Peptide-Based Drug Design

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Solid-Phase Synthesis of O-Phosphopeptides 221 absorption additionally in the 280- to 300-nm region. For larger peptide amounts producing UV signals out of range at 220 nm, use higher wave lengths at 226 or 230 nm. Thereby, impurities eluting close to the target peptide can be distinguished. 12. To our experience, it is difficult to couple more than one phosphoamino acid derivative in one peptide. This is due to a reduced reactivity and/or sterical hindrance, especially if two phosphoamino acids are in close proximity. 13. Do not use higher concentrations of the amino acid derivatives to prevent the risk of precipitation of some derivatives, which may block the instrument. 14. If amino acids with unprotected hydroxyl groups are incorporated, it is better to use carbodiimide activation (such as DIC/HOBt-activation) instead of more reactive procedures to avoid acylation of the hydroxyl-groups. 15. The N-terminal position can be incorporated as a Boc-protected amino acid to omit piperidine cleavage. Otherwise, the Fmoc group is cleaved prior to the global phosphorylation procedure, as fully protected phosphoserine and -threonine are base labile yielding dehydroalanine and dehydrobutyric acid, respectively. Although the N-terminal amino group is also modified by the reagent, this phosphoramidite is cleaved quantitatively during the peptide workup. Usually we phosphorylate peptides with free N-termini. 16. The size of the tube depends on the amount of resin to be phosphorylated resulting in certain volumes of the reagents. For appropriate mixing the reagent volume should not exceed half of the total tube volume. 17. All reagents are applied through the septum using syringes. To keep a constant nitrogen pressure a balloon filled with nitrogen is connected to the tube via a needle through the septum. 18. It is obligatory to use clean and dry syringes. Add first the required volume of DMF (dry, free of amines and oxygen) to the syringe and discharge any gas bubbles from the syringe. Before adding the amidite reagent, place the bottle containing the amidite under nitrogen atmosphere (see Note 17). Add the required volume of amidite reagent and mix the solution by rotating or shaking the syringe. 19. If the tetrazol precipitates (formation of clear white crystals) after addition of the amidite, add more DMF until the precipitate is completely dissolved. 20. <strong>Peptide</strong>s containing no amino acids prone to oxidation (i.e., neither Cys nor Met) the stronger oxidation reagent meta-chloroperbenzoic acid (MCPBA, 20 eq) is preferred as a 0.3 M solution in dry acetonitrile. 21. If the peptide is not completely dissolved add some acetonitrile. 22. If polar sequences or short phosphopeptides fail to bind to the stationary phase upon loading, the acetonitrile content of the eluent could be reduced to 3%. A further decrease is not possible for standard C18-columns, as the stationary phase could collapse reducing the loading capacity and the reproducibility. Instead, TFA could be replaced by the more hydrophobic ion-pair reagent HFBA (heptafluorobutyric acid), or a stationary phase with a special coating compatible with pure aqueous eluents could be used (e.g., Aqua-column from Phenomenex).
222 Singer and Hoffmann Acknowledgment We thank the Deutsche Forschungsgemeinschaft (DFG) and the European Regional Development Fund (EFRE, European Union and Free State Saxonia) for financial support. References 1. McMurray, J.S., Coleman IV, D.R., Wang, W., and Campbell, M.L. (2001) The synthesis of phosphopeptides. Biopolymers 60, 1, 3–31. 2. Bannwarth, W. and Trzeciak, A. (1987) A simple and effective dhemical phosphorylation procedure for biomolecules. Helv. Chim. Acta 70, 175–186. 3. Perich, J.W. and Johns, R.B. (1988) Di-tert-butyl N,N-diethylphosphoramidite. A new phosphitylating agent for the efficient phosphorylation of alcohols. Synthesis 142–144. 4. Handa, K.B. and Hobbs, C.J. (1998) An efficient and convenient procedure for the synthesis of Nα-Fmoc-O-monobenzyl phosphonotyrosine. J. Pept. Sci, 4, 138. 5. Kitas, E.A., Perich, J.W., Wade, J.D., Johns, R.B., and Treagear, G.W. (1989) Fmoc-polyamide solid phase synthesis of an O-phosphotyrosine-containing tridecapeptide. Tetrahedron Lett. 30, 45, 6229–6232. 6. Vorherr, T. and Bannwarth, W. (1995) Phospho-serine and phospho-threonine building blocks for the synthesis of phosphorylated peptides by the Fmoc solid phase strategy. Bioorg. Med. Chem. Lett. 5, 2661–2664. 7. Singer, D., Lehmann, J., Hanisch, K., Härtig, W., and Hoffmann, R. (2006) Neighbored phosphorylation sites as PHF-tau specific markers in Alzheimer’s disease. Biochem. Biophys. Res. Comm. 346, 819–828. 8. Zeller, M. and König, S. (2004) The impact of chromatography and mass spectrometry on the analysis of protein phosphorylation sites. Anal. Bioanal. Chem. 378, 898–909. 9. Hoffmann, R., Wachs, O.W., Berger, G.R., et al. (1995) Chemical phosphorylation of the peptides GGXA (X = S,T,Y): an evaluation of different chemical approaches. Int. J. Pept. Prot. Res. 45, 26–34. 10. Wakamiya, T., Saruta, K., Yasuoka, J., and Kusumoto, S. (1994) An efficient procedure for solid-phase synthesis of phosphopeptides by the Fmoc strategy. Chem. Lett. 1099–1102.
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Peptide-Based Drug Design
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METHODS IN MOLECULAR BIOLOGY TM Pep
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Preface Natural products chemistry
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Contents Preface...................
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Contributors Nikolinka Antcheva •
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Contributors xi Alessandro Tossi
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2 Otvos advance of computer power a
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4 Otvos see derivatives active in b
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6 Otvos was designed based on ligan
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8 Otvos 27. Borghouts, C., Kunz, C.
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10 Bulet Key Words: Invertebrate im
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12 Bulet 6. 5 �L or higher volume
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14 Bulet 2.5.2.1. MALDI-TOF-MS 1. M
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16 Bulet 7. Small-volume low-protei
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18 Bulet 3. Centrifuge between 8000
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20 Bulet internal diameter). Increa
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22 Bulet interest. As no instrument
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24 Bulet 3. Incubate the plates in
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26 Bulet bioactive peptides from th
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28 Bulet 21. Chernysh, S., Kim, S.I
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3 Sequence Analysis of Antimicrobia
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Sequence Analysis of Antimicrobial
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4 The Spot Technique: Synthesis and
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The Spot Technique 49 3. For amine
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The Spot Technique 51 2. Biotinylat
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The Spot Technique 53 the solutions
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The Spot Technique 55 solution. Ens
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The Spot Technique 57 (see Fig. 3)
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The Spot Technique 59 Fig. 5. Princ
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The Spot Technique 61 library, the
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The Spot Technique 63 7. Regenerati
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The Spot Technique 65 following rul
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The Spot Technique 67 20. Atherton,
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The Spot Technique 69 50. Bolger, G
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5 Analysis of A� Interactions Usi
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Analysis of Aβ Interactions 73 are
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Analysis of Aβ Interactions 75 Ana
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Analysis of Aβ Interactions 77 3.
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6 NMR in Peptide Drug Development J
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NMR of Peptides 89 usually require
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NMR of Peptides 91 limiting the siz
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NMR of Peptides 93 and STD NMR expe
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NMR of Peptides 95 The basis of the
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NMR of Peptides 97 Fig. 5. Overlay
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NMR of Peptides 99 Fig. 7. Schemati
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NMR of Peptides 101 4.4.2. Saturati
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NMR of Peptides 103 4.6. Diffusion
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NMR of Peptides 105 Fig. 13. Propos
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NMR of Peptides 107 protein prevent
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NMR of Peptides 109 6. Wuthrich, K.
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NMR of Peptides 111 45. Morris, K.
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NMR of Peptides 113 78. Aumailley,
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116 Copps et al. Fig. 1. Potential
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118 Copps et al. Fig. 2. Flowchart
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120 Copps et al. 10. In accordance
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122 Copps et al. Fig. 3. DSSP for g
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124 Copps et al. ingly with the sam
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126 Copps et al. 19. Essman, U., Pe
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128 Hilpert et al. Key Words: Scree
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130 Hilpert et al. Table 1 (Continu
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132 Hilpert et al. different natura
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134 Hilpert et al. wt A C D E F …
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136 Hilpert et al. methods primaril
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144 Hilpert et al. Table 3 Summary
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148 Hilpert et al. of substituting
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150 Hilpert et al. in models that c
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152 Hilpert et al. than control. Gi
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154 Hilpert et al. Table 4 Accuracy
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156 Hilpert et al. 25. Pini, A., Gi
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158 Hilpert et al. 55. Giacometti,
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9 Investigating the Mode of Action
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Proline-Rich Antimicrobial Peptides
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Page 180 and 181: Proline-Rich Antimicrobial Peptides
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Page 196 and 197: 11 Preparation of Glycosylated Amin
Page 198 and 199: Glycosylated Amino Acid Synthesis 1
Page 218 and 219: 12 Synthesis of O-Phosphopeptides o
Page 220 and 221: Solid-Phase Synthesis of O-Phosphop
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Page 234 and 235: Peptidomimetics 225 O O R S N H Pep
Page 236 and 237: Peptidomimetics 227 not without its
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Page 240 and 241: Peptidomimetics 231 of peptidosulfo
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Page 248 and 249: Peptidomimetics 239 nitrogen (73).
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Page 252 and 253: Peptidomimetics 243 efficient synth
Page 254 and 255: Peptidomimetics 245 55. Alsina, J.,
Page 256 and 257: 14 Synthesis of Toll-Like Receptor-
Page 258 and 259: Lipopeptides 249 2. Materials 2.1.
Page 260 and 261: Lipopeptides 251 Fig. 1. Structural
Page 262 and 263: Lipopeptides 253 8. To enable lipid
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Page 266 and 267: Lipopeptides 257 Fig. 2. Immunogeni
Page 268 and 269: Lipopeptides 259 8. A large plastic
Page 270 and 271: Lipopeptides 261 26. Nardin, E.H.,
Page 272 and 273: 264 Otvos response, synthetic pepti
Page 274 and 275: 266 Otvos Fig. 3. Schematic present
Page 276 and 277: 268 Otvos 3. Methods The main goal
Page 278 and 279: 270 Otvos 3.2.3. Purification and Q
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272 Otvos 6. Meyer, D., and Torres,
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16 Cysteine-Containing Fusion Tag f
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Targeted Therapeutic and Imaging Ag
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Index A A� peptides aggregation a
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Index 297 phosphopeptides influenci
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Index 299 for genetically synthetic
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Index 301 peptidosulfonamide, disad
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Index 303 solid phase, 180, 214 sul
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