Peptide-Based Drug Design

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The Spot Technique 51 2. Biotinylation solution: 10 mM solution of the activated biotin in dimethyl sulfoxide (DMSO). Prepare this mixture always fresh before use. 3. Purification columns: PD-10 columns (Amersham). 4. HRP-labeled streptavidin. 3. Methods If not noted elsewhere, all washing, incubation, and reaction steps could be performed using a rocking shaker. 3.1. Spot Synthesis of Macroarrays Spot synthesis may be carried out manually, semi- or fully automated (see Fig. 1). Manual spot synthesis is convenient for the synthesis and screening of a relative small number of peptides (up to 100) and a large pipetting volume (>0.5 �L). For all other cases a semiautomated or fully automated synthesis is recommended. For synthesis of small spots with a pipetting volume of about 0.1 �L, spot synthesizers from Intavis AG (Köln, Germany) are very useful (3). If not noted elsewhere, all data correspond to the preparation of a 10 × 12 cm cellulose membrane (96-well plate size). For larger membranes preparation of related amounts of reagents is required. If we write about the use of amino acids, it should always include the use of other organic building blocks (e.g., PNA monomers, peptoidic elements, heterocycles) (22,23), which can be used under spot synthesis conditions. Here we describe only the basic procedures for spot synthesis of linear peptides. For the synthesis of modified peptides, such as cyclization or side-chain modifications, see ref. (24). 3.1.1. Preparation of Coupling Solutions Preparation of the activated amino acid solutions can be performed according to two different methods. One method involves the use of preactivated Fmoc-protected amino acids (e.g., pentafluorophenyl ester). This method has the advantage that only one reagent is necessary since preparation of the amino acid solutions is very simple and the likelihood of mistakes is low. One disadvantage is the higher price of the amino acid derivatives, but due to the small amount of activated amino acids used the absolute difference falls in the range of a few dollars for synthesis of an entire peptide membrane array. Another disadvantage lies in the fact that activated esters are only commercially available for the standard amino acids. The other method requires the use of in situ activated amino acids. Activation of the amino acids is carried out by adding an activator and coupling reagent to the unactivated Fmoc-protected amino acid derivative. This method is more time
52 Winkler and Campbell Fig. 1. Outline of spot synthesis (shown without washing steps). consuming than the firstmethod butcan be applied forallbuilding blocks available for use in peptide synthesis according to the Fmoc-protecting group strategy. 3.1.1.1. METHOD 1: PREPARATION OF 0.3 M SOLUTIONS WITH PREACTIVATED AMINO ACID DERIVATIVES Except for the arginine derivative, solutions of preactivated amino acids can be prepared and used for an entire week. Dissolve most of the amino acid derivatives in amine-free NMP at a concentration of 0.3 M. Due to poor solubility, derivatives of serine, threonine, tryptophan, and proline must be dissolved in amine-free DMF. The solutions can be stored at −20◦C for at least 1 wk. Use
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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 •
Page 12 and 13: Contributors xi Alessandro Tossi
Page 14 and 15: 2 Otvos advance of computer power a
Page 16 and 17: 4 Otvos see derivatives active in b
Page 18 and 19: 6 Otvos was designed based on ligan
Page 20 and 21: 8 Otvos 27. Borghouts, C., Kunz, C.
Page 22 and 23: 10 Bulet Key Words: Invertebrate im
Page 24 and 25: 12 Bulet 6. 5 �L or higher volume
Page 26 and 27: 14 Bulet 2.5.2.1. MALDI-TOF-MS 1. M
Page 28 and 29: 16 Bulet 7. Small-volume low-protei
Page 30 and 31: 18 Bulet 3. Centrifuge between 8000
Page 32 and 33: 20 Bulet internal diameter). Increa
Page 34 and 35: 22 Bulet interest. As no instrument
Page 36 and 37: 24 Bulet 3. Incubate the plates in
Page 38 and 39: 26 Bulet bioactive peptides from th
Page 40 and 41: 28 Bulet 21. Chernysh, S., Kim, S.I
Page 42 and 43: 3 Sequence Analysis of Antimicrobia
Page 44 and 45: Sequence Analysis of Antimicrobial
Page 58 and 59: 4 The Spot Technique: Synthesis and
Page 60 and 61: The Spot Technique 49 3. For amine
Page 64 and 65: The Spot Technique 53 the solutions
Page 66 and 67: The Spot Technique 55 solution. Ens
Page 68 and 69: The Spot Technique 57 (see Fig. 3)
Page 70 and 71: The Spot Technique 59 Fig. 5. Princ
Page 72 and 73: The Spot Technique 61 library, the
Page 74 and 75: The Spot Technique 63 7. Regenerati
Page 76 and 77: The Spot Technique 65 following rul
Page 78 and 79: The Spot Technique 67 20. Atherton,
Page 80 and 81: The Spot Technique 69 50. Bolger, G
Page 82 and 83: 5 Analysis of A� Interactions Usi
Page 84 and 85: Analysis of Aβ Interactions 73 are
Page 86 and 87: Analysis of Aβ Interactions 75 Ana
Page 88 and 89: Analysis of Aβ Interactions 77 3.
Page 98 and 99: 6 NMR in Peptide Drug Development J
Page 100 and 101: NMR of Peptides 89 usually require
Page 102 and 103: NMR of Peptides 91 limiting the siz
Page 104 and 105: NMR of Peptides 93 and STD NMR expe
Page 106 and 107: NMR of Peptides 95 The basis of the
Page 108 and 109: NMR of Peptides 97 Fig. 5. Overlay
Page 110 and 111: 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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10 Serum Stability of Peptides Håv
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Serum Stability of Peptides 179 2.
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Serum Stability of Peptides 183 �
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11 Preparation of Glycosylated Amin
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Glycosylated Amino Acid Synthesis 1
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12 Synthesis of O-Phosphopeptides o
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Solid-Phase Synthesis of O-Phosphop
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13 Peptidomimetics: Fmoc Solid-Phas
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Peptidomimetics 225 O O R S N H Pep
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Peptidomimetics 227 not without its
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Peptidomimetics 229 Oxidation step:
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Peptidomimetics 231 of peptidosulfo
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Peptidomimetics 233 8. Allow the re
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Peptidomimetics 235 3.3.2. Solid-Ph
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Peptidomimetics 237 On the other ha
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Peptidomimetics 239 nitrogen (73).
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Peptidomimetics 241 3. Cover the re
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Peptidomimetics 243 efficient synth
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Peptidomimetics 245 55. Alsina, J.,
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14 Synthesis of Toll-Like Receptor-
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Lipopeptides 249 2. Materials 2.1.
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Lipopeptides 251 Fig. 1. Structural
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Lipopeptides 253 8. To enable lipid
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Lipopeptides 255 Representative res
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Lipopeptides 257 Fig. 2. Immunogeni
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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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