Standardized chemical synthesis of Pseudomonas aeruginosa pyocyanin

Graphical abstract

1. 100 mg PMS added to 100 ml of 10 mM TRIS-HCl in a 100 ml capacity thin-stemmed round bottomed glass flask 2. pH to 7.4 using 0.5 M NaOH 3. The reaction mixture kept 25 cm from daylight Phillips TLD 18 W/54 fluorescent tube light for 2.5 h. 4. Chloroform added to the reaction mixture in a separation funnel kept in a fume hood 5. The lower chloroform (organic) phase with pyocyanin transferred to a pear-shaped flask 6. Nitrogen bubbled through the contents of the pear-shaped flask till a blue pyocyanin sludge remains after all the chloroform has been evaporated 7. Blue pyocyanin sludge resuspended in 50 ml chloroform 8. Pyocyanin chloroform solution acidified with 50 ml of 0.1 M HCl -pyocyanin becomes red 9. 50 ml of chloroform added 10. A few drops of 500 mM NaOH added to convert red pyocyanin to blue pyocyanin 11. Chloroform extraction done twice with 50 ml chloroform 12. Pyocyanin chloroform solution kept at À20 8C freezer overnight 13. Chloroform evaporated by gentle nitrogen insufflation 14. Pyocyanin resuspended in small amounts of more chloroform 15. Pyocyanin chloroform (concentrated) solution transferred to a vertical Pyrex glass tube 16. Chloroform evaporated 17. Hexane wash done by adding hexane (pyocyanin is insoluble in hexane), swirling the tube, and aspirating the hexane out 18. Chloroform (1 ml) added to pyocyanin 19. Hexane (5-8 ml) added SLOWLY, DROP by DROP to the pyocyanin chloroform solution 20. 10 min waiting period mandated 21. Pyocyanin crystallized automatically 22. Pyocyanin in hexane and chloroform in 50 ml polypropylene tubes is centrifuged at 5000 rpm for 10 min Table 1 Procedural differences between the original procedure and our procedure.
Procedure step (S) First description [1] Our protocol [2,[4][5][6][7]  Purification steps + ++++ 17 Total time for extraction and purification ++++ + The key procedural differences between the original protocol and our exhaustively revised protocol are tabulated here. Our procedure substantially decreases the total time taken for the synthesis, extraction and purification of pyocyanin, and increases the pyocyanin proportion produced in the final step. Our procedure involves multiple extraction/re-extraction steps, and more purification steps. It removes a rotary evaporation step and multiple lyophilization steps.
23. Supernatant pipetted out from the 50 ml tubes and discarded 24. Lower phase with the pyocyanin crystals transferred to a polypropylene syringe (without a piston) with its tip compactly fitted into a the top nozzle of a filter apparatus containing a type EH 0.5 m filter 25. Pyocyanin crystals trapped by filter 26. Methanol elution into a screw-capped glass bottle done 27. Methanol evaporated by nitrogen bubbling 28. Small amount of methanol used to dissolve the pyocyanin 29. Pyocyanin methanol solution kept at À20 8C freezer overnight 30. Methanol evaporated by nitrogen bubbling 31. Pyocyanin reconstituted in 0.5 ml of chloroform 32. Silica glass TLC plate activated: (i) Excess silica from 3 edges scraped out (ii) TLC plate kept vertically in 10 ml methanol in a glass cage without paper lining. The most jagged/damaged edge placed inferior and in contact with methanol in the glass cage. (iii) Glass plate used to cover the glass cage (iv) TLC plate is taken out of the methanol (and the glass cage) as soon as the solvent (methanol) front reaches 2 cm from the top edge (of the TLC plate) (v) TLC plate kept outside the glass cage, inside the hood, for 10 min (vi) TLC plate kept on a folded A4 size paper and heated with silica side up in a microwave at minimum power setting for 5 min (vii) TLC plate heated in the microwave at power setting 3 for 10 more min (viii) TLC plate kept in the dark or inside a desiccator till use 33. Sample applicator (Camag Nanomat) fitted with a applicator syringe fitted with a 1 ml (preferably) or a 5 ml glass tip, utilized to apply 0.25 ml pyocyanin chloroform solution to the silica part of the silica glass TLC plate 34. TLC plate with loaded pyocyanin kept in chloroform methanol mixture (12.5 ml: 12.5 ml) inside a glass cage with a paper lining 35. TLC plate removed from the glass cage when the solvent front reaches 2 cm from the top edge 36. TLC plate computer-scanned and image saved (Fig. 1) 37. Silica layer with the pyocyanin carefully scraped from the glass part of the TLC plate 38. Scraped silica with pyocyanin dissolved in 2 ml methanol in a screw capped glass bottle 39. Silica pyocyanin methanol solution transferred to small glass tubes (compatible in the slots of the centrifuge to be described soon) 40. Glass tubes centrifuged twice in the centrifuge available inside the walk-in refrigerator (the sealing lid is not shut, only the topmost trap lid is shut) 41. Quantification using spectrophotometer: 318 nm 239 nm (vii) The typically used absorption wavelength of pyocyanin for calculation of Molar concentration is at 318 nm, because that is where the highest peak is seen (viii) The Extinction Coefficient E of pyocyanin in methanol at 318 nm is 30,199. 5 (ix) Therefore the millimolar concentration of pyocyanin is 1 mM

Additional information
The Gram-negative bacterium P. aeruginosa is the most common cause of chronic and recurrent lung infections in patients with cystic fibrosis whose sputa contain copious quantities of P. aeruginosa toxin, pyocyanin. Pyocyanin is a blue-green, phenazine pigment, which triggers tissue damage mainly via its redox cycling, and induction of reactive oxygen species [2]. Pyocyanin is expensive to purchase commercially, and the sellers are reticent in giving out the minutiae of the extraction procedure. The classical method of preparation involves multi-step bacterial culturing and solvent transfer extraction, as first spelt out [3]. A modified chemical synthesis was first presented (1979) with a few details in the journal ''Analytical Biochemistry'' [1]. However, the methodology was not described in detail, and the technical intricacies not posited. This paper does exactly that. We present an extensively revamped version of the modified procedure, spelling out the steps in detail.
The blue band on the TLC plate is formed by pyocyanin (Fig. 1), and the pink band by impurities. The final purified pyocyanin peaked at 718 nm, 318 nm and 239 nm (Fig. 2), typical for pyocyanin synthesized from bacterial cultures.
We have extracted pyocyanin by both microbial extraction and chemical synthesis, and we acknowledge and endorse the relative ease, safety, and convenience of using the chemical synthesis described here. Our changes to the previous procedure involve the following parameters (Table 1): (i) Initial concentration of PMS used (ii) Starting pH (iii) Photolysis time (iv) Rotary evaporation (v) Lyophilization of photolysed products (vi) Sequential acidification/alkalinization, and chloroform extraction-purification (vii) Hexane precipitation from chloroform, filtration, methanol elution from filter TLC plate (viii) Chloroform:methanol proportion for TLC plate equilibration (ix) Solvent for pyocyanin loading on TLC plate (x) Chloroform:methanol proportion for pyocyanin elution (xi) Lyophilization after pyocyanin elution (xii) Pyocyanin yield from TLC plate Purification steps (xiii) Total time for extraction and purification With the pyocyanin extracted using the protocol above, we had generated the following data (previously published).
(1) Pyocyanin reacted with reduced glutathione (GSH) non-enzymatically at 37 8C resulting in the production of red-brown products (480 nm absorption peaks) [2]. The reaction between pyocyanin and GSH was concentration-dependent on reduced glutathione, but not on pyocyanin. Catalase circumvented the reaction. The presence of a previously unidentified non-enzymatic GSHdependent metabolic pathway for pyocyanin has thus been identified. The formation of H 2 O 2 as an intermediate and the thiol group in GSH is crucial to this reaction. (2) Pyocyanin treatment induced a dose-dependent reduction in fenestrations in isolated LSECs [4]. In isolated LSECs, pyocyanin induced a catalase-preventable loss of sieve plate organization with significant reductions in LSEC porosity. In the intact liver [4], within 30 min of intraportal injection of a pathophysiologically relevant dose of pyocyanin, there was a significant reduction in porosity The ''original'' method, and the most common method of pyocyanin preparation involves complicated multi-step solvent extractions from P. aeruginosa cultures [3]. We present a clear-cut chemical synthesis procedure, positing the smallest of steps in exquisite detail. Our exhaustively overhauled procedure (Table 1) markedly decreased the total time taken for the synthesis, extraction and purification [1]. Importantly, our procedure substantially increased the proportion of pure pyocyanin obtained during the final step, although it entailed more purification steps. It also cut short a rotary evaporation step and multiple lyophilization steps at 2 different points of time. Crucially, pyocyanin could be synthesized with ease from phenazine methosulfate, without using infectious bacteria. It is also ''naturally endotoxin-free''. We hope that this paper will benefit interested scientists immensely.

Source of support
National Health and Medical Research Council (NHMRC).

Conflicts of interest
None to report.