You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Solid-Phase Synthesis of O-Phosphopeptides 211<br />
( tBu) for aspartic acid (Asp), glutamic acid (Glu), serine (Ser), threonine (Thr),<br />
and tyrosine (Tyr), tert-butyloxycarbonyl (Boc) for lysine (Lys) and tryptophan<br />
(Trp), triphenylmethyl (trityl, Trt) for cysteine (Cys), histidine (His), asparagine<br />
(Asn), and glutamine (Gln), as well as 2,2,4,6,7-pentamethyldihydrobenzofuran-<br />
5-sulfonyl (Pbf) for arginine (Arg) (ORPEGEN Pharma, Heidelberg, Germany).<br />
2. As solid support use Rink-amide MBHA resin (0.6 mmol/g, 100–200 mesh,<br />
MultiSynTec, Bochum, Germany) to obtain C-terminal peptide amides and<br />
Wang-resin (1.2 mmol/g, 100–200 mesh, MultiSynTec) or 2-chlorotrityl chloride<br />
resin (1.5 mmol/g, 100–200 mesh, Novabiochem, Merck KGaA, Darmstadt,<br />
Germany) to produce C-terminal free acids.<br />
3. Instrument: Multiple peptide synthesizer SYRO (MultiSynTec) equipped with a<br />
48-reaction vessel block (see Note 1).<br />
4. Carbodiimide activation:<br />
a) N,N´-Diisopropylcarbodiimide (DIC), 2 M in DMF (Fluka Neu-Ulm,<br />
Germany).<br />
b) 1-Hydroxybenzotriazole hydrate (HOBt), 0.5 M in DMF (Fluka).<br />
5. Uronium-reagents:<br />
a) O-(benzotriazol-1-yl)-N,N,N´,N´-Tetramethyluronium hexafluorophosphate<br />
(HBTU), 0.5 M in DMF (MultiSynTec).<br />
b) 1-Hydroxybenzotriazole hydrate (HOBt), 0.5 M in DMF (Fluka).<br />
c) N,N-Diisopropylethylamine (DIPEA), 1.33 M in NMP (Fluka).<br />
6. Fmoc cleavage:<br />
a) Mixture of 20% piperidine in DMF (Fluka).<br />
b) Mixture of 40% piperidine in DMF (Fluka).<br />
7. Dimethylformamide (DMF, Biosolve, Valkenswaard, the Netherlands; see<br />
Note 2).<br />
8. N-Methylpyrrolidone (NMP, Biosolve).<br />
9. Dichloromethane (DCM, Biosolve).<br />
10. Methanol (Biosolve).<br />
11. Ether (Merck KGaA, Darmstadt, Germany).<br />
12. Reagents for peptide cleavage (Fluka):<br />
a) Trifluoroacetic acid (TFA) 10 mL (see Note 3).<br />
b) Scavenger mixture: 500 �L water, 500 �L thioanisole, 500 �L m-cresol,<br />
250 �L 1,2-ethanedithiol.<br />
2.2. Direct Incorporation of Fmoc Phosphoamino Acid Derivatives<br />
1. Partially protected phosphoamino acids (Novabiochem, see Note 4):<br />
a) N-�-Fmoc-O-benzyl-l-phosphoserine (Fmoc-Ser(PO(OBzl)OH)-OH) (see<br />
Note 5).