RESUMO
Four novel heterobimetallic ate complexes containing cis-2,6-dimethylpiperidide (cis-DMP) have been prepared and characterised. Two contain one cis-DMP ligand, namely the bisalkyl-amido lithium, and sodium zincates [(TMEDA) x MZn(cis-DMP)(tBu)2] (M = Li for 1, Na for 2). Both 1 and 2 are synthesised by co-complexation of the respective alkali metal amide with di-tert-butylzinc in the presence of a molar equivalent of N,N,N',N'-tetramethylethylenediamine (TMEDA) in a hydrocarbon medium. The third complex, containing two cis-DMP ligands, is the alkyl-bisamido sodium zincate [(TMEDA) x NaZn(cis-DMP)2(tBu)] 3. Complex 3 is prepared from 2 via a disproportionation reaction where the by-product is [(TMEDA) x NaZn(tBu)3]. Another alkyl-diamido sodium zincate, [(TMEDA) x NaZn(DIBA)2(tBu)] 4 is synthesised by utilising diisobutylamine [DIBA(H)]. This reaction emphasises the generality of this disproportionation process. Complex 5 contains three cis-DMP ligands and is a tris-amido sodium magnesiate [(TMEDA) x NaMg(cis-DMP)3]. It is prepared by treating an equimolar mixture of butylsodium and dibutylmagnesium with three and one molar equivalents of cis-DMP(H) and TMEDA respectively, in hydrocarbon solution. By comparison of 1-5 with appropriate complexes from the literature, it has been possible to experimentally determine that the steric bulk of cis-DMP closely resembles that of DA but is considerably less bulky than 2,2,6,6-tetramethylpiperidide (TMP).
RESUMO
Recent crystal structures of cysteine dioxygenase (CDO) suggest the presence of two posttranslational modifications adjacent to the catalytic iron center: a thioether cross-link between Cys93 and Tyr157 and extra electron density at Cys164 which was variously explained as cystine or cysteine sulfinic acid. Purification of recombinant rat CDO yields "mature" and "immature" forms with distinct electrophoretic mobilities. We have positively identified and characterized the two modifications in the products of three sequential proteolytic digestions using liquid chromatography coupled with tandem mass spectrometry. The cross-link is unique to the mature form and was identified in an ion of m/z 3,225.403, consistent with a Tyr-Cys cross-link of peptides Gly80-Phe94 with His155-Phe167. The cross-link is liable to cleavage by in-source decay and the resulting separate peptides were sequenced by collision-induced dissociation tandem mass spectrometry. Mass-spectrometric analysis of these same and overlapping peptides in the presence or absence of reductants and alkylating agents identified the second modification to be a cystine formed between Cys164 and exogenous cysteine as proposed earlier. Both modifications have been shown to form in the presence of high levels of cysteine and iron. This and the presence of small amounts of an apparently off-pathway cystine at position Cys93 suggest that although these conditions promote CDO maturation, they may actually arise via nonenzymatic, nonphysiological processes.
