Phenylthiohydantoine Phosphate

Abbreviations AllocOSu, allyloxycarbonyl-A-hydroxysuccinimide BCIP, 5-bromoA-chloro-3-indolyl Boc, tert-butyloxycarbonyl tBu, tert-butyl DCM, dichloromethane Dde, l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)ethyl Ddz, a, a-dimethyl-3,5-dimethoxyben-zyloxycarbonyl DIC, A,A -diisopropylcarbodiimide DIEA, A,fV-diisopropylethylamine DMF, iV,./V-dimethylformamide FmocOSu, 9-fluorenylmethyloxycarbonyl-AMiydroxysucci-nimide OBOC, one-bead one-compound PBS, phosphate-buffered saline Pmc, 2,2,5,7,8-pentamethylchroman-6-sulfonyl PTH, phenylthiohydantoin RR, relative reactivities Teoc, 2-(trimethylsilyl)ethoxycarbonyl TFA, trifluoroacetic acid Trt, trityl. 

This protocol has several shortcomings. Besides being a potential radioactive hazard, the entire procedure is tedious, labor intensive, prone to sample losses, and often fails to identify the exact site of phosphorylation. In some cases, complete incorporation of [P] fails to occur. Also, the multiplicity of the phosphorylated species within a cell creates problems in achieving the steady state. The identification of phosphorylated amino acids by the Edman procedure is also problematic. Under the harsh conditions employed in the Edman degradation, the phosphate ester bonds to serine and threonine are less stable. Both residues undergo -elimination of H3PO4 to form dehydroalanine and P-methyldehydroalanine, respectively, and phenylthiohydantoin (PTH)-dithioerythreitol as by-products. 

The release of free [ P]phosphate at a particular cycle, which results from P elimination during cyclization, indicates the presence a P.Ser or P.Thr residue. P.Tyr is stable to cyclization and is released as the anilinothiazolinone derivative of P.Tyr. This can be converted to the phenylthiohydantoin (PTH) derivative of P.Tyr by incubation in 0.1 N HCl for 20 min at 80°C. Marker PTH-P.Tyr is readily synthesized by reacting P.Tyr with phenylisothiocyanate as described above in step 2, and can be detected as a dark spot when the TLC plate is examined under a hand-held UV light. In addition to the cycle at which free p P]phosphate or p P]PTH-P.Tyr is released, information on the sequence of the peptide may be obtained by the electrophoretic mobility shifts detected at each cycle. Thus, if a positive or negatively charged amino acid is removed at a cycle before the phosphoamino acid, there will be a corresponding shift in the mobility of the peptide. Remember that if the peptide contains a C-terminal lysine this will react with phenylisothiocyanate at cycle 1, which will cause the loss of a positive charge. It may be difficult to determine the position of a second, more C-terminal phosphorylated residue present in the same peptide. 

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