Amino Acids

, Volume 46, Issue 12, pp 2733–2744 | Cite as

Impact of fluorination on proteolytic stability of peptides: a case study with α-chymotrypsin and pepsin

  • Vivian Asante
  • Jérémie Mortier
  • Gerhard Wolber
  • Beate KokschEmail author
Original Article


Protease stability is a key consideration in the development of peptide-based drugs. A major approach to increase the bioavailability of pharmacologically active peptides is the incorporation of non-natural amino acids. Due to the unique properties of fluorine, fluorinated organic molecules have proven useful in the development of therapeutically active small molecules as well as in materials and crop science. This study presents data on the ability of fluorinated amino acids to influence proteolytic stability when present in peptide sequences that are based on ideal protease substrates. Different model peptides containing fluorinated amino acids or ethylglycine in the P2, P1′or P2′ positions were designed according to the specificities of the serine protease, α-chymotrypsin (EC or the aspartic protease, pepsin (EC The proteolytic stability of the peptides toward these enzymes was determined by an analytical RP-HPLC assay with fluorescence detection and compared to a control sequence. Molecular modeling was used to support the interpretation of the structure–activity relationship based on the analysis of potential ligand-enzyme interactions. Surprisingly, an increase in proteolytic stability was observed only in a few cases. Thus, this systematic study shows that the proteolytic stability of fluorinated peptides is not predictable, but rather is a very complex phenomenon that depends on the particular enzyme, the position of the substitution relative to the cleavage site and the fluorine content of the side chain.


Chymotrypsin Pepsin Aminobutyric acid Difluoroethylglycine Trifluoroethylglycine 



o-Aminobenzoic acid


High performance liquid chromatography


Fluorenylmethoxy carbonyl


Molecular dynamics








Trifluoroacetic acid




Solid phase peptide synthesis



We are grateful to the Rosa-Luxemburg-Stiftung (RLS) and the Deutsche Forschungsgemeinschaft (Research Training Group “Fluorine as Key Element”) for financial support of Vivian Asante. Jérémie Mortier is grateful to WBI—Wallonie-Bruxelles-International for the award of a postdoctoral grant. We also express our sincere thanks to Dr. Allison Berger for her suggestions and careful editing of the text.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

726_2014_1819_MOESM1_ESM.docx (140 kb)
Supplementary material 1 (DOCX 139 kb)


  1. Aguilar MI (2004) HPLC of peptides and proteins: methods and Protocols. Humana Press, TotowaGoogle Scholar
  2. Antal J, Pál G, Asbóth B, Buzás Z, Patthy A, Gráf L (2001) Specificity assay of serine proteinases by reverse-phase high-performance liquid chromatography analysis of competing oligopeptide substrate library. Anal Biochem 288:156–167. doi: 10.1006/abio.2000.4886 PubMedCrossRefGoogle Scholar
  3. Antonov VK, Ginodman LM, Kapitannikov YV, Barshevskaya TN, Gurova AG, Rumsh LD (1978) Mechanism of pepsin catalysis: general base catalysis by the active site carboxylate ion. FEBS Lett 88:87–90PubMedCrossRefGoogle Scholar
  4. Antonov VK, Ginodman LM, Rumsh LD, Kapitannikov YV, Barshevskaja TN, Yavashev LP, Gurova AG, Volkova LI (1981) Studies on mechanism of action of proteolytic enzymes using heavy oxygen exchange. Eur J Biochem 117:195–200PubMedCrossRefGoogle Scholar
  5. Asante V, Mortier J, Schlüter H, Koksch B (2013) Impact of fluorination on proteolytic stability of peptides in human blood plasma. Bioorg Med Chem 21:3542–3546. doi: 10.1016/j.bmc.2013.03.051 PubMedCrossRefGoogle Scholar
  6. Baker PJ, Montclare JK (2011) Enhanced refoldability and thermoactivity of fluorinated phosphotriesterase. ChemBioChem 12:1845–18481. doi: 10.1002/cbic.201100221 PubMedCrossRefGoogle Scholar
  7. Berman HL, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) The protein data bank. Nucl Acids Res 28:235–242PubMedCentralPubMedCrossRefGoogle Scholar
  8. Blow DM, Birktoft JJ, Hartley BS (1969) Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 221:337–340PubMedCrossRefGoogle Scholar
  9. Böhm HJ, Banner D, Bendels S, Kansy M (2004) Fluorine in medicinal chemistry. ChemBioChem 5:637–643PubMedCrossRefGoogle Scholar
  10. Brady K, Abeles RH (1990) Inhibition of chymotrypsin by peptidyltrifluoromethyl: determinants of slow-binding kinetics. Biochemistry 29:7608–7617PubMedCrossRefGoogle Scholar
  11. Buer BC, Marsh ENG (2012) Fluorine: a new element in protein design. Prot Sci 21:453–462CrossRefGoogle Scholar
  12. Chen L, Erickson JW, Rydel TJ, Park CH, Neidhart D, Luly J, Abad-Zapatero C (1992) Structure of a pepsin/renin inhibitor complex reveals a novel crystal packing induced by minor chemical alterations in the inhibitor. Acta Crystallogr B 48(4):476–488PubMedCrossRefGoogle Scholar
  13. Cornish-Bowden AJ, Knowles JR (1969) The pH-dependence of pepsin-catalysed reactions. Biochem J 113:353–362PubMedCentralPubMedGoogle Scholar
  14. Coughlin SR (2000) Thrombin signalling and protease-activated receptors. Nature 407:258–264PubMedCrossRefGoogle Scholar
  15. Czapinska H, Otlewski J (1999) Structural and energetic determinants of the S1-site specificity in serine proteases. Eur J Biochem 260:571–595PubMedCrossRefGoogle Scholar
  16. Dasgupta P, Singh A, Mukherjee R (2002) N-terminal acylation of somatostatin analog with long chain fatty acids enhances its stability and anti-proliferative activity in human breast adenocarcinoma cells. Biol Pharm Bull 25:29–36PubMedCrossRefGoogle Scholar
  17. Davies DR (1990) The structure and function of the aspartic proteinases. Annu Rev Biophys Chem 19:189–215CrossRefGoogle Scholar
  18. Derewenda ZS, Derewenda U, Kobos PM (1994) (His) Cε-H…O = C < hydrogen bond in the active sites of serine. J Mol Biol 241:83–93PubMedCrossRefGoogle Scholar
  19. Dougherty DA (2000) Unnatural amino acids as probes of protein structure and function. Curr Opin Chem Biol 4:645–652PubMedCrossRefGoogle Scholar
  20. Dunn BM (2002) Structure and mechanism of the pepsin-like family of aspartic peptidases. Chem Rev 102:4431–4458PubMedCrossRefGoogle Scholar
  21. Dunn BM, Hung S (2000) The two sides of enzyme-substrate specificity: lessons from the aspartic proteinases. Biochim Biophys Acta 1477:231–240PubMedCrossRefGoogle Scholar
  22. Dunn BM, Jimenez M, Parten BF, Valler MJ, Rolph CE, Kay J (1986) A systematic series of synthetic chromophoric substrates for aspartic proteinases. Biochem J 237:899–906PubMedCentralPubMedGoogle Scholar
  23. Dunn BM, Valler MJ, Rolph CE, Foundling SI, Jimenez M, Kay J (1987) The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO2) Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S3 and S2. Biochim Biophys Acta 913:122–130PubMedCrossRefGoogle Scholar
  24. Erickson JA, McLoughlin JI (1995) Hydrogen bond donor properties of the difluoromethyl group. J Org Chem 60:1626–1631. doi: 10.1021/jo00111a021 CrossRefGoogle Scholar
  25. Ferrie JJ, Gruskos JJ, Goldwaser AL, Decker ME, Guarracino DA (2013) A comparative protease stability study of synthetic macrocyclic peptides that mimic two endocrine hormones. Bioorg Med Chem Lett 23:989–995PubMedCrossRefGoogle Scholar
  26. Filler R, Saha R (2009) Fluorine in medicinal chemistry: a century of progress and a 60-years retrospective of selected highlights. Future Med Chem 1:777–791PubMedCrossRefGoogle Scholar
  27. Frackenpohl J, Arvidsson PI, Schreiber JV, Seebach D (2001) The outstanding biological stability of β- and γ-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases. ChemBioChem 2:445–455PubMedCrossRefGoogle Scholar
  28. Fruton JS (1970) The specificity and mechanism of pepsin action. Adv Enzymol Relat Areas Mol Biol 33:401–443PubMedGoogle Scholar
  29. Fujinaga M, Chernaia MM, Tarasova NI, Mosimann SC, James MNG (1995) Crystal-structure of human pepsin and its complex with pepstatin. Protein Sci 4:960–972PubMedCentralPubMedCrossRefGoogle Scholar
  30. Fujinaga M, Cherney MM, Tarasova NI, Bartlett PA, Hanson JE, James MNG (2000) Structural study of the complex between human pepsin and a phosphorus-containing peptidic—transition-state analog. Acta Crystallogr Sect D56:272–279. doi: 10.1107/S0907444999016376Wiley Google Scholar
  31. Geurink PP, Liu N, Spaans MP, Downey SL, van den Nieuwendijk AMCH, van der Marel GA, Kisselev AF, Florea BI, Overkleeft HS (2010) Incorporation of fluorinated phenylalanine generates highly specific inhibitor of proteasome’s chymotrypsin-like sites. J Med Chem 53:2319–2323PubMedCentralPubMedCrossRefGoogle Scholar
  32. Giannis A, Kolter T (1993) Peptidomimetics for receptor ligands-discovery, development and medicinal perspectives. Angew Chem Int Ed Engl 32:1244–1267CrossRefGoogle Scholar
  33. Gottler LM, Lee HY, Shelburne CE, Ramamoorthy A, Marsh ENG (2008) Modulating the biological activity of an antimicrobial peptide using fluorous amino acids. ChemBioChem 9:370–373PubMedCrossRefGoogle Scholar
  34. Hedstrom L (2002) Serine protease mechanism and specificity. Chem Rev 102:4501–4523PubMedCrossRefGoogle Scholar
  35. Hruby VJ (2002) Designing peptide receptor agonists and antagonists. Nat Rev Drug Discov 1:847–858PubMedCrossRefGoogle Scholar
  36. Hua J, Huang K-L (2010) A reversed phase HPLC method for the analysis of nucleotides to determine 5′-PDE enzyme activity. Bull Chem Soc Ethiop 24:167–174CrossRefGoogle Scholar
  37. Hummel G, Reineke U, Reimer U (2006) Translating peptides into small molecules. Mol BioSyst 2:499–508PubMedCrossRefGoogle Scholar
  38. Isanbor C, O’Hagan D (2006) Fluorine in medicinal chemistry: a review of anticancer agent. J Fluorine Chem 127:303–319CrossRefGoogle Scholar
  39. Jäckel C, Koksch B (2005) Fluorine in peptide design and protein engineering. Eur J Org Chem 21:4482–4503Google Scholar
  40. Jäckel C, Seufert W, Thust S, Koksch B (2004) Evaluation of the molecular interactions of fluorinated amino acids with native polypeptides. ChemBioChem 5:717–720PubMedCrossRefGoogle Scholar
  41. Jäckel C, Salwiczek M, Koksch B (2006) Fluorine in a native protein environment—how space filling and polarity of fluorinated alkyl groups affect protein folding. Angew Chem Int Ed 45:4198–4203CrossRefGoogle Scholar
  42. Jones G, Willett P, Glen RC (1995) Molecular recognition of receptor sites using a genetic algorithm with a description of desolvation. J Mol Biol 245:43–53. doi: 10.1016/S0022-2836(95)80037-9 PubMedCrossRefGoogle Scholar
  43. Jones G, Willett P, Glen RC, Leach AR, Taylor R (1997) Development and validation of a genetic algorithm for flexible docking. J Mol Biol 267:727–748. doi: 10.1006/jmbi.1996.0897 PubMedCrossRefGoogle Scholar
  44. Kageyama T (2002) Pepsinogens, progastricsins, and prochymosins: structure, function, evolution, and development. Cell Mol Life Sci 59:288–306PubMedCrossRefGoogle Scholar
  45. Keil B (1992) Specificity of proteolysis. Springer, Berlin 335CrossRefGoogle Scholar
  46. Koksch B, Sewald N, Burger K, Jakubke H-D (1996a) Peptide modification by incorporation of α-trifluoromethyl substituted amino acids. Amino Acids 11:425–434PubMedCrossRefGoogle Scholar
  47. Koksch B, Sewald N, Jakubke H-D, Burger K (1996b) Synthesis and incorporation of α-trifluoromethyl-substituted amino acids into peptides. In: Ojima I, McCarthy JR, Welch JT (eds) Biomedical frontiers of fluorine chemistry, ACS Symposium Series, vol 639. American Chemical Society, Washington DC, pp 42–58Google Scholar
  48. Koksch B, Sewald N, Hofmann H-J, Burger K, Jakubke H-D (1997) Proteolytically stable peptides by incorporation of alpha-Tfm amino acids. J Peptide Sci 3:157–167CrossRefGoogle Scholar
  49. Lee H, Jang IH, Ryu SH, Park TG (2003) N-terminal site-specific mono-PEGylation of epidermal growth factor. Pharm Res 20:818–825PubMedCrossRefGoogle Scholar
  50. Łęgowska A, Dębowski D, Lesner A, Wysocka M, Rolka K (2009) Introduction of non-natural amino acid residues into the substrate-specific P1 position of trypsin inhibitor SFTI-1 yields potent chymotrypsin and cathepsin G inhibitors. Bioorg Med Chem 17:3302–3307PubMedCrossRefGoogle Scholar
  51. March TL, Johnston MR, Duggan PJ, Gardiner J (2012) Synthesis, structure, and biological applications of α-fluorinated β-amino acids and derivatives. Chem Biodiversity 9:2410–2441. doi: 10.1002/cbdv.201200307 CrossRefGoogle Scholar
  52. McGregor DP (2008) Discovering and improving novel peptide therapeutics. Curr Opin Pharmacol 8:616–619PubMedCrossRefGoogle Scholar
  53. Meng H, Kumar K (2007) Antimicrobial activity and protease stability of peptides containing fluorinated amino acids. J Am Chem Soc 129:15615–15622PubMedCrossRefGoogle Scholar
  54. 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 51:7303–7307. doi: 10.1021/jm8008579 PubMedCentralPubMedCrossRefGoogle Scholar
  55. Muller K, Faeh C, Diederich F (2007) Fluorine in pharmaceuticals: looking beyond intuition. Science 317:1881–1886PubMedCrossRefGoogle Scholar
  56. Northrop JH (1930) Crystalline pepsin. I. Isolation and tests of purity. J Gen Physiol 13:739–766PubMedCentralPubMedCrossRefGoogle Scholar
  57. Polgár L (2005) The catalytic triad of serine peptidases. Cell Mol Life Sci 62:2161–2172. doi: 10.1007/s00018-005-5160-x PubMedCrossRefGoogle Scholar
  58. Powers JC, Harley AD, Myers DV (1977) Subsite specificity of porcine pepsin. Adv Exp Med Biol 95:141–157PubMedCrossRefGoogle Scholar
  59. Saeki K, Ozaki K, Kobayashi T, Ito S (2007) Enzymatic properties, genes, and crystal structures. J Biosci Bioeng 103:501–508PubMedCrossRefGoogle Scholar
  60. Saffran M, Kumar G, Savariar C, Burnham J, Williams F, Neckers D (1986) A new approach to the oral administration of insulin and other peptide drugs. Science 233:1081–1084PubMedCrossRefGoogle Scholar
  61. Salwiczek M, Samsonov S, Vagt T, Baldauf C, Nyakatura E, Fleige E, Numata J, Cölfen H, Pisabarro MT, Koksch B (2009a) Position dependent effects of fluorinated amino acids on hydrophobic core formation of a coiled coil heterodimer. Chem Eur J 15:7628–7636PubMedCrossRefGoogle Scholar
  62. Salwiczek M, Vagt T, Koksch B (2009b) Artificial model systems designed to investigate fluorinated amino acids in native protein environments. In: Ojima I (ed) Fluorine in bioorganic and medicinal chemistry. Blackwell Publishers, pp 391–409Google Scholar
  63. Salwiczek M, Nyakatura EK, Gerling UIM, Ye S, Koksch B (2012) Fluorinated amino acids: compatibility with native protein structures and effects on protein–protein interactions. Chem Soc Rev 41:2135–2171. doi: 10.1039/C1CS15241F PubMedCrossRefGoogle Scholar
  64. Samsonov SA, Salwiczek M, Anders G, Koksch B, Pisabarro MT (2009) Fluorine in protein environments: a QM and MD study. J Phys Chem B 113:16400–16408PubMedCrossRefGoogle Scholar
  65. Sani M, Sinisi R, Viani F (2006) Peptidyl fluoro-ketones as proteolytic enzyme inhibitors. Curr Top in Med Chem 6:1545–1566CrossRefGoogle Scholar
  66. Sato AK, Viswanathan M, Kent RB, Wood CR (2006) Therapeutic peptides: technological advances driving peptides into development. Curr Opin Biotechnol 17:638–642PubMedCrossRefGoogle Scholar
  67. Schechter I, Berger A (1967) On the size of the active site in proteases. Biochem Biophys Res Com 27:157–162PubMedCrossRefGoogle Scholar
  68. Schellenberger V, Jakubke HD (1986) A spectrophotometric assay for the characterization of the S’ subsite specificity of alpha-chymotrypsin. Biochim Biophys Acta 869:54–60PubMedCrossRefGoogle Scholar
  69. Schellenberger V, Turck CW, Rutter WJ (1994) Role of the S’ subsites in serine protease catalysis-active-site mapping of rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma-Mansoni. Biochem 33:4251–4257CrossRefGoogle Scholar
  70. Seidel T, Ibis G, Bendix F, Wolber G (2010) Strategies for 3D pharmacophore-based virtual screening. Drug Discov Today Tecnol 7:221–228. doi: 10.1016/j.ddtec.2010.11.004 CrossRefGoogle Scholar
  71. Shaji J, Patole V (2008) Protein and peptide drug delivery: oral approaches. Indian J Pharm Sci 70:269–277PubMedCentralPubMedCrossRefGoogle Scholar
  72. Shrimpton CN, Abbenante G, Lew RA, Smith AI (2000) Development and characterization of novel potent and stable inhibitors of endopeptidase EC Biochem J 345:351–356PubMedCentralPubMedCrossRefGoogle Scholar
  73. Smits R, Koksch B (2006) How C-alpha-fluoroalkyl amino acids and peptides interact with enzymes: studies concerning the influence on proteolytic stability, enzymatic resolution and peptide coupling. Curr Top Med Chem 16:1483–1498CrossRefGoogle Scholar
  74. Suguna K, Padlan EA, Smith CW, Carlson WD, Davies DR (1987) Binding of a reduced peptide inhibitor to the aspartic proteinase from Rhizopus chinensis: implications for a mechanism of action. Proc Natl Acad Sci USA 84:7009–7013PubMedCentralPubMedCrossRefGoogle Scholar
  75. Tsukada H, Blow DM (1985) Structure of α-chymotrypsin refined at 1.6 Å resolution. J Mol Biol 184:703–711PubMedCrossRefGoogle Scholar
  76. Vincent HL, Satish DK, George MG, Werner R (1991) Oral route of protein and peptide drug delivery. In: Vincent HL (ed) Peptide and protein drug delivery. Marcel Dekker, New York, pp 691–738Google Scholar
  77. Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M (2010) Synthetic therapeutic peptides: science and market. Drug Discov Today 15:40–56PubMedCrossRefGoogle Scholar
  78. Voloshchuk N, Zhu AY, Snydacker D, Montclare JK (2009) Positional effects of monofluorinated phenylalanines on histone acetyltransferase stability and activity. Bioorg Med Chem Lett 19:5449–5451PubMedCrossRefGoogle Scholar
  79. Werle M, Bernkop-Schnürch A (2006) Strategies to improve plasma half life time of peptide and protein drugs. Amino Acids 30:351–367. doi: 10.1007/s00726-005-0289-3 PubMedCrossRefGoogle Scholar
  80. Wolber G, Langer T (2005) LigandScout: 3-D pharmacophores derived from protein-bound ligands and their use as virtual screening filters. J Chem Inf Model 45:160–169. doi: 10.1021/ci049885e PubMedCrossRefGoogle Scholar
  81. Wolber G, Dornhofer AA, Langer T (2006) Efficient overlay of small molecules using 3-D pharmacophores. J Comput-Aid Mol Des 20:773–788. doi: 10.1007/s10822-006-9078-7 CrossRefGoogle Scholar
  82. Zhou P, Tian F, Lv F, Shang Z (2009) Geometric characteristics of hydrogen bonds involving sulfur atoms in proteins. Proteins 76:151–163. doi: 10.1002/prot.22327 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Vivian Asante
    • 1
  • Jérémie Mortier
    • 2
  • Gerhard Wolber
    • 2
  • Beate Koksch
    • 1
    Email author
  1. 1.Institute of Chemistry and Biochemistry, Freie Universität BerlinBerlinGermany
  2. 2.Institute of Pharmacy, Department Pharmaceutical and Medicinal ChemistryFreie Universität BerlinBerlinGermany

Personalised recommendations