Unusual odd-electron fragments from even-electron protonated prodiginine precursors using positive-ion electrospray tandem mass spectrometry

  • Kan Chen
  • Nalaka S. Rannulu
  • Yang Cai
  • Pat Lane
  • Andrea L. Liebl
  • Bernard B. Rees
  • Christophe Corre
  • Gregory L. Challis
  • Richard B. ColeEmail author


Reports of anticancer and immunosuppressive properties have spurred recent interest in the bacterially produced prodiginines. We use electrospray tandem mass spectrometry (ES-MS/MS) to investigate prodigiosin, undecylprodiginine, and streptorubin B (butyl-meta-cycloheptylprodiginine) and to explore their fragmentation pathways to explain the unusual methyl radical loss and consecutive fragment ions that dominate low-energy collision-induced dissociation (CID) mass spectra. The competition between the formation of even-electron ions and radical ions is examined in detail. Theoretical calculations are used to optimize the structures and calculate the energies of both reactants and products using the Gaussian 03 program. Results indicate that protonation occurs on the nitrogen atom that initially held no hydrogen, thus allowing formation of a pseudo-seven-membered ring that constitutes the most stable ground state [M+H]+ structure. From this precursor, experimental data show that methyl radical loss has the lowest apparent threshold but, alternatively, even-electron fragment ions can be formed by loss of a methanol molecule. Computational modeling indicates that methyl radical loss is the more endothermic process in this competition, but the lower apparent threshold associated with methyl radical loss points to a lower kinetic barrier. Additionally, this characteristic and unusual loss of methyl radical (in combination with weaker methanol loss) from each prodiginine is useful for performing constant neutral loss scans to quickly and efficiently identify all prodiginines in a complex biological mixture without any clean-up or purification. The feasibility of this approach has been proven through the identification of a new, low-abundance prodigiosin analog arising from Hahella chejuensis.


Neutral Loss Fragmentation Pathway Tandem Mass Spectrum Prodigiosin Electrospray Tandem Mass Spectrometry 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Williams, R. P. Biosynthesis of Prodigiosin, a Secondary Metabolite of Serratia marcescens. Appl. Microbiol. 1973, 25, 7102–7109.Google Scholar
  2. 2.
    Wasserman, H. H.; McKeon, J.; Santer, U. V. Studies Related to the Biosynthesis of Prodigiosin in Serratia marcescens. Biochem. Biophys. Res. Commun. 1960, 3, 146–149.CrossRefGoogle Scholar
  3. 3.
    Rapoport, H.; Holden, K. G. The Synthesis of Prodigiosin. J. Am. Chem. Soc. 1962, 82, 5510–5511.CrossRefGoogle Scholar
  4. 4.
    Gerber, N. N. Prodigiosin-like Pigments. CRC Crit. Rev. Microbiol. 1975, 3, 469–485.CrossRefGoogle Scholar
  5. 5.
    Laatsch, H.; Kellner, M.; Weyland, H. Butyl-meta-cycloheptylprodiginine — a revision of the structure of the former ortho-isomer. J. Antibiot. 1991, 44, 187–191.Google Scholar
  6. 6.
    Gerber, N. N. Prodigiosin-like Pigments from Actinomadura (Nocardia) pelletieri. J. Antibiot. 1971, 24, 636–640.Google Scholar
  7. 7.
    Kawauchi, K.; Shibutani, K.; Yagisawa, H.; Kamata, H.; Nakatsuji, S.; Anzai, H.; Yokoyama, Y.; Ikegami, Y.; Moriyama, Y.; Hirata, H. A Possible Immunosuppressant, Cycloprodigiosin Hydrochloride, Obtained from Pseudoalteromonas denitrificans. Biochem. Biophys. Res. Commun. 1997, 237, 543–547.CrossRefGoogle Scholar
  8. 8.
    Bennett, J. W.; Bentley, R. Seeing Red: The Story of Prodigiosin. Adv. Appl. Microbiol. 2000, 47, 1–32.CrossRefGoogle Scholar
  9. 9.
    Furstner, A. Chemistry and Biology of Roseophilin and the Prodigiosin Alkaloids: A Survey of the Last 2500 Years. Angew. Chem. Int. Ed. Engl. 2003, 42, 3582–3603.CrossRefGoogle Scholar
  10. 10.
    Cerdeno, A. M.; Bibb, M. J.; Challis, G. L. Analysis of the Prodiginine Biosynthesis Gene Cluster of Streptomyces coelicolor A3(2): New Mechanisms for Chain Initiation and Termination in Modular Multienzymes. Chem. Biol. 2001, 8, 817–829.CrossRefGoogle Scholar
  11. 11.
    Llagostera, E.; Soto-Cerrato, V.; Montaner, B.; Perez-Tomas, R. Prodigiosin Induces Apoptosis by Acting on Mitochondria in Human Lung Cancer Cells. Ann. N. Y. Acad. Sci. 2003, 1010, 178–181.CrossRefGoogle Scholar
  12. 12.
    Manderville, R. A. Synthesis, Proton-Affinity and Anti-Cancer Properties of the Prodigiosin-Group Natural Products. Curr. Med. Chem. Anticancer Agents. 2001, 1, 195–218.CrossRefGoogle Scholar
  13. 13.
    Montaner, B.; Castillo-Avila, W.; Martinell, M.; Ollinger, R.; Aymami, J.; Giralt, E.; Perez-Tomas, R. DNA Interaction and Dual Topoisomerase I and II Inhibition Properties of the Anti-Tumor Drug Prodigiosin. Toxicol. Sci. 2005, 85, 870–879.CrossRefGoogle Scholar
  14. 14.
    Perez-Tomas, R.; Montaner, B.; Llagostera, E.; Soto-Cerrato, V. The Prodigiosins, Proapoptotic Drugs with Anticancer Properties. Biochem. Pharmacol. 2003, 66, 1447–1452.CrossRefGoogle Scholar
  15. 15.
    Montaner, B.; Perez-Tomas, R. The Prodigiosins: A New Family of Anticancer Drugs. Curr. Cancer Drug Targets. 2003, 3, 57–65.CrossRefGoogle Scholar
  16. 16.
    Soto-Cerrato, V.; Llagostera, E.; Montaner, B.; Scheffer, G. L.; Perez-Tomas, R. Mitochondria-Mediated Apoptosis Operating Irrespective of Multidrug Resistance in Breast Cancer Cells by the Anticancer Agent Prodigiosin. Biochem. Pharmacol. 2004, 68, 1345–1352.CrossRefGoogle Scholar
  17. 17.
    Stepkowski, S. M.; Nagy, Z. S.; Wang, M. E.; Behbod, F.; Erwin-Cohen, R.; Kahan, B. D.; Kirken, R. A. PNU156804 Inhibits Jak3 Tyrosine Kinase and Rat Heart Allograft Rejection. Transplant. Proc. 2001, 33, 3272–3273.CrossRefGoogle Scholar
  18. 18.
    Mortellaro, A.; Songia, S.; Gnocchi, P.; Ferrari, M.; Fornasiero, C.; D’Alessio, R.; Isetta, A.; Colotta, F.; Golay, J. New Immunosuppressive Drug PNU156804 Blocks IL-2-dependent Proliferation and NF-kappa B and AP-1 Activation. J. Immunol. 1999, 162, 7102–7109.Google Scholar
  19. 19.
    Furstner, A.; Grabowski, J.; Lehmann, C.W. Total Synthesis and Structural Refinement of the Cyclic Tripyrrole Pigment Nonylprodigiosin. J. Org. Chem. 1999, 64, 8275–8280.CrossRefGoogle Scholar
  20. 20.
    Dairi, K.; Tripathy, S.; Attardo, G.; Lavallee, J. F. Two Step Synthesis of the Bipyrrole Precursor of Prodigiosins. Tetrahedron Lett. 2006, 47, 2605–2606.CrossRefGoogle Scholar
  21. 21.
    Stanley, A. E.; Walton, L. J.; Kourdi Zerikly, M.; Corre, C.; Challis, G. L. Elucidation of the Streptomyces coelicolor Pathway to 4-Methoxy-2,2′-bipyrrole-5-carboxaldehyde, an Intermediate in Prodiginine Biosynthesis. Chem. Commun. (Camb.) 2006, 38, 3981–3983.CrossRefGoogle Scholar
  22. 22.
    Mo, S. J.; Sydor, P. K.; Corre, C.; Alhamadsheh, M. M.; Stanley, A. E.; Haynes, S. W.; Song, L.; Reynolds, K. A.; Challis, G. L. Elucidation of the Streptomyces coelicolor Pathway to 2-Undecylpyrrole, a Key Intermediate in Undecylprodiginine and Streptorubin B Biosynthesis. Chem. Biol. 2008, 15, 137–148.CrossRefGoogle Scholar
  23. 23.
    Odulate, O.; Barona-Gomez, F.; Corre, C.; Challis, G. L. Unpublished results.Google Scholar
  24. 24.
    Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A. J.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A.J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. J. B.; Ortiz, V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, M. W.; Wong, W.; Gonzalez, C.; Pople, J. A. Gaussian 03, Revision B. 03; Gaussian Inc.: Pittsburgh, PA, 2003.Google Scholar
  25. 25.
    Becke, A. D. Density-Functional Thermochemistry: III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98, 5648–5652CrossRefGoogle Scholar
  26. 26.
    Perdew, J. P.; Wang, Y. Pair-Distribution Function and Its Coupling-Constant Average for the Spin-Polarized Electron Gas. Phys. Rev. B. 1992, 46, 12947.CrossRefGoogle Scholar
  27. 27.
    Rajkarnikar, A.; Kwon, H. J.; Suh, J. W. Role of Adenosine Kinase in the Control of Streptomyces Differentiations: Loss of Adenosine Kinase Suppresses Sporulation and Actinorhodin Biosynthesis While Inducing Hyperproduction of Undecylprodigiosin in Streptomyces lividans. Biochem. Biophys. Res. Commun. 2007, 363, 322–328.CrossRefGoogle Scholar
  28. 28.
    Sigsby, M. L.; Day, R. J.; Cooks, R. G. Fragmentation of Even Electron Ions: Protonated Ketones and Ethers. Org. Mass. Spectrom. 1979, 14, 273.CrossRefGoogle Scholar
  29. 29.
    Fenselau, C.; Brown, P.; Patterson, D. G. Fragmentation of [M+H]+ to Form Ion Radicals Following Chemical Ionization. Spectroscopy (Amsterdam). 1983, 2, 348–351.Google Scholar
  30. 30.
    Bowie, J. H.; Stringer, M. B.; Duus, F.; Lawesson, S. O.; Larsson, F. C. V.; Madsen, J. O. Fragmentations of Organic Negative-Ions, Mercapto Acids and Esters: A Reinvestigation. Aust. J. Chem. 1984, 37, 1619–1624.CrossRefGoogle Scholar
  31. 31.
    Nikolic, D.; Li, Y.; Chadwick, L. R.; Grubjesic, S.; Schwab, P.; Metz, P.; van Breemen, R.B. Metabolism of 8-Prenylnaringenin, a Potent Phytoestrogen from Hops (Humulus lupulus), by Human Liver Microsomes. Drug Metab. Dispos. 2004, 32, 272–279.CrossRefGoogle Scholar
  32. 32.
    Levsen, K.; Schiebel, H. M.; Terlouw, J. K.; Jobst, K. J.; Elend, M.; Preiss, A.; Thiele, H.; Ingendoh, A. Even-Electron Ions: A Systematic Study of the Neutral Species Lost in the Dissociation of Quasi-Molecular Ions. J. Mass Spectrom. 2007, 42, 1024–1044.CrossRefGoogle Scholar
  33. 33.
    Williams, J. P.; Nibbering, N. M.; Green, B. N.; Patel, V. J.; Scrivens, J. H. Collision-Induced Fragmentation Pathways Including Odd-Electron Ion Formation from Desorption Electrospray Ionisation Generated Protonated and Deprotonated Drugs Derived from Tandem Accurate Mass Spectrometry. J. Mass Spectrom. 2006, 41, 1277–1286.CrossRefGoogle Scholar
  34. 34.
    Jeong, H.; Yim, J. H.; Lee, C.; Choi, S. H.; Park, Y. K.; Yoon, S. H.; Hur, C. G.; Kang, H. Y.; Kim, D.; Lee, H. H.; Park, K. H.; Park, S. H.; Park, H. S.; Lee, H. K.; Oh, T. K.; Kim, J. F. Genomic Blueprint of Hahella chejuensis, a Marine Microbe Producing an Algicidal Agent. Nucleic Acids Res. 2005, 33, 7066–7073.CrossRefGoogle Scholar
  35. 35.
    Chen, K.; Lane, P.; Cai, Y.; Rees, B. B.; Challis, G. L.; Cole, R. B. Unusual Odd-Electron Fragments from Even-Electron Protonated Prodiginine Precursors Using Positive Ion Electrospray Tandem Mass Spectrometry. Proceedings of the 55th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis IN, June 3–7, 2007.Google Scholar

Copyright information

© American Society for Mass Spectrometry 2008

Authors and Affiliations

  • Kan Chen
    • 1
  • Nalaka S. Rannulu
    • 1
  • Yang Cai
    • 1
    • 2
  • Pat Lane
    • 1
  • Andrea L. Liebl
    • 3
  • Bernard B. Rees
    • 3
  • Christophe Corre
    • 4
  • Gregory L. Challis
    • 4
  • Richard B. Cole
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of New OrleansNew OrleansUSA
  2. 2.The Research Institute for ChildrenChildren’s Hospital of New OrleansNew OrleansUSA
  3. 3.Department of Biological SciencesUniversity of New OrleansNew OrleansUSA
  4. 4.Department of ChemistryUniversity of WarwickCoventryUnited Kingdom

Personalised recommendations