Modulating the Antimicrobial Activity of Temporin L Through Introduction of Fluorinated Phenylalanine

  • Subbaiah Chennam Setty
  • Soyar Horam
  • Mukesh Pasupuleti
  • Wahajul Haq


Antimicrobial peptides (AMPs) are the promising future therapeutic candidates because of their multifunctional roles and unique mode of action against microbes. Despite several advantages, developing AMPs into therapeutic antibiotics is often associated with limitations, such as thermal and enzymatic stability, moderate antimicrobial activity and higher toxicity. We here report the synthesis of 2-fluoro- and 2,6-difluorophenyalanine, their introduction into naturally occurring antimicrobial peptide Temporin L (TL). We also report the antimicrobial and hemolytic activity of parent TL as well as the fluorinated variant in plasma and buffer conditions. Circular dichroism studies clearly show that fluorination reduces the helical propensity, thus accounting for lower activity. We further demonstrated that fluorinated TL can act as antimicrobial agents in creams and gels used for treating skin infections.

Graphical Abstract


Antimicrobial peptides Temporin L Fluorination Toxicity Fluorinated phenylalanine Helical propensity 



C.S.S. and S.H thanks UGC for award of senior and junior research fellowship respectively. The authors gratefully acknowledge the SAIF Division of CSIR-CDRI for providing the spectroscopic data. Dr M.P. thank the Director, Central Drug Research Institute (CDRI), Lucknow, and SERB, DST India, for his encouragement and providing the seed money to establish the lab and carry out the work. This manuscript has CDRI Communication No.56/2015/WH.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain studies with human or animal subjects. However, human blood was used in the hemolysis studies for which, Institutional human ethical committee, CSIR-CDRI, Lucknow, has approved the use of human blood for the experiments (CDRI/IEC/2014/A1). Written informed consent was obtained from the all the blood donor. For the pig skin experiment, the required skin was collected from the adult male animals, which were sacrificed for human consumption at local slaughter house. As the animals were not housed for the experimental purpose, no animal ethical permission was needed as per Indian animal ethics code.

Supplementary material

10989_2016_9553_MOESM1_ESM.docx (498 kb)
Supplementary material 1 (DOCX 497 kb)


  1. Aiyelabola T, Ojo I, Adebajo A, Ogunlusi G, Oyetunji O, Akinkunmi E, Adeoye A (2012) Synthesis, characterization and antimicrobial activities of some metal(II) amino acids’ complexes. Adv Biol Chem 2:268–273CrossRefGoogle Scholar
  2. Arai T, Maruo N, Sumida Y, Korosue C, Nishino N (1999) Spatially close porphyrin pair linked by the cyclic peptide Gramicidin S. Chem Commun 16:1503–1504CrossRefGoogle Scholar
  3. Balducci D, Contaldi S, Lazzari I, Porzi G (2009) A highly efficient stereocontrolled synthesis of (S)-2′ 6′-dimethyltyrosine [(S)-DMT]. Tetrahedron Asymmetry 20:1398–1401CrossRefGoogle Scholar
  4. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3:238–250CrossRefPubMedGoogle Scholar
  5. de Souza Mendes C, de Souza Antunes A (2013) Pipeline of known chemical classes of antibiotics. Antibiotics 2:500–534CrossRefPubMedGoogle Scholar
  6. Godballe T, Nilsson LL, Petersen PD, Jenssen H (2011) Antimicrobial beta-peptides and alpha-peptoids. Chem Biol Drug Des 77:107–116CrossRefPubMedGoogle Scholar
  7. Grieco P et al (2013) The effect of d-amino acid substitution on the selectivity of temporin L towards target cells: identification of a potent anti-Candida peptide. Biochim Biophys Acta 1828:652–660CrossRefPubMedGoogle Scholar
  8. Haney EF, Nazmi K, Bolscher JG, Vogel HJ (2012) Influence of specific amino acid side-chains on the antimicrobial activity and structure of bovine lactoferrampin. Biochem Cell Biol 90:362–377CrossRefPubMedGoogle Scholar
  9. Jiang Z, Vasil AI, Hale J, Hancock RE, Vasil ML, Hodges RS (2009) Effects of net charge and the number of positively charged residues on the biological activity of amphipathic alpha-helical cationic antimicrobial peptides. Adv Exp Med Biol 611:561–562CrossRefPubMedGoogle Scholar
  10. Konai MM, Ghosh C, Yarlagadda V, Samaddar S, Haldar J (2014) Membrane active phenylalanine conjugated lipophilic norspermidine derivatives with selective antibacterial activity. J Med Chem 57:9409–9423CrossRefPubMedGoogle Scholar
  11. Lai JR, Epand RF, Weisblum B, Epand RM, Gellman SH (2006) Roles of salt and conformation in the biological and physicochemical behavior of protegrin-1 and designed analogues: correlation of antimicrobial, hemolytic, and lipid bilayer-perturbing activities. Biochemistry 45:15718–15730CrossRefPubMedGoogle Scholar
  12. Lee E et al (2014) Role of phenylalanine and valine10 residues in the antimicrobial activity and cytotoxicity of piscidin-1. PLoS One 9:e114453CrossRefPubMedPubMedCentralGoogle Scholar
  13. Lehrer RI, Barton A, Ganz T (1988) Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry. J Immunol Methods 108:153–158CrossRefPubMedGoogle Scholar
  14. Lewis K (2013) Platforms for antibiotic discovery. Nat Rev Drug Discov 12:371–387CrossRefPubMedGoogle Scholar
  15. Loureiro JA et al (2014) Fluorinated beta-sheet breaker peptides. J Mater Chem 2:2259–2264CrossRefGoogle Scholar
  16. Mae M, Amii H, Uneyama K (2000) First synthesis of 3,3-difluoroserine and cysteine derivatives via Mg(0)-promoted selective Cî—¸F bond cleavage of trifluoromethylimines. Tetrahedron Lett 41:7893–7896CrossRefGoogle Scholar
  17. Mahalka AK, Kinnunen PK (2009) Binding of amphipathic alpha-helical antimicrobial peptides to lipid membranes: lessons from temporins B and L. Biochim Biophys Acta 1788:1600–1609CrossRefPubMedGoogle Scholar
  18. Maisetta G et al (2013) pH-dependent disruption of Escherichia coli ATCC 25922 and model membranes by the human antimicrobial peptides hepcidin 20 and 25. FEBS J 280:2842–2854CrossRefPubMedGoogle Scholar
  19. Malmsten M, Kasetty G, Pasupuleti M, Alenfall J, Schmidtchen A (2011) Highly selective end-tagged antimicrobial peptides derived from PRELP. PLoS One 6:e16400CrossRefPubMedPubMedCentralGoogle Scholar
  20. Mangoni ML et al (2011) Structure-activity relationship, conformational and biological studies of temporin L analogues. J Med Chem 54:1298–1307CrossRefPubMedGoogle Scholar
  21. McCloskey AP, Gilmore BF, Laverty G (2014) Evolution of antimicrobial peptides to self-assembled peptides for biomaterial applications. Pathogens 3:791–821CrossRefPubMedPubMedCentralGoogle Scholar
  22. Meng H, Kumar K (2007) Antimicrobial activity and protease stability of peptides containing fluorinated amino acids. J Am Chem Soc 129:15615–15622CrossRefPubMedGoogle Scholar
  23. Meng H, Krishnaji ST, Beinborn M, Kumar K (2008) Influence of selective fluorination on the biological activity and proteolytic stability of glucagon-like peptide-1. J Med Chem 51:7303–7307CrossRefPubMedPubMedCentralGoogle Scholar
  24. Mercer DK, O’Neil DA (2013) Peptides as the next generation of anti-infectives. Future Med Chem 5:315–337CrossRefPubMedGoogle Scholar
  25. Molhoek EM et al (2010) Chicken cathelicidin-2-derived peptides with enhanced immunomodulatory and antibacterial activities against biological warfare agents. Int J Antimicrob Agents 36:271–274CrossRefPubMedGoogle Scholar
  26. Moncla BJ, Pryke K, Rohan LC, Graebing PW (2011) Degradation of naturally occurring and engineered antimicrobial peptides by proteases. Adv Biosci Biotechnol 2:404–408CrossRefPubMedPubMedCentralGoogle Scholar
  27. Niemz A, Tirrell DA (2001) Self-association and membrane-binding behavior of melittins containing trifluoroleucine. J Am Chem Soc 123:7407–7413CrossRefPubMedGoogle Scholar
  28. Pal T, Sonnevend A, Galadari S, Conlon JM (2005) Design of potent, non-toxic antimicrobial agents based upon the structure of the frog skin peptide, pseudin-2. Regul Pept 129:85–91CrossRefPubMedGoogle Scholar
  29. Pandurangan K, Kitchen JA, Blasco S, Paradisi F, Gunnlaugsson T (2014) Supramolecular pyridyl urea gels as soft matter with antibacterial properties against MRSA and/or E. coli. Chem Commun (Camb) 50:10819–10822CrossRefGoogle Scholar
  30. Papareddy P et al (2010) Proteolysis of human thrombin generates novel host defense peptides. PLoS Pathog 6:e1000857CrossRefPubMedPubMedCentralGoogle Scholar
  31. Pasupuleti M, Walse B, Nordahl EA, Morgelin M, Malmsten M, Schmidtchen A (2007) Preservation of antimicrobial properties of complement peptide C3a, from invertebrates to humans. J Biol Chem 282:2520–2528CrossRefPubMedGoogle Scholar
  32. Pasupuleti M, Walse B, Svensson B, Malmsten M, Schmidtchen A (2008) Rational design of antimicrobial C3a analogues with enhanced effects against staphylococci using an integrated structure and function-based approach. Biochemistry 47:9057–9070CrossRefPubMedGoogle Scholar
  33. Pasupuleti M, Chalupka A, Morgelin M, Schmidtchen A, Malmsten M (2009a) Tryptophan end-tagging of antimicrobial peptides for increased potency against Pseudomonas aeruginosa. Biochim Biophys Acta 1790:800–808CrossRefPubMedGoogle Scholar
  34. Pasupuleti M, Schmidtchen A, Chalupka A, Ringstad L, Malmsten M (2009b) End-tagging of ultra-short antimicrobial peptides by W/F stretches to facilitate bacterial killing. PLoS ONE 4:e5285CrossRefPubMedPubMedCentralGoogle Scholar
  35. Pasupuleti M, Schmidtchen A, Malmsten M (2012) Antimicrobial peptides: key components of the innate immune system. Crit Rev Biotechnol 32:143–171CrossRefPubMedGoogle Scholar
  36. Salwiczek M, Nyakatura EK, Gerling UI, Ye S, Koksch B (2012) Fluorinated amino acids: compatibility with native protein structures and effects on protein-protein interactions. Chem Soc Rev 41:2135–2171CrossRefPubMedGoogle Scholar
  37. Saviello MR, Malfi S, Campiglia P, Cavalli A, Grieco P, Novellino E, Carotenuto A (2010) New insight into the mechanism of action of the temporin antimicrobial peptides. Biochemistry 49:1477–1485CrossRefPubMedGoogle Scholar
  38. Schmidtchen A, Pasupuleti M, Morgelin M, Davoudi M, Alenfall J, Chalupka A, Malmsten M (2009) Boosting antimicrobial peptides by hydrophobic oligopeptide end tags. J Biol Chem 284:17584–17594CrossRefPubMedPubMedCentralGoogle Scholar
  39. Schmidtchen A, Pasupuleti M, Malmsten M (2014) Effect of hydrophobic modifications in antimicrobial peptides. Adv Colloid Interface Sci 205:265–274CrossRefPubMedGoogle Scholar
  40. Stromstedt AA, Pasupuleti M, Schmidtchen A, Malmsten M (2009) Evaluation of strategies for improving proteolytic resistance of antimicrobial peptides by using variants of EFK17, an internal segment of LL-37. Antimicrob Agents Chemother 53:593–602CrossRefPubMedGoogle Scholar
  41. Subbaiah CS, Haq W (2014) Efficient stereocontrolled synthesis of sitagliptin phosphate. Tetrahedron Asymmetry 25:1026–1030CrossRefGoogle Scholar
  42. Thirumalai MK, Roy A, Sanikommu S, Arockiaraj J, Pasupuleti M (2014) A simple, robust enzymatic-based high-throughput screening method for antimicrobial peptides discovery against Escherichia coli. J Pept Sci 20:341–348CrossRefPubMedGoogle Scholar
  43. Waters ML (2002) Aromatic interactions in model systems. Curr Opin Chem Biol 6:736–741CrossRefPubMedGoogle Scholar
  44. Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat Protoc 3:163–175CrossRefPubMedGoogle Scholar
  45. Wolny M, Batchelor M, Knight PJ, Paci E, Dougan L, Peckham M (2014) Stable single alpha-helices are constant force springs in proteins. J Biol Chem 289:27825–27835CrossRefPubMedPubMedCentralGoogle Scholar
  46. Yeaman MR, Gank KD, Bayer AS, Brass EP (2002) Synthetic peptides that exert antimicrobial activities in whole blood and blood-derived matrices. Antimicrob Agents Chemother 46:3883–3891CrossRefPubMedPubMedCentralGoogle Scholar
  47. Zaknoon F, Goldberg K, Sarig H, Epand RF, Epand RM, Mor A (2012) Antibacterial properties of an oligo-acyl-lysyl hexamer targeting Gram-negative species. Antimicrob Agents Chemother 56:4827–4832CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Division of Medicinal and Process ChemistryCentral Drug Research InstituteLucknowIndia
  2. 2.Division of MicrobiologyCentral Drug Research InstituteLucknowIndia

Personalised recommendations