Peptide-Based Drug Design

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Solid-Phase Synthesis of O-Phosphopeptides 211 ( tBu) for aspartic acid (Asp), glutamic acid (Glu), serine (Ser), threonine (Thr), and tyrosine (Tyr), tert-butyloxycarbonyl (Boc) for lysine (Lys) and tryptophan (Trp), triphenylmethyl (trityl, Trt) for cysteine (Cys), histidine (His), asparagine (Asn), and glutamine (Gln), as well as 2,2,4,6,7-pentamethyldihydrobenzofuran- 5-sulfonyl (Pbf) for arginine (Arg) (ORPEGEN Pharma, Heidelberg, Germany). 2. As solid support use Rink-amide MBHA resin (0.6 mmol/g, 100–200 mesh, MultiSynTec, Bochum, Germany) to obtain C-terminal peptide amides and Wang-resin (1.2 mmol/g, 100–200 mesh, MultiSynTec) or 2-chlorotrityl chloride resin (1.5 mmol/g, 100–200 mesh, Novabiochem, Merck KGaA, Darmstadt, Germany) to produce C-terminal free acids. 3. Instrument: Multiple peptide synthesizer SYRO (MultiSynTec) equipped with a 48-reaction vessel block (see Note 1). 4. Carbodiimide activation: a) N,N´-Diisopropylcarbodiimide (DIC), 2 M in DMF (Fluka Neu-Ulm, Germany). b) 1-Hydroxybenzotriazole hydrate (HOBt), 0.5 M in DMF (Fluka). 5. Uronium-reagents: a) O-(benzotriazol-1-yl)-N,N,N´,N´-Tetramethyluronium hexafluorophosphate (HBTU), 0.5 M in DMF (MultiSynTec). b) 1-Hydroxybenzotriazole hydrate (HOBt), 0.5 M in DMF (Fluka). c) N,N-Diisopropylethylamine (DIPEA), 1.33 M in NMP (Fluka). 6. Fmoc cleavage: a) Mixture of 20% piperidine in DMF (Fluka). b) Mixture of 40% piperidine in DMF (Fluka). 7. Dimethylformamide (DMF, Biosolve, Valkenswaard, the Netherlands; see Note 2). 8. N-Methylpyrrolidone (NMP, Biosolve). 9. Dichloromethane (DCM, Biosolve). 10. Methanol (Biosolve). 11. Ether (Merck KGaA, Darmstadt, Germany). 12. Reagents for peptide cleavage (Fluka): a) Trifluoroacetic acid (TFA) 10 mL (see Note 3). b) Scavenger mixture: 500 �L water, 500 �L thioanisole, 500 �L m-cresol, 250 �L 1,2-ethanedithiol. 2.2. Direct Incorporation of Fmoc Phosphoamino Acid Derivatives 1. Partially protected phosphoamino acids (Novabiochem, see Note 4): a) N-�-Fmoc-O-benzyl-l-phosphoserine (Fmoc-Ser(PO(OBzl)OH)-OH) (see Note 5).
212 Singer and Hoffmann b) N-�-Fmoc-O-benzyl-l-phosphothreonine (Fmoc-Thr(PO(OBzl)OH)-OH). c) N-�-Fmoc-O-benzyl-l-phosphotyrosine (Fmoc-Tyr(PO(OBzl)OH)-OH). d) N-�-Fmoc-O-benzyl-d-phosphoserine (Fmoc-D-Ser(PO(OBzl)OH)-OH). e) N-�-Fmoc-O-benzyl-d-phosphothreonine (Fmoc-D-Thr(PO(OBzl)OH)-OH). f) N-�-Fmoc-O-benzyl-d-phosphotyrosine (Fmoc-D-Tyr(PO(OBzl)OH)-OH). g) -N-�-Fmoc-O-(bis-dimethylamino-phosphono)-l-tyrosine (Fmoc-Tyr(PO (NMe2)2-OH). h) N-�-Fmoc-O-phospho-l-tyrosine (Fmoc-Tyr(PO3H2)-OH). 2.3. Global Phosphorylation of Partially Protected <strong>Peptide</strong>s 1. Side-chain–unprotected hydroxyamino acids (Novabiochem) (see Note 4): a) Fmoc-Ser-OH / Fmoc-D-Ser-OH (see Note 5). b) Fmoc-Thr-OH / Fmoc-D-Thr-OH. c) Fmoc-Tyr-OH. 2. Hydroxyamino acids with selectively cleavable side-chain–protecting groups (Novabiochem) (see Note 4): a) Fmoc-Ser(Trt)-OH / Fmoc-D-Ser(Trt)-OH (see Note 5). b) Fmoc-Thr(Trt)-OH / Fmoc-D-Thr(Trt)-OH. c) Fmoc-Tyr(2-ClTrt)-OH / Fmoc-D-Tyr(2-ClTrt)-OH. 3. Dibenzyl-N,N-diisopropylphosphoramidite (Fluka). 4. 1H-Tetrazole solution in acetonitrile (purum, ∼0.45 M, Fluka; see Note 6). 5. tert-Butylhydroperoxide solution, 5.5 M in decan, anhydrous (Fluka). 6. Acetonitrile (Biosolve; see Note 1). 7. Fifteen- or 50-mL polypropylene tubes, septa, syringes, and cannulae (6 and 12 cm). 2.4. Liquid Chromatography 1. Instrument: ÄKTA purifier 10 HPLC System (GE Healthcare, Munich, Germany) consisting of pump P-900, detector UV-900, autosampler A-905, and fraction collector Frac-950. 2. Analytical columns: a) ZORBAX Eclipse XDB-C8 (C8 stationary phase, 5 �m particle size, 80 ˚A pore size, 4.6 mm internal diameter and 150 mm long; Agilent Technologies GmbH, Böblingen, Germany). b) Jupiter C18 (C18 stationary phase, 5 �m particle size, 300 ˚A pore size, 4.6 mm internal diameter and 150 mm long; Phenomenex Inc, Torrance, CA). 3. Preparative column: Jupiter C18 (C18 stationary phase, 15 �m particle size, 300 ˚A pore size, 21.2 mm internal diameter and 250 mm long; Phenomenex Inc). 4. Eluents:
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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,
Page 170 and 171: 9 Investigating the Mode of Action
Page 172 and 173: 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
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Page 222 and 223: Solid-Phase Synthesis of O-Phosphop
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Page 234 and 235: Peptidomimetics 225 O O R S N H Pep
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Page 238 and 239: Peptidomimetics 229 Oxidation step:
Page 240 and 241: Peptidomimetics 231 of peptidosulfo
Page 242 and 243: Peptidomimetics 233 8. Allow the re
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Page 248 and 249: Peptidomimetics 239 nitrogen (73).
Page 250 and 251: Peptidomimetics 241 3. Cover the re
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Page 254 and 255: Peptidomimetics 245 55. Alsina, J.,
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Page 258 and 259: Lipopeptides 249 2. Materials 2.1.
Page 260 and 261: Lipopeptides 251 Fig. 1. Structural
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Page 266 and 267: Lipopeptides 257 Fig. 2. Immunogeni
Page 268 and 269: Lipopeptides 259 8. A large plastic
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Lipopeptides 261 26. Nardin, E.H.,
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264 Otvos response, synthetic pepti
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266 Otvos Fig. 3. Schematic present
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268 Otvos 3. Methods The main goal
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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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