Biosynthetic origin of natural products isolated from marine microorganism–invertebrate assemblages

Simmons et al. 10.1073/pnas.0709851105.

Supporting Information

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SI Figure 8
SI Figure 9
SI Figure 10
SI Methods and Data




SI Figure 8

Fig. 8. (A) Partial schematic of capillary LC-MS instrument. (B) Photograph of the actual capillary column (Lower) adjacent to a penny (Right).





SI Figure 9

Fig. 9. Nanospray-FT-ICRMS analysis of extract containing iejimalide A (8). (A) Detection of iejimalide A in a methylene chloride/methanol extract from a mixed assemblage of cyanobacteria collected in Papua New Guinea. (B) Detection of iejimalide A from an HPLC fractionated sample. Hydroxylated or otherwise oxidized iejimalide derivatives are annotated as B and C in Inset. The calculated mass increase for the addition of one oxygen atom is 15.9950 Da and for two oxygen atoms is 31.9900 Da.





SI Figure 10

Fig. 10. MALDI-TOF-MS analysis of a single filament of Lyngbya bouillonii showing the presence of apratoxin A (9), a new one carbon shortened analog, and sodium adducts.





SI Methods and Data

General Experimental Procedures. Optical rotations were measured on a JASCO P1010 polarimeter. IR and UV spectra were recorded on a Nicolet IR-100 FT-IR and Beckman-Coulter DU800 spectrophotometer, respectively. NMR spectra were recorded on Varian Unity 300 and 500-MHz spectrometers with the solvent CDCl3 (dH at 7.26, dC at 77.0) used as an internal standard. HPLC was carried out by using a Waters system consisting of a Rheodyne 7725i injector, two 515 pumps, a module, and a 996 photodiode array detector, running under control of Excalibur Software. ESI mass spectra were obtained on a Thermo-Finnigan LCQ Advantage mass spectrometer, FTMS on a Thermo-Finnegan LTQ-FTMS, and MALDI on a Voyager MALDI instrument. TLC grade (10-40 mm) Si gel was used for vacuum chromatography. All solvents were purchased as HPLC grade.

Isolation of Iejimalide A (8). A collection of the cyanobacterium Lyngbya sp. was obtained on 06/30/2002 from the Milne Bay Province of Papua New Guinea (10°36.938S ´ 150°45.400E). »500 ml of fresh cyanobacterial tissue was collected by hand from a depth of 50 ft by using SCUBA. This material was strained to remove excess seawater and preserved in an EtOH/sea water (1:1) solution at 20°C until shipment back to the labs at SIO/UCSD. Extraction of the material by CH2Cl2:CH3OH (2:1) yielded a crude oil (2.0 gm), which was subjected to NP Si VLC fractionation. The resultant nine fractions (A-I) were then submitted to a cancer cell cytotoxicity assay. Fraction H (25% CH3OH in EtOAc) was shown to be toxic against the OVCAR-5 cell line. Repetitive RP-HPLC (70% CH3CN in water, Phenomenex Jupiter RP-18 column, 250 ´ 10 mm) yielded a pure oil with a LR LC-ESI pseudomolecular ion m/z 701.68 [M+Na]+ (Thermo-Finnigan LCQ Advantage Max, scanning m/z 15-2,000; HPLC conditions used a Phenomenex Fusion RP18 250 ´ 4.6 mm; gradient 7:3 CH3N/H2O to 100% CH3N over 30 min at 0.8 ml/min), which combined with NMR data analysis gave the molecular formula C40H58O7N2. Detailed comparison of the UV, MS, and NMR data as well as optical rotation values with the literature indicated that this active cytotoxin was identical with iejimalide A (8) as reported in ref. 1.

Isolation of Curazole (11). Following the m/z 372 peak observed first by MALDI of filaments and subsequently by LTQ FTMS (m/z 372.2372, +1.1 mmu deviation) of a CH2Cl2/MeOH (2:1) extract of cultured Lyngbya majuscula strain "3L," curazole (11) was partially purified by VLC (5% EtOAc/hexanes elution) followed by HPLC (10% EtOAc/hexanes, 11 ml/min, Phenomenex Primesphere, 500 ´21.2 mm, 10 mm, UV detection at 254 nm, 6 fractions taken from 4-60 min). The resulting fractions were analyzed by LCMS (70-100% MeOH/H2O over 30 min; Lichrospher RP-100 C18, 125 ´4 mm, 5 mm; retention time = 23 min). The early-eluting fraction (44-132 ml retention volume on the above chromatography) containing the m/z 372 peak was subjected to brief treatment with CH2N2 (Et2O) followed by HPLC (isocratic 80% MeOH/H2O, 4 ml/min, Phenomenex Synergi-Hydro 250 ´21.2 mm, 4 mm, UV detection at 240 nm) to give pure curazole (11, 0.4 mg). The 1H NMR (CDCl3, 500 MHz) and LCQ LCMS data matched those of authentic curazole (2).

Culture of Cyanobacteria and Preparation for Microscopy. Lyngbya majuscula strain "3L" was collected at Las Palmas beach near the CARMABI Research Station in Curacao, The Netherlands. Lyngbya majuscula strain "JHB" was collected in Hector's Bay, Jamaica. Both cultures were subsequently isolated to a monoclonal culture by using standard microbiological isolation techniques. Approximately 3 g of L. majuscula strains "3L" and "JHB" were separately inoculated into 2-liter Fernbach flasks containing 1 liter of SWBG11 medium. These static cultures were grown at 28° C under uniform illumination (4.67 mmol photon/sec/m2) with a 16-h/8-h light/dark cycle for 62 days. For MALDI analysis, individual filaments were removed from these cultures and rinsed in deionized H2O and placed directly onto the MALDI target plate. For DAPI stain analysis, multiple filaments were removed from the cultures and placed on a microscope slide, and a single drop of VECTASHIELD Mounting Medium with DAPI was applied. Photomicrographs were taken at ´1,000 magnification by using a Zeiss Axioskop (filter set #2 , emission 420 nm+).

Cyanobacterial Collection, Taxonomy, DNA Extraction, 16S rRNA Gene PCR-Amplification, and Cloning. Cyanobacterial specimens were collected by SCUBA or snorkeling at shallow depths (<70 ft) and rinsed in filtered seawater. The samples collected for genetic analysis were immediately preserved in Falcon tubes with 5 ml of RNA-later (Ambion) at -20°C. Genomic DNA was extracted from 40 mg of cleaned algal tissue by using the Wizard Genomic DNA Purification Kit (Promega, cat. no. A1120) according to the manufacturer's specifications. The isolated genomic DNA was further purified by using a Genomic-tip 20/G kit from Qiagen (cat. no. 10223).

The 16S rDNA genes were PCR-amplified from isolated DNA using the primer set, 106F and 1509R, as described in ref. 1. The PCR reactions were performed in an Eppendorf Mastercycler gradient as follows: initial denaturation for 2 min at 95°C, 30 cycles of amplification: 20 sec at 95°C, 20 sec at 50°C, and 15 sec at 72°C, and final elongation for 3 min at 72°C. The reaction volumes were 25 ml containing 0.5 ml of DNA (50 ng), 2.5 ml of 10´ PfuUltra II reaction buffer, 0.5 ml of dNTP mix (25 mM each dATP, dTTP, dGTP, and dCTP), 0.5 ml of each primer (10 mM), 0.5 ml of PfuUltra II fusion HS DNA polymerase (cat. no. 600760), and 20.25 ml of dH2O. The PCR products were subcloned by using the Zero Blunt TOPO PCR Cloning Kit from Invitrogen (cat. no. K2800-20SC) into the pCR-Blunt II TOPO vector and then transformed into TOPO cells and cultured on LB-kanamycin plates. Plasmid DNA was isolated by using the QIAprep Spin Miniprep Kit from Qiagen (cat. no. 27106) and sequenced with pCR-Blunt II TOPO vector specific primers M13F/M13R and internal middle primers 359F and 781R as described in ref. 3.

The bi-directional 16S rRNA gene sequences from individual 16S clones were combined and the resulting consensus sequence visually inspected to confirm reliability and sequence coverage. BLAST analysis was performed in GenBank/EMBL/DDBJ (www.ncbi.nlm.nih.gov) and the Ribosomal Database Project II (http://rdp8.cme.msu.edu/html) to determine the phylogeny of the species yielding these 16S rRNA genes. All cyanobacterial morphological characterization was made in accordance with traditional phycological and bacteriological taxonomic systems (4-6).

Bioinformatics Analysis of Curacin A (10) and Bryostatin (2) Gene Clusters. The curacin A pathway nucleotide sequence was cut after the stop codon of the CurN ORF. This downstream nucleotide sequence was translated by using ORF finder (www.ncbi.nlm.nih.gov/gorf/gorf.html). The ensuing ORFs were aligned with the GenBank database by using BlastP (www.ncbi.nlm.nih.gov/blast/Blast.cgi?PAGE=Proteins&PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on). The B. neritina KSa sequence was used to search the NCBI (National Center for Biotechnology Information) BLAST server. BlastP analysis revealed that the KSa sequence has 98% identity to the first KS domain in BryB and 96% identity to the first KS domain in BryC.

General Capillary LC Protocol. Nano-capillary columns were prepared by drawing a 360-mm O.D., 100-mm I.D. deactivated, fused silica tubing (Agilent) with a Model P-2000 laser puller (Sutter Instruments) (Heat: 330, 325, 320; Vel, 45; Del, 125) and were packed at ~600 psi to a length of ~10 cm with C18 reverse-phase resin suspended in methanol. The column was equilibrated with 90% of solvent A (water, 0.1% AcOH) and loaded with 10 ml of a 1:100 dilution in water of the crude extract (<0.1 mg of extract) by flowing 90% of solvent A and 10% of solvent B (CH3CN, 0.1% AcOH) at 20 ml/min for 5 min, 15 ml/min for 3 min, and 10 ml/min for 12 min. At 12 min the flow rate was increased to 200 ml/min and infused into a split-flow so that ~200-500 nl/min went through the capillary column while the remainder of the flow was diverted to waste. A gradient for eluting iejimalide was established with a time-varying solvent mixture [(min, % of solvent A): (20, 90), (23, 75), (43,10)] and directly electrosprayed into the LTQ-FT MS inlet (source voltage, 1.8 kV; capillary temperature, 180°C). The resulting data were analyzed by using QualBrowser (Thermo-Finnigan).

FT-ICR-MS of Iejimalide A (8). A 1-ml sample of iejimalide A was diluted 100-fold with a 50% methanol/1% formic acid/water solution. Infusion into the Fourier transform ion cyclotron resonance mass spectrometer was performed by nano-electrospray ionization with a Biversa Nanomate (Advion Biosystems) on a Finnigan LTQ-MS (Thermo-Electron) running Tune Plus software version 1.0 (Thermo). The Biversa Nanomate parameters were the following: airgap between chip, no; contact closure, after; voltage timing, after; equalization delay, 0 s; aspiration delay, 0 s; contact closure delay, 1 s; voltage timing delay, 0 s; aspiration depth, 0.6 nm; gas pressure, 30-60 psi; voltage, 1.40-1.80 kV, positive ion mode; begin spray sensing, 30 s; sample volume, 5 ml. The ionized sample was first visualized on the LTQ-MS broadband spectrum and then the ions were passed to the FT-ICR-MS where the final spectrum was obtained and the mass of iejimalide A was identified. The FT-ICR-MS parameters were the following: number of microscans set at 1-5 and mass inject time kept at 8,000 ms. For the autogain values, the full MS target was set to 3 ´106, the SIM target was set to 1 ´ 105. The signal was maximized using the automated tuning option. Mass ranges were typically kept at m/z 150-2,000. Typical resolution of the mass spectrometer was set at 100,000. The resulting spectra was then analyzed on QualBrowser software version 1.4 SR1 (Thermo).

MALDI/Epifluorescence Sponge Protocol. Sample collection and storage. Dysidea herbaceae was collected in Papua New Guinea in 2002 and provided by Prof. P Crews and K. Tenney (Department of Chemistry, University of California, Santa Cruz). It was stored frozen in 1:1 EtOH/seawater.

Cryosectioning. The sample was thawed and precut, and then embedded in 1´ Dulbecco's PBS and placed in the cryostat at -20°C. Once the tissue and embedding block had frozen completely, 10- to 20-mm-thick coronal sections were cut and mounted onto a semithawed MALDI plate. The plate was desiccated at 38°C for 10 min.

Epifluorescence imaging. The MALDI plate containing the sponge tissue slices was imaged on a Zeiss Axioskop. Two sets of images were collected on the same region of the sponge tissue slice at ´400 (Image B: Zeiss filter set #14, emission 590 nm+; Image C: Zeiss filter set #2, emission 420 nm+).

General protocol for MALDI imaging. After gathering photomicrographs of the samples using a Nikon digital camera (2 megapixels), a matrix composed of 35 mg/ml a-cyano-hydroxycinnamic acid, 35 mg/ml DHB, 78% CH3CN, and 0.1% TFA was coated onto the MALDI plate by using an airbrush (www.paascheairbrush.com). The Bruker MSP 96 anchor target plate containing sample and matrix was then placed in an empty Petri dish until the sample was analyzed.

MALDI-MS and imagining. The Bruker MSP 96 anchor target plate containing the sample was introduced into a Microflex Bruker Daltonics mass spectrometer and repeated side-to-side strokes until an even, thin crystalline layer occluded the background of the plate (7). The sample was run on the MALDI MS (Bruker microflex outfitted with flexImaging 2.0 software) in positive mode, with 100-mm raster intervals in XY and approximately 35-62% laser power. Briefly, a photomicrograph of the sample to be imaged by mass spectrometry was loaded onto the flexImaging command window. Three teach points were selected to align the background image with the sample target plate. After the target plate calibration was complete, the AutoXecute command was used to analyze the samples: 350-2,000 m/z window, reflector mode, positive ion mode, 100-mV electronic gain, and real-time smooth off. The instrument was calibrated externally by using a digest of BSA as a standard. In the resulting spectra, different colors were designated to the masses associated with curacin A (10) and jamaicamide B (16), allowing the visualization of their respective distribution in the rastering window.

MALDI-TOF of Apratoxin (9). Single filaments of Lyngbya bouillonii were removed from the culture flask by using blunt-nose tweezers and placed in a Petri dish containing deionized water as prewash. The filaments were then laid down on the MALDI MSP 96 Anchor Target Plate and manually spotted with a matrix composed of 35 mg/ml a-cyano-hydroxycinammic acid, 35 mg/ml DHB, 0.1% TFA in 78% aq. CH3CN, and H2O (7). The target plate was desiccated for 5 min at 38°C. The remaining MALDI parameters were identical to those described in the general procedure.

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This Article

  1. Published online before print February 4, 2008, doi: 10.1073/pnas.0709851105 PNAS March 25, 2008 vol. 105 no. 12 4587-4594
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