Amino Acids

, Volume 43, Issue 1, pp 195–206 | Cite as

Helix formation and capping energetics of arginine analogs with varying side chain length

  • Richard P. ChengEmail author
  • Yi-Jen Weng
  • Wei-Ren Wang
  • Marc J. Koyack
  • Yuta Suzuki
  • Cheng-Hsun Wu
  • Po-An Yang
  • Hao-Chun Hsu
  • Hsiou-Ting Kuo
  • Prashant Girinath
  • Chun-Jen Fang
Original Article


Arginine (Arg) has been used for recognizing negatively charged biological molecules, cell penetration, and oligosaccharide mass signal enhancement. The versatility of Arg has inspired the need to develop Arg analogs and to research the structural effects of incorporating Arg analogs. Accordingly, we investigated the effect of Arg side chain length on helix formation by studying 12 Ala-based peptides containing the Arg analogs (S)-2-amino-6-guanidino-hexanoic acid (Agh), (S)-2-amino-4-guanidinobutyric acid (Agb), and (S)-2-amino-3-guanidinopropionic acid (Agp). Solid phase guanidinylation with orthogonal protection strategies was necessary to synthesize Agb- and Agp-containing peptides using Fmoc-based chemistry. The fraction helix for the peptides was determined by circular dichroism spectroscopy, and used to derive the statistical mechanical parameters and energetics for N-capping, C-capping, and helix propagation (propensity). All four Arg analogs were unfavorable for N-capping. The C-cap parameter followed the trend Agp < Agb < Arg < Agh, showing more favorable C-cap energetics with increasing side chain length. In contrast, helix propensity followed the trend Agp < Agb < Arg > Agh, highlighting the uniqueness of the Arg side chain length in helix formation. Molecular mechanics calculations and a survey on protein structures were consistent with the experimental results. Furthermore, calculations and survey both showed that the g– conformation for the χ1 dihedral was present for the first two residues at the N-terminus of helices, but not favored in the center or C-terminus of helices due to sterics. These results should serve as the foundation for developing Arg-related bioactive compounds and technologies.


Arginine Side chain length Peptide Helix 



This work was supported by NYSTAR James D. Watson Investigator Program (R.P.C.), Kapoor funds (R.P.C.), The State University of New York at Buffalo (R.P.C.), National Science Foundation (R.P.C., CHE0809633), National Taiwan University (R.P.C.), National Science Council (R.P.C., NSC-97-2113-M-002-019-MY2, NSC-98-2119-M-002-025, NSC-99-2113-M-002-002-MY2; Y.J.W., NSC-98-2815-C-002-056-M). The authors would like to thank the Computer and Information Networking Center at National Taiwan University for the support of the high-performance computing facilities. The authors would like to thank Professor Cheu-Pyeng Cheng (Department of Chemistry, National Tsing Hua University) for helpful discussions, and Ms. Chia-Wen Kuo for proofreading the manuscript.

Supplementary material

726_2011_1064_MOESM1_ESM.pdf (265 kb)
Supplementary material 1 (PDF 265 kb)


  1. Argos P (1988) An investigation of protein subunit and domain interfaces. Protein Eng 2:101–113PubMedCrossRefGoogle Scholar
  2. Aurora R, Rose GD (1998) Helix capping. Protein Sci 7:21–38PubMedGoogle Scholar
  3. Barlow DJ, Thornton JM (1988) Helix geometry in proteins. J Mol Biol 201(3):601–619PubMedCrossRefGoogle Scholar
  4. Baumgart S, Lindner Y, Kühne R, Oberemm A, Wenschuh H, Krause E (2004) The contributions of specific amino acid side chains to signal intensities of peptides in matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 18:863–868PubMedCrossRefGoogle Scholar
  5. Blagdon DE, Goodman M (1975) Mechanisms of protein and polypeptide helix initiation. Biopolymers 14:241–245PubMedCrossRefGoogle Scholar
  6. Blaum BS, Deakin JA, Johansson CM, Herbert AP, Barlow PN, Lyon M, Uhrin D (2010) Lysine and arginine side chains in glycosaminoglycan–protein complexes investigated by NMR, cross-linking, and mass spectrometry: a case study of the factor H-heparin interaction. J Am Chem Soc 132:6374–6381PubMedCrossRefGoogle Scholar
  7. Bogan AA, Thorn KS (1998) Anatomy of hot spots in protein interfaces. J Mol Biol 280:1–9PubMedCrossRefGoogle Scholar
  8. Chakrabarti P (1994) Conformations of arginine and lysine side chains in association with anions. Int J Peptide Protein Res 43:284–291CrossRefGoogle Scholar
  9. Chakrabartty A, Kortemme T, Baldwin RL (1994) Helix propensities of the amino-acids measured in alanine-based peptides without helix-stabilizing side-chain interactions. Protein Sci 3(5):843–852PubMedCrossRefGoogle Scholar
  10. Cheng RP, Girinath P, Ahmad R (2007) Effect of lysine side chain length on intra-helical glutamate–lysine ion pairing interactions. Biochemistry 46:10528–10537PubMedCrossRefGoogle Scholar
  11. Cheng RP, Girinath P, Suzuki Y, Kuo H-T, Hsu H-C, Wang W-R, Yang P-A, Gullickson D, Wu C-H, Koyack MJ, Chiu H-P, Weng Y-J, Hart P, Kokona B, Fairman R, Lin T-E, Barrett O (2010) Positional effects on helical Ala-based peptides. Biochemistry 49:9372–9384PubMedCrossRefGoogle Scholar
  12. Chhabra SR, Hothi B, Evans DJ, White PD, Bycroft BW, Chan WC (1998) An appraisal of new variants of Dde amine protecting group for solid phase peptide synthesis. Tetrahedron Lett 39:1603–1606CrossRefGoogle Scholar
  13. Chiu H-P, Cheng RP (2007) Chemoenzymatic synthesis of (S)-hexafluoroleucine and (S)-tetrafluoroleucine. Org Lett 9:5517–5520PubMedCrossRefGoogle Scholar
  14. Chiu HP, Suzuki Y, Gullickson D, Ahmad R, Kokona B, Fairman R, Cheng RP (2006) Helix propensity of highly fluorinated amino acids. J Am Chem Soc 128(49):15556–15557PubMedCrossRefGoogle Scholar
  15. Chou PY, Fasman GD (1974) Conformational parameters for amino acids in helical, β-sheet, and random coil regions calculated from proteins. Biochemistry 13(2):211–222PubMedCrossRefGoogle Scholar
  16. Diss ML, Kennan AJ (2008) Orthogonal recognition in dimeric coiled coils via buried polar-group modulation. J Am Chem Soc 130:1321–1327PubMedCrossRefGoogle Scholar
  17. Doig AJ, Baldwin RL (1995) N- and C-capping preferences for all 20 amino acids in α-helical peptides. Protein Sci 4:1325–1336PubMedCrossRefGoogle Scholar
  18. Doig AJ, Chakrabartty A, Klingler TM, Baldwin RL (1994) Determination of free-energies of N-capping in a-helices by modification of the Lifson–Roig helix-coil theory to include N-capping and C-capping. Biochemistry 33(11):3396–3403PubMedCrossRefGoogle Scholar
  19. Drake B, Patek M, Lebl M (1994) A convenient preparation of monosubstituted N,N’-di(Boc)-protected guanidines. Synthesis:579–582Google Scholar
  20. Dunbrack RL, Karplus M (1993) Backbone-dependent rotamer library for proteins—application to side-chain prediction. J Mol Biol 230(2):543–574PubMedCrossRefGoogle Scholar
  21. Edelhoch H (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6(7):1948–1954PubMedCrossRefGoogle Scholar
  22. Edsall JT, Flory PJ, Kendrew JC, Liquori AM, Némethy G, Ramachandran GN, Scheraga HA (1966) A proposal of standard conventions and nomenclature for the description of polypeptide conformations. J Mol Biol 15:399–407PubMedCrossRefGoogle Scholar
  23. Efron B, Gong G (1983) A leisurely look at the bootstrap, the jackknife, and cross-validation. Am Stat 37(1):36–48Google Scholar
  24. Engel DE, De Grado WF (2004) Amino Acid propensities are position-dependent throughout the length of α-helices. J Mol Biol 337:1195–1205PubMedCrossRefGoogle Scholar
  25. Feichtinger K, Sings HL, Baker TJ, Matthews K, Goodman M (1998a) Triurethane-protected guanidines and triflyldiurethane-protected guanidines: new reagents for guanidinylation reactions. J Org Chem 63:8432–8439CrossRefGoogle Scholar
  26. Feichtinger K, Zapf C, Sings HL, Goodman M (1998b) Diprotected triflyguanidines: a new class of guanidinylation reagents. J Org Chem 63:3804–3805CrossRefGoogle Scholar
  27. Fields GB, Noble RL (1990) Solid-phase peptide-synthesis utilizing 9-fluorenylmethoxycarbonyl amino-acids. Int J Pept Protein Res 35(3):161–214PubMedCrossRefGoogle Scholar
  28. Griep S, Hobohm U (2010) PDBselect 1992–2009 and PDBfilter-select. Nucleic Acids Res 38:D318–D319PubMedCrossRefGoogle Scholar
  29. Gunasekaran K, Nagarajaram HA, Ramakrishnan C, Balaram P (1998) Stereochemical punctuation marks in protein structures: glycine and proline containing helix stop signals. J Mol Biol 275:917–932PubMedCrossRefGoogle Scholar
  30. Hirsch AKH, Fischer FR, Diederich F (2007) Phosphate recognition in structural biology. Angew Chem Int Ed 46:338–352CrossRefGoogle Scholar
  31. Ho K-C, Sun C-M (1999) Liquid phase parallel synthesis of guanidines. Bioorg Med Chem Lett 9:1517–1520PubMedCrossRefGoogle Scholar
  32. Hobohm U, Sander C (1994) Enlarged representative set of protein structures. Protein Sci 3(3):522–524PubMedCrossRefGoogle Scholar
  33. Hu Z, Ma B, Wolfson H, Nussinov R (2000) Conservation of polar residues as hot spots at protein interfaces. Proteins Struct Funct Genet 39:331–342PubMedCrossRefGoogle Scholar
  34. Janin J, Miller S, Chothia C (1988) Surface, subunit interfaces and interior of oligomeric proteins. J Mol Biol 204:155–164PubMedCrossRefGoogle Scholar
  35. Jones S, Thornton JM (1995) Protein–protein interactions: a review of protein dimer structures. Prog Biophys Mol Biol 63:31–65PubMedCrossRefGoogle Scholar
  36. Katritzky AR, Rogovoy BV (2005) Recent developments in guanylating agents. ARKIVOC iv:49–87Google Scholar
  37. Klugh HE (1970) Statistics-the essentials for research. In: Statistics-The essentials for research. John Wiley & Sons Inc, New York. pp 350Google Scholar
  38. Kuebler RR, Smith H (1976) Statistics-A beginning. In: Statistics-A beginning. John Wiley & Sons, Inc, New York. pp 302Google Scholar
  39. Kumar S, Bansal M (1998) Dissecting α-helices: position-specific analysis of α-helices in globular proteins. Proteins Struct Funct Genet 31:460–476PubMedCrossRefGoogle Scholar
  40. Lifson S, Roig A (1961) The theory of helix-coil transition in polypeptides. J Chem Phys 34:1963–1974CrossRefGoogle Scholar
  41. McGregor MJ, Islam SA, Sternberg MJE (1987) Analysis of the relationship between side-chain conformation and secondary structure in globular-proteins. J Mol Biol 198(2):295–310PubMedCrossRefGoogle Scholar
  42. Mitchell DJ, Kim DT, Steinman L, Fathman CG, Rothbard JB (2000) Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 56:318–325PubMedCrossRefGoogle Scholar
  43. Moroni M, Koksch B, Osipov SN, Crucianelli M, Frigerio M, Bravo P, Burger K (2001) First synthesis of totally orthogonal protected α-(trifluoromethyl)- and α-(difluoromethyl)arginines. J Org Chem 66:130–133PubMedCrossRefGoogle Scholar
  44. Nishikaze T, Takayama M (2006) Cooperative effect of factors governing molecular ion yields in desorption/ionization mass spectrometry. Rapid Commun Mass Spectrom 20:376–382PubMedCrossRefGoogle Scholar
  45. Northern TR, Lee J-C, Hoang L, Raymond J, Hwang D-R, Yannone SM, Wong CH, Siuzdak G (2008) A nanostructure-initiator mass spectrometry-based enzyme activity assay. Proc Natl Acad Sci USA 105:3678–3683CrossRefGoogle Scholar
  46. Pace CN, Vajdos F, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption-coefficient of a protein. Protein Sci 4(11):2411–2423PubMedCrossRefGoogle Scholar
  47. Padmanabhan S, Baldwin RL (1991) Straight-chain non-polar amino acids are good helix-formers in water. J Mol Biol 219:135–137PubMedCrossRefGoogle Scholar
  48. Padmanabhan S, Marqusee S, Ridgeway T, Laue TM, Baldwin Robert L (1990) Relative helix-forming tendencies of nonpolar amino acids. Nature 344:268–270PubMedCrossRefGoogle Scholar
  49. Padmanabhan S, York EJ, Stewart JM, Baldwin RL (1996) Helix propensities of basic amino acids increase with the length of the side-chain. J Mol Biol 257(3):726–734PubMedCrossRefGoogle Scholar
  50. Poss MA, Iwanowicz E, Reid JA, Lin J, Gu Z (1992) A mild and efficient method for the preparation of guanidines. Tetrahedron Lett 40:5933–5936CrossRefGoogle Scholar
  51. Pradhan TK, Joosten A, Vasse J-L, Bertus P, Karoyan P, Szymoniak J (2009) A concise stereoselective synthesis of 2-substituted 1-aminocyclopropanecarboxyllic acids. Eur J Org Chem 5072–5078Google Scholar
  52. Presta LG, Rose GD (1988) Helix signals in proteins. Science 240(4859):1632–1641PubMedCrossRefGoogle Scholar
  53. Richardson JS, Richardson DC (1988) Amino acid preferences for specific locations at the ends of α helices. Science 240:1648–1652PubMedCrossRefGoogle Scholar
  54. Robinson S, Roskamp EJ (1997) Solid phase synthesis of guanidines. Tetrahedron 53:6697–6705CrossRefGoogle Scholar
  55. Sali D, Bycroft M, Fersht AR (1988) Stabilization of protein structure by interaction of alpha-helix dipole with a charged side chain. Nature 355:740–743Google Scholar
  56. Schneider JP, DeGrado WF (1998) The design of efficient α-helical C-capping auxiliaries. J Am Chem Soc 120:2764–2767CrossRefGoogle Scholar
  57. Shey J-Y, Sun C-M (1998) Soluble polymer-supported synthesis of N,N-di(Boc)-protected guanidines. Synlett 1423–1425Google Scholar
  58. Shinohara Y, Furukawa J, Niikura K, Miura N, Nishimura S-I (2004) Direct N-glycan profiling in the presence of tryptic peptides on MALDI-TOF by controlled ion enhancement and suppression upon glycan-selective derivatization. Anal Chem 76:6989–6997PubMedCrossRefGoogle Scholar
  59. Tsai C-J, Lin SL, Wolfson HJ, Nussinov R (1997) Studies of protein–protein interfaces: a statistical analysis of the hydrophobic effect. Protein Sci 6:53–64PubMedCrossRefGoogle Scholar
  60. Umezawa N, Gelman MA, Haigis MC, Raines RT, Gellman SH (2001) Translocation of a β-peptide across cell membranes. J Am Chem Soc 124:368–369CrossRefGoogle Scholar
  61. Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci USA 97: 13003–13008PubMedCrossRefGoogle Scholar
  62. Wender PA, Rothbard JB, Jessop TC, Kreider EL, Wylie BL (2002) Oligocarbamate molecular transporters: design, synthesis, and biological evaluation of a new class of transporters for drug delivery. J Am Chem Soc 124:13382–13383PubMedCrossRefGoogle Scholar
  63. Wender PA, Galliber WC, Goun EA, Jones JR, Pillow TH (2008) The design of guanidinium-rich transporters and their internalization mechanisms. Adv Drug Deliv Rev 60:452–472PubMedCrossRefGoogle Scholar
  64. Yong YF, Kowalski JA, Lipton MA (1997) Facile and efficient guanylation of amines using thioureas and Mukaiyama’s reagent. J Org Chem 62:1540–1542CrossRefGoogle Scholar
  65. Yong YF, Kowalski JA, Thoen JC, Lipton MA (1999) A new reagent for solid and solution phase synthesis of protected guanidines from amines. Tetrahedron Lett 40:53–56CrossRefGoogle Scholar
  66. Zhang Y, Kennan AJ (2001) Efficient introduction of protected guanidines in BOC solid phase peptide synthesis. Org Lett 3:2341–2344PubMedCrossRefGoogle Scholar
  67. Zhao Y, Kent SBH, Chait BT (1997) Rapid, sensitive structure analysis of oligosaccharides. Proc Natl Acad Sci USA 94:1629–1633PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Richard P. Cheng
    • 1
    Email author
  • Yi-Jen Weng
    • 1
  • Wei-Ren Wang
    • 1
  • Marc J. Koyack
    • 2
  • Yuta Suzuki
    • 2
  • Cheng-Hsun Wu
    • 1
  • Po-An Yang
    • 1
  • Hao-Chun Hsu
    • 1
  • Hsiou-Ting Kuo
    • 1
  • Prashant Girinath
    • 2
  • Chun-Jen Fang
    • 1
  1. 1.Department of ChemistryNational Taiwan UniversityTaipeiTaiwan
  2. 2.Department of ChemistryUniversity at Buffalo, The State University of New YorkBuffaloUSA

Personalised recommendations