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Intens.x108Dppa-Gly-Arg-Thr-Leu (L) + Cu 2+Pba-Gly-Arg-Thr-Leu (L) + Cu 2+1.51.00.5x10 4cal[L-H+Cu(II)] +386.1858.3198[2L-2H+2Cu(II)] 2+[L+Cu(II)] 2+[L+H]858.3196797.403859.328860.316[2L-H+2Cu(II)] 3+*[L+Cu(II)] 2+572.546573.212858.821859.820430.165313.279501.288622.042572.535572.881861.326 573.547860.818862.329858.298573.881cal574.213859.301 860.296 574.547574.880575.216922.033861.300573.201862.303572.535572.869573.535858.298859.297858.800859.799573.869860.301574.201860.798574.536574.870861.300861.801572 573 574 575 m/z858 860 862 m/z1524.948 1707.438 1843.0441372.6271053.8260.0200 400 600 800 1000 1200 1400 1600 1800 m/zloss of CO 2and Leu backbone342.6[L-2H+2Cu(II)] 2+formation. The observed fragments result from deterioration of peptide chain and,according to isotopic pattern analysis, all contain the copper ion, suggesting the relativestability of the complex and localization of metal ion in heterocyclic part of the conjugate.The results of CD and NMR experiments imply that the interactions with Cu(II) ion affectthe structure of peptide conjugate with a shift to a more ordered conformation. The primaryinteraction site is located in the bipyridyl-like motif of the heterocyclic moiety.The immunomodulatory activity of the investigated conjugates and their complexeswas examined in vitro in the mouse model of secondary humoral immune response to sheeperythrocytes (SRBC) using the antibody forming cells (AFC) test. The effect of theheterocyclic motif was established after comparison with the original ubiquitin sequence(peptide I, 83% immunosuppression at the dose of 100 μg/ml), whereas the sequenceinvolvement was examined in relation to respective dipeptides (Xaa-Gly). The unusuallyhigh immunostimulatory effect of conjugate III, containing a rigid phenazine skeleton(280% stimulation of immune response), seems to be unique for this combination ofheterocycle and peptide sequence, as a respective dipeptide Dppa-Gly was a strongimmunosuppressor (87% at the highest dose). All copper complexes of investigatedconjugates evoked significant immunosuppression (77 – 90% at the highest dose).ConclusionsThe combination of the immunomodulatory fragment DGRTL of the 50-59 loop ofubiquitin with heterocyclic motifs resulted in peptides with significant metal ion affinityand interesting biological properties. The MS experiments and NMR analysis indicate thecoordination of metal ion mostly by the heterocyclic component of the conjugate.Introducing the heterocyclic modifications to bioactive peptides may result in novelbioactive compounds - targeted metal carriers.AcknowledgmentsThis work was supported in part by grant No. N N401 222734 from the Ministry of Science andHigher Education (Poland).References1. Hershko, A., Ciechanover, A. Annu. Rev. Biochem. 67, 425-479 (1998).2. Pasikowski, P., Cydzik, M., Kluczyk, A., Stefanowicz, P., Szewczuk Z. Biomolecular Concepts 1,67-84 (2010).3. Szewczuk, Z., Stefanowicz, P., Wilczynski, A., Staszewska, A., Siemion, I.Z., Zimecki, M.,Wieczorek, Z. Biopolymers 74, 352-362 (2004).4. Jaremko, L., Jaremko, M., Pasikowski, P., Cebrat, M., Stefanowicz, P., Lisowski, M., Artym, J.,Zimecki, M., Zhukov, I., Szewczuk, Z. Biopolymers 91, 423-431 (2009).5. Staszewska, A., Stefanowicz, P., Szewczuk, Z. Tetrahedron Lett. 46, 5525-5528 (2005).6. Koprowska-Ratajska, M., et al. Amino Acids 36, 309-315 (2009).Intens.420323.2[L+H]355.7416.6446.7386.145387.145386.648387.647388.149386.147387.147386.649387.648388.149387 388 386 389416.606417.605417.108418.108418.605419.121416.604417.604417.106418.105418.604419.104416 417 418 419 420300 400 500 600 700Fig. 2. High resolution mass spectra of Cu(II) complexes of selected heterocyclic peptideconjugates. Isotopic patterns simulated for proposed complex formulas in red (gray).Cal – internal calibration was used to increase m/z precision.201