We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.

Biotransformation of quinoline, isoquinoline, quinoxaline and quinazoline using growing cultures of Pseudomonas putida UV4 yielded cis-dihydro diols from the oxidation of the carbocyclic aromatic ring. Aromatic hydroxylation was observed in both carbocyclic and heterocyclic rings. Ring cleavage of the quinoline skeleton to yield anthranilic acid, and cis-diol formation (with alkene bond reduction) to yield cis-5,6,7,8-tetrahydroquinazoline-5.6-diol from quinazoline were observed. The cis-dihydro diol metabolites of quinoline (5,6and 7,8-) and quinoxaline (5,6-) were found to be optically pure, while metabolism of isoquinoline gave one homochiral (5,6-) and one racemic (7.8-) cis-dihydro diol product. The absolute configurations of the cis-dihydro diol metabolites have been determined using ' H N M R analyses, stereochemical correlations and X-ray crystallography methods.Azaarenes (e.g. quinoline 1 and isoquinoline 2) are widely distributed throughout the global environment, mainly as a result of partial combustion of plant material and fossil fuels. 'Quinoline I, the most abundant of the azaarenes in the environment, is both carcinogenic and mutagenic. The latter effects probably result from the derived metabolites of quinoline 1. Known and potential metabolites of quinoline l 2 and isoquinoline 23 have recently been synthesised as part of a Paper 2/06567C

Metabolism of the aza-arenes quinoline, isoquinoline, quinazoline, and quinoxaline by a mutant strain of the bacterium Pseudomonas putida resulted in attack at the carbocyclic ring (to yield stable cis-dihydrodiols and phenols) and at the heterocyclic ring (to yield phenols and ring cleavage products).

Chemical synthesis in combination with precision polymer modification allows the systematic exploration of the effect of protein properties, such as charge and hydrodynamic radius, on potency using defined, homogeneous conjugates. A series of polymer-modified synthetic erythropoiesis proteins were constructed that had a polypeptide chain similar to the amino acid sequence of human erythropoietin but differed significantly in the number and type of attached polymers. The analogs differed in charge from +5 to -26 at neutral pH and varied in molecular weight from 30 to 54 kDa. All were active in an in vitro cell proliferation assay. However, in vivo potency was found to be strongly dependent on overall charge and size. The trends observed in this study may serve as starting points for the construction of more potent synthetic EPO analogs in the future.

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