由於實驗結果顯示白胺酸腦啡之酪胺酸側鏈上4’端的OH置換為CONH2 (簡稱Cpa修飾)，會增加其與δ受體結合的親和力。因此，為了進一步了解此一修飾分子對δ類鴉片受體有更好的結合親和力，本文將利用分子動力學(molecular dynamics；MD)模擬方法來探討其結構與活性關係。模擬結果發現Cpa修飾之白胺酸腦啡在骨幹上之Leu5—NH與Gly2—CO間有氫鍵產生，使得分子於水溶液中呈現穩定之單折疊構形。此外，我們發現另一重要之氫鍵作用力存在於修飾端Cpa1側鏈之C=O原子與Phe4—NH原子之間。我們進一步分析分子間的半徑分佈函數，結果顯示的確有氫鍵的產生。我們也進一步分析第一個與第四個殘基側鏈的χ1扭轉角，結果顯示它們會個別偏愛 ±180度以及_ 60度。最後，我們比較經修飾的白胺酸腦啡、自然的白胺酸腦啡以及前人提出的δ藥效基團模型的兩環距離間的機率分佈函數。結果顯示，白胺酸腦啡經修飾後會增加對δ類鴉片受體的結合效能與選擇性，而此一研究結果亦與實驗吻合。

Motivated by recent experimental work on Leu-Enkephalin modification with (4-Carboxamido)phenylalanine (Cpa), we perform MD simulations to study the structure–activity relationships of the [Cpa1, Leu5]-enkephalin (Cpa-LE) for better understandings of the binding affinity in δ-selective opioid ligands. Our simulations show the Cpa-LE structure in aqueous solution revealed that the occurrence of single-bend packed state can be stabilized by an intramolecular hydrogen bond from Leu5-NH to Gly2-CO (5→2). In addition, an intramolecular sidechain to backbone hydrogen bond, i.e., hydrogen bond binding between the sidechain carbonyl CO group of the Cpa residue and backbone amide NH group of the Phe residue was examined. Furthermore, the hydration effects of carboxamido group (CONH2) for Cpa residue and 5→2 hydrogen bond were calculated via the solute-solvent radial distribution functions gα-β (r), providing direct evidence of strong hydrogen bond interactions. Our simulation results further reveal the χ1 rotamers of the Cpa1 and Phe4 that show preferences for trans and gauche(-), respectively. Finally, we elucidate the probability distributions of two aromatic rings among the Cpa-LE, Leu-enkephalin, and δ pharmacophore model. The results show that modified the Tyr1 to Cpa1 can lead to increase the potency and selectivity for δ-opioid receptor (DOR), consistent with experimental findings.

[1] Evans, C. K.; Keith, D.E.; Morrison, H.; Magendzo, K.; Edwards, R.H. “Cloning of a delta opioid receptor by functional expression” Science, 258, 19521955, 1992.
[2] Kieffer, B. L.; Befort, K.; Gaveriaux-Guff, C.; Hirth, C. G. “The delta opioid receptor:. isolation of a cDNA by expression cloning and pharmacological characterization” Proc Natl Acad Sci, 89, 1204812052, 1992.
[3] Knapp, R. J.; Malatynska, E.; Fang, L.; Li, X.; Babin, E.; Nguyen, M.; Santoro, G.; Varga, E. V.; Hruby, V. J.; Roeske, W. R.; Yamamura, H. I. “Identification of a human delta opioid receptor: cloning and expression” Life Sci, 54, L463L469, 1994.
[4] Hughes, J.; Smith, T. W.; Kosterlitz, H. W,; Fothergill, L. A.; Morgan, B. A.; Morris, H. R. “Identification of two related pentapeptides from the brain with potent opiate agonist activity” Nature, 258, 577579, 1975.
[5] Li, C. H. “Beta-Endorphin” Cell, 31, 504505, 1982.
[6] Goldstein, A.; Tachibana, S.; Lowney, L. I.; Hunkapillar, M.; Hood, L. “Dynorphin (1 - 13) an extraordinarily potent opioid peptide” Natl Acad Sci, 76, 66666670, 1979.
[7] Zadina, J. E.; Hackler, L.; Ge, L. J.; Kastin, A. J. “A potent and selective endogenous agonist for the mu-opiate receptor”Nature, 386, 499502, 1997.
[8] Hruby, V. J. “Design in topographical space of peptide and peptidomimetic ligands that affect behavior. A chemist's glimpse at the mind–body problem” Accarbonyl oxygenunts Chem Res, 34, 389397, 2001.
[9] Smith, D.; Griffin, J. F. “Conformation of [Leu5]enkephalin from X-ray diffraction: features important for recognition at opiate receptor” Science, 199, 12141216, 1978.
[10] Aubry, A.; Birlirakis, N.; Sakarellos-Daitsiotis, M.; Sakarellos, C.; Marraud, M. “A crystal molecular-conformation of leucine-enkephalin related to the morphine molecule” Biopolymers, 28, 2740, 1989.
[11] Karle, I. L.; Karle, J.; Mastropaolo, D.; Camerman, A.; Camerman, N. “[Leu-5]Enkephalin - 4 cocrystallizing confor-. mers with extended backbones that form an antiparallel beta- sheet” Acta Crystallogr Sect B-Struct Sci, B39, 625637, 1983.
[12] Khaled, M. A.; Long, M. M.; Thompson, W.D.; Bradley, R. J.; Brown, G. B.; Urry, D. W. “Conformational states of enkephalins in solution” Biochem Biophys Res Commun, 76, 224231, 1977.
[13] Stimson, E. R.; Meinwald, Y. C.; Scheraga, H. A. “Solution conformation of enkephalin. A nuclear magnetic resonance study of 13C-enriched carbonyl carbons in [Leu5]-enkephalin” Biochemistry, 18, 16611671, 1979.
[14] Gupta, G. ; Sarma, M. H. ; Dhingra, M. M. “NOE data at 500 MHz reveal the proximity of phenyl and tyrosine rings in enkephalin” FEBS Lett, 198, 245250, 1986.
[15] Abdali, S.; Jensen, M. O.; Bohr, H. J “Energy levels and quantum states of [Leu]enkephalin conformations based on theoretical and experimental investigations” Phys-Carbonyl oxygenndes Matter, 15, S1853S1860, 2003.
[16] Abdali, S. ; Jalkanen, K. J. ; Cao, X. ; Nafie, L. A. ; Bohr, H. “Conformational determination of [Leu]enkephalin based on theoretical and experimental VA and VCD spectral analyses” Phys Chem Chem Phys, 6, 24342439, 2004.
[17] Smith, P. E.; Pettitt, B. M. “Modeling solvent in biomolecular systems” J Phys Chem, 98, 97009711, 1994.
[18] van der Spoel, D.; Berendsen, H. J. “Molecular dynamics simulations of Leu-enkephalin in water and DMSO” Biophys J, 72(5), 20322041, 1997.
[19] Nielsen, B. G.; Jensen, M. O.; Bohr, H. G. “The probability distribution of side-chain conformations in [Leu] and [Met]enkephalin determines the potency and selectivity to mu and delta opiate receptors” Biopolymers, 71(5), 577592, 2003.
[20] Karvounis, G.; Nerukh, D.; Glen, R. C. “Water Network dynamics at the critical moment of the peptide's beta turn formation: A Molecular dynamics study” J Chem Phys, 121, 49254935, 2004.
[21] Dolle, R. E.; Machaut, M.; Martinez-Teipel, B.; Belanger, S.; Cassel, J.A.; Stabley, G.J.; Graczyk, T. M.; DeHaven, R. N. “(4-Carboxamido)phenylalanine is a surrogate for tyrosine in opioid receptor peptide ligands” Bioorg Med Chem Lett, 14, 35453548, 2004.
[22] Shenderovich, M. D.; Liao, S.; Qian, X.; Hruby, V. J. “A Three Dimensional Model of the d Opioid Pharmacophore: Comparative Molecular Modeling of Peptide and Non-Peptide Ligands” Biopolymers, 53, 565-580, 2000.
[23] Loew, G. H. “Molecular modeling of opioid analgesics” Mod Drug Discov, 2, 2430, 1999.
[24] Marrone, T. J.; Briggs, J. M.; McCammon, J. A. “Structure-based Drug Design: Computational Advances”, Ann. Rev. Pharm. Toxicology” Annu Rev Pharmacarbonyl oxygenl Toxicarbonyl oxygenl, 37, 7190, 1997.
[25] Hansson, T.; Oostenbrink, C.; van Gunsteren, W. “Molecular dynamics simulations” Curr Opin Struct Biol, 12, 190196, 2002.
[26] Karplus, M.; McCammon, J. A. “Molecular dynamics simulations of biomolecules” Nat Struct Mol Biol, 9(9), 646652, 2002.
[27] Lindahl, E.; Hess, B.; van der Spoel, D. “GROMACS 3.0:a package for molecular simulation and trajectory analysis” J Mol Model, 7, 306317, 2001.
[28] Scott, W. R. P.; Huenenberger, P.; Tironi, I.; Mark, A.; Billeter, S.; Fennen, J.; Torda, A.; Huber, T.; Krueger, P.; van Gunsteren, W. F. “The GROMOS biomolecular simulation program” J Phys Chem A, 103, 35963607, 1999.
[29] Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.; Hermans, J. “Intermolecular Forces” Intermol Forces, 331–342, 1981.
[30] Schuettelkopf, A. W.; van Aalten, D. M. F. “PRODRG: a tool for high-throughput crystallography of protein-ligand complexes” Acta Cryst, D60, 1355–1363, 2004.
[31] Ryckaert, J. P.; Ciccotti, G.; Berendsen, H. J. C. “Numerical integration of the cartesian equations of motion of a system with constrains: molecular dynamics of n-alkanes” J Comput Phys, 23, 327–341, 1977.
[32] Berendsen, H.J. C.; Postma, J. P.M.; Vangunsteren, W. F.; DiNola, A.; Haak, J. R. “Molecular Dynamics with Coupling to an External Bath” J Chem Phys, 81, 36843690, 1984.
[33] Darden, T.; York, D.; Pedersen, L. “Particle mesh Ewald (PME): a N log(N) method for Ewald sums in large systems” J Chem Phys, 98, 1008910092, 1993.
[34] Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. “A smooth particle mesh Ewald method” J Chem Phys, 103, 85778593, 1995.
[35] Aburi, M.; Smith, P. E. “A conformational analysis of leucine enkephalin as a function of pH” Biopolymers, 64, 177–188, 2002.
[36] Wentland, M. P.; Lou, R.; Dehnhardt, C. M.; Duan, W.; Cohen, D. J.; Bidlack, J. M. “3-Carboxamido analogues of morphine and naltrexone: Synthesis and opioid receptor binding properties” Bioorg Med Chem Lett, 11, 17171721, 2001.
[37] Zhang, A.; Xiong, W.; Bidlack, J. M.; Hilbert, J. E.; Knapp, B. I.; Wentland, M. P.; Neumeyer, J. L. “2-Aminothiazole-Derived Opiates - Bioisosteric Replacement of Phenols” J Med Chem, 47, 165174, 2004.
[38] Wentland, M. P.; Lou, R.; Ye, Y.; Cohen, D. J.; Richardson, G. P.; Bidlack, J. M. “Carboxamidocyclazocine analogues: Redefining the structure-activity relationships of 2,6-methano-3-benzazocines” Bioorg Med Chem Lett, 11, 623626, 2001.
[39] Schiller, P. W. The peptide: analysis, synthesis, biology. Academic Press: Orlando, 1984.
[40] Flippen-Anderson, J. L.; Deschamps, J. R.; Ward, K. B.; George, C.; Houghten, R. “The crystal structure of deltakephalin: a δ-selective opioid peptide with a novel β-bend-like conformation” Int J Pept Protein Res, 44(2), 97104, 1994.
[41] Stimson, E. R.; Meinwald, Y. C.; Scheraga, H. A. “Solution conformation of enkephalin. A nuclear magnetic resonance study of 13C-enriched carbonyl carbons in [Leu5]-enkephalin” Biochemistry, 18(9), 16611671, 1979.
[42] Picone, D. ; D’Ursi, A. ; Motta, A. ; Tancredi, T. ; Temussi, P. A. “Conformational preferences of [Leu5]enkephalin in biomimetic media. Investigation by 1H NMR” Eur J Biochem, 192, 433439, 1990.
[43] Milon, A.; Miyazawa, T.; Higashijima, T. “Transferred nuclear Overhauser effect analyses of membrane-bound enkephalin analogues by 1H nuclear magnetic resonance: correlation between activities and membrane-bound conformations” Biochemistry, 29, 6575,1990.
[44] Dudowicz, J.; Freed, K. F.; Shen, M. Y. “Hydration Structure of Met-enkephalin: A Molecular Dynamics Study” J Chem Phys, 118, 19891995,2003.
[45] Wang, Y.; Kuczera, K. “Molecular dynamics simulations of cyclic and linear DPDPE: Influence of the disulfide bond on peptide flexibility” J Phys Chem, 100, 25552563, 1996.
[46] Mosberg, H. I.; Fowler, C. B. “Development and validation of opiod ligand−receptor interaction models: The structural basis of mu vs. delta selectivity” J. Pept Res, 60, 329335, 2002.
[47] Przydzial, M. J.; Pogozheva, I. D.; Bosse, K. E.; Andrews, S. M.; Tharp, T. A.; Traynor, J. R.; Mosberg, H. I. “Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the kappa- opioid receptor” J Pept Res, 65, 333342, 2005.
[48] Waldhoer, M; Bartlett S. E.; Whistler J. L. “Opioid receptors” Annu. Rev. Biochem. 73, 953-990, 2004.