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We are currently developing strategies to synthesize bisubstrate analogs as potential inhibitors of serine and tyrosine protein kinases; several such analogs have been synthesized. The initial target proteins were the cAMP dependent protein kinase (cAPK) and the Ca(+2)/calmodulin dependent protein kinase (CaM kiiase II). These bisubstrate analogs were based on either known peptide substrates such as kemptide, a seven amino acid peptide substrate of cAPK, or on inhibitory peptides such as a seventeen amino acid peptide encompassing the autoinhibitory domain of CaM kinase II. Peptides containing a single phosphoserine group were first synthesized and then adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), or adenosine 5'-triphosphate (ATP) was coupled through the serine phosphate with prior activation by 1,1-carbonyldiimidazole using either a solution or solid phase reaction scheme. In this current study, we report the characterization of the bisubstrate analogs by liquid secondary ionization mass spectrometry (LSIMS), matrix-assisted laser desorption mass spectrometry (MALDI), and tandem mass spectrometry (MS/MS).In the positive-ion mode, the LSIMS spectra of the bisubstrate analogs yielded a series of molecular ions containing mono-, di-, and trivalent cation adducts. Cation adducts were absent in the negative-ion mode where the dominant species were deprotonated molecular ions, [M - H](-), making this latter technique more useful for confirming product identity and assessing purity. Analysis of these compounds by MALDI in both the positive- and negative-ion modes yielded molecular ions which also contained metal ion adducts, although they were limited primarily to Fe(+2) adducts. Unlike LSIMS, the MALDI spectra showed no evidence for the elimination of the phosphoadenosine or other structural moieties. When these compounds were subjected to high energy collision-induced dissociation (CID), the dominant fragmentation pathways under positive-ion MS/MS conditions resulted from cleavage of the phosphate linkages to the adenosine moiety with charge retention on the peptide, although a major peak for 5'-deoxyadenosine was also seen at m/z 250. Charge retention in the negative-ion mode was most pronounced for ion fragments containing the highly acidic phosphate moieties and yielded phosphoadenosine related ions, for example, (AMP-H)(-), (AMP-H-H2O)(-), (ADP-H)(-), etc., as well as ions originating from the phosphate linker such as PO3 (-), H2PO4 (-), HP2O6 (-), H3P2O7 (-), and H2P3O9 (-). The largest phosphoadenosine ion in the negative-ion CID spectra for each bisubstrate analog, for example, m/z 426 (ADP-H)(-), m/z 506 (ATP-H)(-), or m/z 586 (AP4-H)(-), indicated that the desired covalent modification had been formed between the phosphoserine and APn moieties.

Mass spectrometric characterization of a series of adenosylated peptides acting as bisubstrate analogs of protein kinases