*2.5. SAR Analysis*

To date, 61 napyradiomycin derivatives have been discovered and elucidated [42]. In general, the chemical structure of the napyradiomycins consists of a semi-napthoquinone core, a prenyl unit attached at C-4a that is cyclized to form a tetrahydropyran ring in all the napyradiomycins reported here (Figure 1), and a monoterpenoid substituent attached at C-10a. The variation of the chemical structure of napyradiomycins is mainly due from the monoterpenoid subunit (10-carbon), which can be linear (as the napyradiomycins **1**–**7**) or cyclized to a 6-membered ring (as napyradiomycins **8**–**12**). As mentioned above, napyradiomycins from the strains PTM-029 and PTM-420 differ by a methyl group at position 7 instead of hydrogen, respectively (Figure 1). Overall, the antifouling activities of (**1**–**12**) ranked in descending order are summarized as: (**1**) > (**8** and **11**) > (**4**) for antibacterial activity, (**4**) > (**5** and **6**) > (**8** and **11**) > (**3** and **7**) for antimicrofouling, and (**10**) > (**9** and **12**) > (**1**) > (**8** and **11**) > (**12**) > (**3** and **7**) for antimacrofouling activity. Interestingly, all the napyradiomycins with antibacterial activity and the three napyradiomycins with highest antimicrofouling activity were isolated from the strain PTM-420 with a hydrogen atom at position 7. Conversely, the methyl containing napyradiomycins from PTM-029 generally had higher antimacrofouling activity.

Based on the biosynthetic scheme for napyradiomycins reported by Moore and co-workers [68], (**1**), (**2**), (**4**–**6**), (**8**), (**11**) lack the methylation of flaviolin by the methyltransferase NapB5. Similar behavior occurs with the 10-carbon monoterpenoid subunit, which is linear in almost all of the napyradiomycins with more potent antibacterial and antimicrofouling activities, with the exception of napyradiomycins (**8** and **11**), and cyclized to a 6-membered ring in most of the most active antimacrofouling napyradiomycins, except for napyradiomycins (**1**) and (**3** and **7**). Therefore, our antifouling results (micro and macro) sugges<sup>t</sup> a correlation with this biosynthetic feature. Napyradiomycins (**8** and **11**) stand out as having the ability to inhibit both micro and macrofouling, this feature appears to be related with the presence of a bromine substitute at C-16 (Figure 1). MNP bromide derivatives seem to play an important role as antifouling agents, as 2-furanone bromine derivatives [26] and bromopyrrole alkaloids from oroidin family [27,28].

### **3. Materials and Methods**

### *3.1. General Experimental Procedures*

The optical rotations were measured using a Bellingham+Stanley (Berlin, Germany), model ADP410 Polarimeter with a 5 dm cell. UV/VIS spectra were recorded using an Ultraspect 3100 Pro Amersham Bioscienses spectrophotometer (Champaign, IL, USA) with a path length of 1 cm, and IR spectra were recorded on a Perkin Elmer Spectrum Two FT-IR Spectrometer (Santa Clara, CA, USA). 1H and 2D NMR spectral data were recorded at 400 or 600 MHz in CDCl3 containing Me4Si as internal standard on Bruker Advance and Bruker BioSpin spectrometers respectively (Ettlingen, Germany). 13C NMR spectra were acquired at 100 MHz on a Bruker Advance spectrometer. High resolution ESI-TOF mass spectra were obtained through acquired services using an Agilent 6230 Accurate-Mass TOFMS spectrometer (Santa Clara, CA, USA) by the mass spectrometry facility at the Department of Chemistry and Biochemistry at the University of California, San Diego, La Jolla, CA. Low-resolution LC/MS data were measured through acquired services at MARINOVA, CIIMAR, Portugal, using a Thermo Finnigan Surveyor HPLC System (Thermo Fisher Scientific, Needham, MA, USA), coupled with Mass Spectrometry LCQ Fleet™ Ion Trap Mass Spectrometer (Thermo Fisher Scientific, Needham, MA, USA), with reversed-phase C18 column (Phenomenex Luna, 100 mm × 1.0 mm, 5 μm), ACN:H2O 10–100% gradient, with 0.1% formic acid, at a flow rate of 0.7 mL/min.

### *3.2. Collection and Isolation of Marine-Derived Actinomycetes*

Sediment samples were collected in June 2012 o ff shore of the Madeira Archipelago of Portugal. Strain PTM-029 was isolated from samples collect near Madeira Island at 728 m using a dredge, while PTM-420 was isolated from samples collected from Desertas Island at 15 m on SCUBA. The sediments were inoculated using a heat-shock method: wet sediment (c. 0.5 g) was diluted with 2 mL of sterile seawater (SSW). After mixing, the diluted samples were allowed to settle for few minutes, heated to 55 ◦C for 6 min. 50 μL of the top layer was spread on an agar plate, with seawater based medium SWA (18 g agar per L) with the antifungal cycloheximide (100 μl/L).

Inoculated Petri dishes were incubated at RT (c. 25–28 ◦C) and monitored periodically over 6 months for actinomycete growth. The PTM-029 and PTM-420 colonies were successively transferred onto new seawater based A1 medium (10 g starch, 4 g yeas<sup>t</sup> extract, 2 g peptone per L) until the attaining of pure strain. PTM-029 and PTM-420 were grown in liquid culture (without agar) and cryopreserved in 10% (v/v) glycerol at −80 ◦C.

### *3.3. Phylogenetic Analysis of Strains PTM-029 and PTM-420*

Strains PTM-029 and PTM-420 were cultured in 4mL of A1 medium, with agitation (200 rpm) at 25 ◦C for 7 days. Genomic DNA was isolated using the Wizard ® Genomic DNA Purification Kit (Promega, Madison, WI, USA) protocol for Gram positive bacteria. The manufacturer recommendations were followed, though with longer incubation periods of the lytic enzyme (i.e., lysozyme) and the RNase solution to obtain su fficient amounts of genomic DNA. The 16S rRNA gene was amplified using the primers 27F (5--AGAGTTTGATCCTGGCTCAG-3-) and 1492R (5--TACGGCTA CCTTGTTACGACTT-3-) [41,69] and purified using SureClean PCR cleanup kit (BioLine, London, UK), using the protocol provided by the manufacturer. Purified PCR reactions were cycle- sequenced with the primers listed above at STABVIDA, Lda (www.stabvida.net), using ABI BigDye ® Terminator v3.1 Cycle Sequencing Kit (Needham, MA, USA). Purified products were run on an ABI PRISM ® 3730xl Genetic Analyzer (Needham, MA, USA) and sequence traces were edited using Sequencing Analysis 5.3.1 from Applied Biosystems ™ (Needham, MA, USA). The sequence was compared to the GenBank database by the blastn algorithm.

PTM-029 and PTM-420 sequences have been deposited in GenBank under accession numbers KP869059 and KP869064 respectively, available at www.ncbi.nlm.nih.gov/genbank.

### *3.4. Growth Conditions and Crude Extract Production*

The actinomycetes (strains PTM-029 and PTM-420) were grown in 20 Erlenmeyer flasks with 2L capacity, each containing 1 L of seawater based A1 medium with agitation (200 rpm) at 30 ◦C. After seven days of incubation, the culture was extracted thrice with half volume of EtOAc and evaporated to dryness in vacuum to yield ~1.0 g of crude extracts.

### *3.5. Isolation of Napyradiomycins*

The PTM-029 (~1.0 g) and PTM-420 crude extracts (~1.0 g) were fractionated by silica flash chromatography, eluted with step gradients of isooctane/EtOAc followed by EtOAc/MeOH. A mixture of five napyradiomycins from PTM-029 and seven from PTM-420 eluted with the 8:2 and 6:4 fractions of isooctane/EtOAc, respectively, and further isolated by reversed phase HPLC (Phenomenex Luna, 250 mm × 4.6 mm, 5 μm, 100 Å, 1.5 mL/min, UV 210, 250 and 360 nm) using a gradient solvent system from 70% to 100% CH3CN in water (0.1% TFA) over 90 min to yield napyradiomycins (**1**, 12.86 mg), (**2**, 5.04 mg), (**3** and **7**, 6.80 mg), (**4**, 11.36 mg), (**5** and **6**, 1.67 mg), (**8** and **11**, 5.21 mg), (**9** and **12**, 7.40 mg), and (**10**, 13.20 mg) as orange oils. The data for structural characterization is described in the Supplementary Materials.
