Spectroscopic Investigation of the Metal Ligation and Reactivity of the Ferrous Active Sites of Bleomycin and Bleomycin Derivatives

Abstract

The geometric and electronic structures of high-spin ferrous complexes of bleomycin (FeIIBLM) and a series of systematically perturbed BLM derivatives have been investigated by optical absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopies. The active site of the unmodified drug complex is six-coordinate with the coordination sphere completed by at least five endogenous ligands including the pyrimidine, imidazole, deprotonated amide, and secondary and primary amine functionalities with either the 3-O-carbamoyl substituent of the mannose sugar or solvent bound at the sixth site. This weak sixth ligand is the exchangeable site of exogenous small molecule binding. Perturbing the carbamoyl substituent alters the coordination environment of the metal and decreases the azide binding affinities of the perturbed complexes. This is correlated with altered DNA cleaving capabilities. Additionally, altering the binding of the axial primary amine significantly affects the iron coordination sphere as evidenced by reduced π-back-bonding interactions specifically with the pyrimidine ligand. This pyrimidine π-back-bonding appears to play a key role in mediating the electron density localized on the ferrous center, which contributes to the unique oxygen chemistry and reactivity exhibited by FeIIBLM relative to other non-heme iron sites. Oxygen binding to derivatives in which the β-aminoalanine fragment has been removed leads to a high-spin ferric complex and no observed DNA strand scission, in contrast to the long-lived low-spin activated BLM intermediate that precedes DNA degradation.

Possible structural role of the disaccharide unit in Fe-bleomycin before and after oxygen activation

“…This conclusion is based on the diminished paramagnetic character exhibited by the carbohydrate moiety in FeAZBLM when compared with its parent metallo-BLM. These results are in agreement with the MCD studies on FeBLM and its derivatives (iso-and decarbamoyl-BLM) performed by Loeb et al 17 In their study it was observed that upon binding of AZ, the MCD spectra of these complexes changed to an identical form, which was found consistent with the assignment of the sixth ligand as the carbamoyl substituent. Although the MCD studies referred to above have already suggested the release of the M moiety from coordination to the metal center upon addition of AZ, the NMR studies of FeAZBLM presented herein give a wider scope of the molecular changes that take place as a consequence of AZ binding, as they extend way beyond the coordination cage of the metal.…”

“…18,19 The results of this modelling work indicated that the most favourable structures for the aforementioned metalloBLMs is six coordinated, with the primary amine and either the carbamoyl group in the mannose (M) moiety or a solvent molecule occupying the axial sites. Our results bolster the arguments offered by Loeb et al 17 in their spectroscopic investigation of the metal ligation of the ferrous active site of BLM through magnetic CD (MCD).…”

“…[6][7][8][9][10] The overall structure of this agent can be thought of as containing four distinct regions ( Figure 1): the metalbinding domain, which is responsible for metal binding, 11,12 oxygen activation 8,11,13,14 and site-selective DNA cleavage; 12,15 the peptide linker; the DNA binding domain, containing a bithiazole moiety, which provides the majority of the DNA binding affinity, 7,16 and the disaccharide moiety, which may influence metal ion binding. 12,[17][18][19][20][21][22][23][24] With the aim of establishing structure-function correlations, much research has been devoted to the elucidation of the three-dimensional structures of some metallo-BLMs. 25 The results of these studies have led to the general agreement that the secondary amine of b-aminoalanine (A) segment, the pyrimidinylpropionamide (P) and imidazole rings, and the amide nitrogen in b-hydroxyhistidine (H) are the equatorial ligands to the metal centers in most of the metal-BLM adducts studied to date.…”

“…AZ is a small molecule that has been widely and successfully used to probe the dioxygen binding site in FeBLM. 14,17,25 Although the coordination chemistry of FeBLM was assessed by us previously, 24 and the NMR features of the components of the BLM mixture found in blenoxane have already been assigned, 29 we decided to re-visit Fe-and apo-blenoxane in the present work. This is our way to ensure that the comparison between the parent and the AZ-bound molecules is done under the exact same NMR and sample preparation conditions, and to guarantee successful assignments through saturation transfer (ST) experiments.…”

Further insights into the mechanism of the reaction of activated bleomycin with DNA

“…Preparation of Fe II BLM-DNA. Ferrous BLM was prepared as previously described (14,(33)(34)(35). Oligonucleotides [10 bp; 5Ј-d(GGAAGCT-TCC) 2-3Ј] from Operon DNA were dissolved in Hepes buffer (100 mM, pH 7.4) and heated in a 90°C sand bath for 5 min to anneal the single strands.…”