Chemistry Techniques

2.10.1 Conversion of desmethylundecylprodiginine to undecylprodiginine

HPLC purified desmethylundecylprodiginine (from 4 R5 agar plates) was resuspended in 1.5 mL of CH2Cl2 containing 48% aqueous fluoroboric acid (0.026 mL) at 0 °C under N2. Then, 3 x 0.05 mL portions of TMSCHN2(2.0M in diethyl ether) were added dropwise at intervals of 20 minutes to the reaction mixture. After the addition was complete, the solution was stirred at 0 °C for a further 30 minutes, followed by a further 30 min stirring at room temperature. The organic layer was separated and evaporated to dryness. The residue was resuspended in 1 mL of MeCN and analysed by LC-MS/MS.

2.10.2 Purification of prodiginines fromS. coelicolorextract

Prodiginine antibiotics were extracted from eight agar plate cultures of S. coeli- colorW31/pOSV556mcpG grown as described above. The solvent was evaporated; the residue was resuspended in CHCl3and washed 3 times with deionised water (adjusted to pH 3 with aqueous HCl). The organic layer was separated and evaporated to dryness. The prodiginines were purified from this crude extract by flash column chromatography on basic alumina eluting with 10% EtOAc/Hexane. Fractions containing prodiginines

were combined and concentratedin vacuo. The residue was partitioned between CHCl3 and deionised water (pH 3) to convert the prodiginines to their HCl salts. The organic layer was separated and evaporated to dryness.

Partialy purified metacycloprodigiosin was further purified using an Agilent 1200 Series preparative HPLC instrument equipped with a binary pump and a diode array detector. The prodiginine mixture obtained from the alumina column was resus- pended in 6 mL of MeOH and passed through a 0.4 μm filter. 5.5 mL of the filtrate were purified on an Agilent Zorbax C18 column (150 x 21.2 mm, 5μm) using the gradient elution profile in Table 2.10 and the compound with a retention time of 7 minutes was collected.

Table 2.10 HPLC conditions used for purification of metacycloprodigiosin on Agilent Zorbax C18

column (150 x 21.2 mm, 5μm)

Time (min) H2O/HCl (pH 3) (%) MeOH (%) Flow Rate (mL/min)

0 30 70 20.0 5 35 75 20.0 7.5 20 80 20.0 10 10 90 20.0 11.25 10 90 20.0 11.50 30 70 20.0

The fraction was extracted with CHCl3 and washed with deionised water (ad- justed to pH 3 with aqueous HCl). The organic layer was separated and evaporated to dryness. The residue was further purified using the same HPLC instrument on an Agilent Zorbax Phenyl column (250 x 21.2 mm, 7μm) using the conditions in Table 2.11 and the compound with a retention time of 6 minutes was collected and the fraction was extracted with CHCl3. The organic layer was separated and evaporated to dryness to

afford metacycloprodigiosin as a pink solid. The identity of this compound as metacy- cloprodigiosin was confirmed by CD and 1H NMR (Bruker Avance spectrometer, 400 MHz, CDCl3) spectroscopic comparisons with authentic samples of metacycloprodigi- osin and streptrorubin B isolated fromS. longispororuberM-3 andS. coelicolorM511, respectively.

Table 2.11 HPLC conditions used to purify metacycloprodigiosin on an Agilent Zorbax Phenyl column

(250 x 21.2 mm, 7μm)

Time (min) H2O/HCl (pH 3) (%) MeOH (%) Flow Rate (mL/min)

0 24 76 20.0

10 18 82 20.0

11 10 90 20.0

13 10 90 20.0

16.50 24 76 20.0

2.10.2.1 Circular Dichroism (CD) spectroscopy

Metacycloprodigiosin purified from S. longispororuber/S. coelicolor

W31/pOSV556mcpG and streptorubin B purified from S. coelicolor M511 were ana- lysed by CD spectroscopy on a Jasco J-815 CD Spectrometer. Samples were dissolved in MeOH and the CD spectrum from 700 to 200 nm of each solution was measured in a 1 mm path length cuvette. Instrument settings were as follows: resolution 0.4 nm, band width 2.0 nm, sensitivity 200 mdeg, response 1 s, speed 200 nm/min.

2.10.3 Purification of Desmethylundecylprodiginine

An Agilent 1100 instrument equipped with a quaternary pump and variable wavelength detector set to monitor absorbance at 490 nm was used to purify desmethy-

lundecylprodiginine. 1 mL of filtered extract was injected onto an Agilent Zorbax C18 column (100 x 21.2 mm, 5μm) using the gradient elution profile shown in Table 2.12 and the compound with a retention time of 20 minutes was collected. The fraction was extracted with CHCl3and the organic layer was separated and washed with deionised water (adjusted with HCl to pH 3). The organic layer was separated and evaporated to dryness.

Table 2.12 HPLC conditions used in desmethylundecylprodiginine purification

Time (min) H2O/HCl (pH 3) (%) Acetonitrile (%) Flow Rate (mL/min)

0 20 80 5.0

10 20 80 5.0

20 0 100 5.0

30 0 100 5.0

2.10.4 LC-MS

Liquid chromatography – Mass Spectrometry (LC-MS) was used to analyse culture extracts/culture supernatants for prodiginines, actinorhodin-related metabolites and coelichelin. 50 L of each extract/supernatant was injected onto an Eclipse XDB- C18 column (150 x 4.6 mm, 5μm, column temperature 25 ºC, Agilent) connected to an Agilent 1100 instrument equipped with a binary pump and a diode array detector. The HPLC outflow was connected via a splitter (10% flow to MS, 90% flow to waste) to a Bruker HCT+ mass spectrometer equipped with an electrospray source operated in positive ion mode with parameters as follows: nebulizer flow 40 psi, dry gas flow 10.0 L/min, dry temperature 300 ºC, capillary – 4 kV, skimmer 40V, capillary exit 106 V, ion charge control target (ICC) 100,000, spectral averages 3. The gradient elution profile

used to analyse 2-UP, MBC, prodiginines and analogues is shown in Table 2.13 and that used to analyse actinorhodin and coelichelin is shown in Table 2.14.

Table 2.13 Gradient elution profile used in LC-MS analyses of prodiginine production Time (mins) Water (pH3 with HCl or

0.1% formic acid MeCN or MeOH Flow Rate

0 50 50 1.0

1 50 50 1.0

4 25 75 1.3

21 20 80 1.4

23 50 50 1.0

Table 2.14 Gradient elution profile used in LC-MS analyses of actinorhodin and coelichelin production Time (mins) Water + 0.1% formic acid MeOH + 0.1% formic acid Flow Rate

0 50 50 1.0

1 50 50 1.0

4 25 75 1.0

21 20 80 1.0

23 50 50 1.0

2.10.5 High Resolution Mass Spectrometry

High Resolution Mass Spectrometry (HRMS) was done bu University of War- wick Mass Spectrometry Service and was used to confirm the molecular formulae of the purified compounds. HRMS was carried out on an ESI-TOF-MS (Bruker MicroTOF) using direct infusion.