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NMR studies of the hydroperoxide form of cobalt deglycoBleomycin A2 (HOO−CodGBLM) and its complex with oligonucleotide d(CCAGGCCTGG) (1, C is the site of cleavage) are presented in an effort to establish deglycoBLM as a prototype for studies with BLM analogues, synthesized without the sugar moieties (Boger et al. Bioorg. Med. Chem. 1995, 3, 1281−1295). The structure determination of free HOO−CodGBLM has been hampered by the lack of NOE or ROE information. By direct comparison of the chemical shifts and coupling constants of HOO−CodGBLM with its glycosylated parent HOO−CoBLM, the former is shown to share many global structural features with the latter, including the nature of axial ligands (a point recently disputed by Caceres-Cortes et al. Eur. J. Biochem. 1997, 244, 818−828), the screw sense of metal-coordinating ligands, and the folding of the peptide linker region. The structure of HOO−CodGBLM bound to 1 is reported based on molecular modeling using NMR constraints (39 intermolecular and 44 intramolecu...

A model for the structure of the aquated form of cobalt bleomycin (H2O-CoBLM or CoBLM A2 brown), free and bound to d(CCAGGCCTGG)2 (1) is reported based on molecular modeling using the constraints obtained from 2D NMR studies. CoBLM A2 brown has a chiral organization of its ligands, including the axial primary amine of β-aminoalanine, identical to the hydroperoxide form of cobalt BLM (HOO-CoBLM or CoBLM A2 green). H2O-CoBLM forms a 1:1 complex with 1 with a Kd of 2 × 10-6 M which is in slow exchange on the NMR time scale. The complex exhibits 44 intermolecular NOEs and 56 intramolecular NOEs within H2O-CoBLM itself. Molecular modeling reveals that H2O-CoBLM's mode of binding, basis for sequence specificity, and chemical specificity are almost identical to that previously reported for HOO-CoBLM (Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281−1294). The bithiazole tail is inserted from the minor groove 3‘ to C6 and the terminal thiazolium ring is well ...

The structure of homogeneous Co·Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) has been determined using 2D NMR methods and molecular dynamics calculations. Previous studies of Xu et al. (Xu, R. X.; Nettesheim, D.; Otvos, J. D.; Petering, D. H. Biochemistry 1994, 33, 907−916) reported several possible structures for CoBLM A2 green compatible with their NMR data acquired on a mixture of CoBLM A2 green and A2 brown forms. The availability of the pure CoBLM A2 green, which is stable for months at neutral pH, has allowed the complete assignments of the 1H and 13C chemical shifts, observation of 55 intramolecular NOEs, and determination of 15 coupling constants allowing the definition of dihedral angles. These results are a prerequisite to determining its structure with duplex DNA of a defined sequence (Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281−1294). Two screw sense isomers each containing two possible axial ligands (the primary amine...

The solution structure of Co·Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) complexed with the self-complementary oligonucleotide d(CCAGGCCTGG) with a cleavage site at C6 has been determined by 2D NMR spectroscopic methods and molecular dynamics calculations. Intermolecular NOEs (60 between CoBLM A2 green and DNA) and intramolecular NOEs (61 within CoBLM A2 green) have defined the position and orientation of CoBLM A2 green with respect to its single binding site in the duplex. CoBLM A2 green is a stable analog of the activated BLM, the Fe3+ hydroperoxide (Sam, J. W.; Tang, X.-J.; Peisach, J. J. Am. Chem. Soc. 1994, 116, 5250−5256). These studies have provided the first structural insight into the mode of binding of the bithiazole tail of CoBLM A2 green to DNA, the basis for specificity of its cleavage at pyrimidines (Py) in d(G-Py) sequences, and the orientation of its terminal oxygen of the hydroperoxide relative to the 4‘ carbon hydrogen bond being cleaved in the DNA. The bithiazole tail i...