Peptide-Based Drug Design

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).