A protocol has been developed far the synthesis of bisubstrate analogs of protein kinases containing adenosine 5’-phosphates (AP(n), n = 2-4) and substrate peptides attached covalently through the hydroxyl group of serine. The procedure involves the coupling of the terminal phosphate group of the phosphoadenosine to phosphorylated substrate peptide(s) after activation with 1,1’-carbonyldiimidazole. The peptide substrates were synthesized by standard Fmoc chemistry and phosphorylated at the target serine using either di-tert-butyl or diallyl N,N-diisopropylphosphoramidite, followed by oxidation with tert-butyl hydroperoxide. Allyl protection was preferred in the solid phase strategy since these groups could be selectively removed and the activated AP(n) moieties coupled to the phosphopeptides while still bound to the resin for a yield of approximate to 40-80%. A less satisfactory approach was initially attempted using di-tert-butyl N,N-diisopropylphosphoramidite as the phosphite transfer reagent to yield free phosphopeptides which were subsequently coupled in a solution phase reaction (less than or equal to 2-3% yield). The high efficiency of the solid phase method affords a general approach for synthesizing peptide-nucleoside analogs as well as potentially other types of covalent peptide-ligand complexes through the formation of phosphodiester linkages. In this study, a series of bisubstrate inhibitor candidates were synthesized using this new approach based on kemptide (LRRASLG), a substrate of cAMP dependent protein kinase (cAPK), and several extended analogs. The bisubstrate analogs containing ADP, ATP, and AP(4) were tested as inhibitors of the catalytic subunit of cAPK. IC50 values were determined and the best inhibitor in this series was the adenosine 5’-tetraphosphate peptide kemptide-AP(4) (IC50 = 68 mu M). The efficiency of inhibition was found to decrease rapidly with shorter phosphate chains, yielding IC50 values of 226 and 935 mu M for kemptide-ATP and kemptide-ADP, respectively. All three inhibitors in this series acted competitively with respect to ATP but not with respect to the peptide substrate, suggesting that larger peptides may be required to generate bisubstrate inhibitors for cAPK.