 1.13 (3H, s), 1.01 (3H, s); 13 C-NMR (100 MHz, CD 3 OD) δ C 




  c 0.57, CHCl 3 ); UV (MeOH) λ max 252 nm; IR (Neat) ν max 1660, 3271 cm -1 ; (+)ESI-MS m/z 283.2 [M+Na] + (calcd.for C 14 H 16 N 2 O 3 ). 1 H-NMR (400 MHz, CD 3 OD) δ H 7.04 (1H, d, J = 8.4 Hz), 7.04 (1H, d, J = 8.4 Hz), 6.70 (1H, d, J = 8.4 Hz), 6.70 (1H, d, J = 8.4 Hz), 4.36 (1H, brt, J = 4.8 Hz), 4.05 (1H, m), 3.59 (1H, m), 3.39 (1H, m), 3.08 (2H, dd, J = 14.0, 4.8 Hz), 1.82 (2H, m), 2.10 (1H, m), 1.23 (1H, m); 




  .1 (c 0.23, MeOH); UV (MeOH) λ max 295, 309, 357 nm; IR (Neat) ν max 3285, 2961, 1655 cm -1 ; (+)ESI-MS m/z 191.0 [M+Na] + (calcd. for C 8 H 12 N 2 O 2 ). 1 H-NMR (400 MHz, CD 3 OD) δ H 4.26 (1H, dd, J = 8 Hz), 4.19 (1H, q, J = 13.3 Hz), 3.52 (2H, m), 2.32 (1H, m), 2.02 (1H, m), 1.95 (1H, m), 2.01(1H, m), 1.38 (3H, d, J = 6.8 Hz); 13 C-NMR (100 MHz, CD 3 OD) δ C 




  c 0.63, CHCl 3 ); UV (MeOH) λ max 241 nm; IR (Neat) ν max 1718 cm -1 ; (+)ESI-MS m/z 701.4 [M+H] + (calcd. for C 28 H 38 O 19 ) 


Figure S6 .

 S6 Figure S6. LR-ESI-MS spectrum of 6-pentyl-2H-pyran-2-one (1) 


Figure S38. 1 H

 1 Figure S38. 1 H NMR spectrum of 6-benzyl-2H-pyran-2-one (S2) 


Figure S42. 1 H

 1 Figure S42. 1 H NMR spectrum of benzyl-4-methyl-2H-pyran-2-one (S3) 


Figure S46. 1 HFigure S50. 1 HFigure S54. 1 H

 111 Figure S46. 1 H NMR spectrum of 6-heptyl-4-methyl-2H-pyran-2-one (S4) 




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  




  


(6-helptyl-2H-pyran-2-one) was

  Structures of the compounds obtained from Trichoderma spp. 


	eluted by methanol to water 45:55 to obtained 5 (1.0 mg). Fr. 6 from n-hexane layer was separated by normal phase column with hexane and chloroform in ration of 5:1 to afford compound 10 (3.8 mg). subjected to sephadex LH20 column and several fractions were collected. F10 was separated by reverse-phase HPLC and eluted with MeOH to Water in ratio of 30:70 to obtained compound 9 (1.1 mg). Moreover, n-hexane layer was subjected to normal phase column and eluted by step gradient from pure hexane to ethyl acetate and methanol (5:1) to obtain several fractions. Fr. 2 was loaded to normal phase column and eluted with Hex: CHCl 3 5:1 to CHCl 3 : MeOH 5:1 to obtained compound 1 (4.6 mg). 1 2 3 4 5 6 7 8 9 10 α-Pyrone derivatives were synthesized by gold-catalysed coupling reaction. Two catalysts, 5% chloro (triphenylphosphine)gold(I) and 5% silver trifluromethanesulphonate (AgOTf) were used in the synthesis (Luo et al. 2011). All the mixtures were dissolved in dichloromethane (DCM) and reactions were carried Figure S1. Synthesis schemes of pyrone-derivatives out at ambient temperature for 12 h. Compound S1
	MeOH layer (2.06 g) and n-hexane (545.8 mg) respectively. MeOH layer (2.06 g) was

3 mg) was subjected to reverse-phase silica gel column chromatography and eluted by the ratio of MeOH to water 60:40 to afford subfraction Fr. 8-2 and Fr. 8-3. Sub-fraction 8-2 and 8-3 were further separated by reverse-phase High Performance Liquid Chromatography (HPLC) and eluted by methanol to water 45:55 to obtained 2 (1.8 mg), 3 (2.1 mg) and 4 (2.4) mg. Fr. 4 (54.1 mg) was separated by HPLC and eluted with MeOH to Water in ratio of 40:60 to obtained compound 6 (1.5 mg) and 7 (1.2 mg). Fr. 11 was separated by HPLC and eluted with MeOH to Water in ratio of 10:90 to obtained compound 8 (1.0 mg). n-Hexane layer was separated by normal phase column and Fr. 3 was obtained by eluting n-hexane to ethyl acetate in ratio of 10:1. Fr. 3 was purified by HPLC and EtOAc layer of TR-13-01 was partitioned between MeOH and of n-hexane to afford 
Table S1 .

 S1 The barnacle inhibition results of synthesized compounds S1 -S6 


	Compounds Conc.	Total	Swim Still Settlement Death Settlement
		(µg/mL)	Individual					rate %
	S1	5	39	0	0	33	6	86.6
	S2	5	41	0	4	5	32	12.3
	S3	5	36	3	0	33	0	91.6
	S4	5	42	3	0	39	0	86.6
	S5	5	45	1	1	12	31	26.6
	S6	5	43	0	5	11	27	25.5

Table S5 :

 S5 The MIC 50 and BIC 50 values of S5 for 48h incubation Compound S5 exhibited inhibitory activity against the growth of L. 


	S5	Antibacterial activity	Antibiofilm formation
	Bacterial Name	(MIC 50 )	activity (BIC 50 )
	L. hongkongensis	12.5 µg/mL	6.25 µg/mL
	S. cohnii	25 µg/mL	1.56 µg/mL
	S. saprophyticus	-	-
	A. macleodii	-	-
	P. angustum	-	-
	Note:		



  ). 


	Trichodermanone A (2): Yellowish viscous oil; [α]	25 D +72.0 (c 0.33, MeOH); UV
	(MeOH) λ max 252, 361 nm; IR (Neat) ν max 3360, 2922, 2852, 1731, 1635, 1599, 1564,
	1446, 1385 cm -1 ; (+)ESI-MS m/z 431.7 [M+Na] + , 839.7 [2M+Na] + (calcd. for
	C 20 H 26 O 8 ).	



  (Neumann, Abdel-Lateff, Wright, Kehraus, Krick and König 2007).CHCl 3 ); UV (MeOH) λ max 315, 364 nm; IR (Neat) ν max3444, 2927, 1731, 1613, 1446, 1359, 1227, 1153cm -1 ; (+)ESI-MS m/z 233.0 [M+Na] + , 255.0 [M+Na] + (calcd. for C 14 H 16 O 3 ). 1 H-NMR (400 MHz, CDCl 3 ) δ H 13.59 (OH), 7.50 (2H, m), 6.95 (1H, m), 6.31 (2H, m), 2.22 (3H, s), 2.15 (3H, s), 1.91 (3H, d, J = 6 Hz); 13 C-NMR (100 MHz, CDCl 3 ) δ C 


	Sorbicillin (5): Yellowish viscous oil; [α] 25 D -16.3 (c 0.14,

DNA Geno Typing data of species identification of the marine bacterium Loktanella hongkongensis

  
	AGACGTCGGGGGGCTTGCCCTTCGGTGTCACACCTAACGGATTAAGCATT
	CCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGG
	GGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGA Staphylococcus saprophyticus
	ACCTTACCAACCCTTGACATCCTCGGACCGTCGCAGAGATGCGTCTTTCTC CTTGCTCCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACC
	GTAAGAGACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGT TACCTATAAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAA
	GAGATGTTCGGTTAAGTCCGGCAACGAGCGCAACCCACACCCCTAGTTGC CATTTGGAACCGCATGGTTCTAAAGTGAAAGATGGTTTTGCTATCACTTAT
	CAGCATTTGGTTGGGCACTCTAGGGGAACTGCCCGTGATAAGCGGGAGGA AGATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAG
	AGGTGTGGATGACGTCAAGTCCTCATGGCCCTTACGGGTTGGGCTACACA GCAACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTG
	CGTGCTACAATGGCATCAACAATGGGTTAATCCCTAAAAGATGTCTCAGT AGACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAA
	TCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAATCGCTAGTAAT TGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGGTTTCG
	CGCGTAACAGCAT GCTCGTAAAACTCTGTTATTAGGGAAGAACAAATGTGTAAGTAACTGTGC
	ACATCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCA
	Staphylococcus cohnii GCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAA
	CTTGCTCTTTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCT AGCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAAC
	ACCTATAAGACTGGAATAACTCCGGGAAACCGGGGCTAATGCCGGATAAC CGTGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAAAGT
	ATTTAGAACCGCATGGTTCTAAAGTGAAAGATGGTTTTGCTATCACTTATA GGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCA
	GATGGACCCGCGCCGTATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGG GTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCG
	CAACGATACGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGA TGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATG
	GACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAAT AGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATT
	GGGCGAAAGCCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGG AAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATT
	ATCGTAAAACTCTGTTATTAGGGAAGAACAAATGTGTAAGTAACTATGCA GACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAAC
	CGTCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAG GCGAAGAACCTTACCAAATCTTGACATCCTTTGAAAACTCTAGAGATAGA
	CCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAA GCCTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAG
	GCGCGCGTAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACC CTCGTGTCGTGAGATGTTGGTTAAGTCCCGC
	GTGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAAAGTG
	GAATTCCATGTGTAGCGGTGAAATGCGCAGAGATATGGAGGAACACCAGT
	GGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAAGCGTG Alteromonas macleodii
	GGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAG TGAGCGTCAGTCTTTGGCCAGGGAGTCGCCTTCGCCACTGATGTTCCTCCA
	TGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAA GATATCTACGCATTTCACCGCTACACCTGGAATTCCACTCCCCTCTCCAAG
	GCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGA ACTCTAGTCTGCCAGTTCTAAATGACCATCCCAGGTTGAGCCCGGGGCTTT
	CGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCG CACATCTAGCTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCG
	AAGAACCTTACCAAATCTTGACATCCTTTGACAACTCTAGAGATAGAGCC ATTAACGCTCGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGC
	TTCCCCTTCGGGGGACAAGTGACAGGTGGTGCATGGCTGTCGTCAGCTCG CGGTGCTTCTTCTGTTGTTAACGTCACGGCTAGCAGGTATTAACTACTAAC
	TGTCGTGAGATGTTGGGTTAAGTCCCGCAAC TTTTCCTCACAACTGAAAGTGCTTTACAACCCGAAGGCCTTCTTCACACAC
	Photobacterium angustum GCGGCATGGCTGCATCAGGGTTTCCCCCATTGTGCAATATTCCCCACTGCT
	CAGATTGATAGCTTGCTATCAATGCTGACGAGCGGCGGACGGGTGAGTAA GCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCTGATCTTC
	TCGGGCTTAGTGGCGGACGGGTTAGTAACGCGTGGGAACGTGCCCTTCTC TGCCTGGGAATATGCCCTGATGTGGGGGATAACTATTGGAAACGATAGCT CTCTCAGAACAGCTAGAGATCGTCGCCTTGGTAAGCCGTTACCTTACCAA
	TACGGAATAGTCCCGGGAAACTGGGTTTAATACCGTATACGCCCTTCGGG AATACCGCATAATCTCTTCGGAGCAAAGAGGGGGACCTTCGGGCCTCTCG CTAGCTAATCTCACTTGGGCCTCTCTTTGCGCCGGAGCCGAAGCCCCGTTT
	GGAAAGATTTATCGGGGAAGGATCGGCCCGCGTCTGATTAGATAGTTGGT CGTCAGGATTAGCCCAGGTGGGATTAGCTTGTTGGTGGGGTAATGGCTCA GGTCCGAAGACATTATGCGGTATTAGCAGTCGTTTCCAACTGTTATCCCCC
	GGGGTAACGGCCTACCAAGTCGACGATCAGTAGCTGGTTTGAGAGGATGA CCAAGGCAACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGA TCGCAAAGGCAAGTTCCCAAGCATTACTCACCCGTCCGCCACTCGTCATCT
	TCAGCAACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGC ACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGC TCTAGCAAGCTAGAAATGTTACCGTTCGACTTGCATGG
	AGTGGGGAATCTTAGACAATGGGCGCAAGCCTGATCTAGCCATGCCGCGT ACAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCT
	GAGTGATGAAGGCCCTAGGGTCGTAAAGCTCTTTCGCTGGGGAAGATAAT TCGGGTTGTAAAGTACTTTCAGTTGTGAGGAAGGCGTTGATGTTAATAGCT
	GACGGTACCCAGTAAAGAAACCCCGGCTAACTCCGTGCCAGCAGCCGCGG TCAGCGTTTGACGTTAGCAACAGAAGAAGCACCGGCTAACTCCGTGCCAG
	TAATACGGAGGGGGTTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCGC CAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGT
	GTAGGCGGATTGGAAAGTTGGGGGTGAAATCCCGGGGCTCAACCCCGGA AAAGCGCATGCAGGTGGTCTGTTAAGCAAGATGTGAAAGCCCGGGGCTCA
	ACGGCCTCCAAAACTATCAGTCTAGAGGTCGAGAGAGGTGAGTGGAATTC ACCTCGGAACAGCATTTTGAACTGGCAGACTAGAGTACTGTAGAGGGGGG
	CGAGTGTAGAGGTGAAATTCGTAGATATTCGGAGGAACACCAGTGGCGA TAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCG
	AGGCGGCTCACTGGCTCGATACTGACGCTGAGGTGCGAAAGCGTGGGGA GTGGCGAAGGCGGCCCCCTGGACAGATACTGACACTCAGATGCGAAAGC
	GCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCC GTG

The GenBank accession number of the ITS sequence is MT781339. The GenBank accession number of the ITS sequence of T. reessei is MT781338. 
Supplementary Information

Spectrum data of compounds from Trichoderma spp.
Mo Aqib, Raza Khan, Bo-Wei Wang, Yih-Yu Chen, Ting-Hsuan Lin, Hsiu- Chin Lin, Yu-Liang Yang, Ka-Lai Pang, Chih-Chuang Liaw
Natural polyketide 6-pentyl-2H-pyrone-2-one and its synthetic analogues efficiently prevent marine biofouling