OxyB is a cytochrome P450 enzyme that catalyzes the first phenol coupling reaction during the biosynthesis of vancomycin-like glycopeptide antibiotics. The phenol coupling reaction occurs on a linear peptide intermediate linked as a C-terminal thioester to a peptide carrier protein (PCP) domain within the multidomain glycopeptide nonribosomal peptide synthetase (NRPS). Using model peptides with the sequence (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP and (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-(S)Dpg-S-PCP (where Hpg = 4-hydroxyphenylglycine, and Dpg = 3,5-dihydroxyphenylglycine), or containing (R)Leu instead of (R)(NMe)Leu, attached to recombinant PCPs derived from modules-6 and -7 in the vancomycin NRPS, we show that cross-linking of Hpg4 and Tyr6 by OxyB can occur in both hexapeptide- and heptapeptide-PCP conjugates. Thus, whereas OxyB may act preferentially on a hexapeptide still linked to the PCP-6 in NRPS subunit-2, it is possible that a linear heptapeptide intermediate linked to PCP-7 in NRPS subunit-3 may also be transformed into monocyclic product. For turnover, OxyB requires electrons, which in vitro can be supplied by spinach ferredoxin and E. coli flavodoxin reductase. Turnover is also dependent upon the presence of molecular oxygen. The model substrate (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP is transformed into cross-linked product by OxyB with a kcat of 0.1 s-1 and Km in the range 4-13 muM. Equilibrium binding of this substrate to OxyB, monitored by UV-vis, is accompanied by a typical low-to-high spin state change in the heme, characterized with a Kd of 17 +/- 5 muM.

During the biosynthesis of glycopeptide antibiotics of the vancomycin family, several oxidative phenol coupling reactions take place. An oxygenase (OxyB) from the vancomycin producer catalyzes the first of these coupling reactions to a significant extent only when the putative hexapeptide substrate is linked as a thioester to a peptide carrier domain (PCD) derived from the non‐ribosomal peptide synthetase (see picture).

Halo‐giant: The title compound at the left in the scheme was constructed by the synthesis and coupling of two stereochemically challenging fragments. A comparison of the NMR data of the synthetic material and the natural product indicates that the configuration had been misassigned in the latter. PT=5‐(1‐phenyltetrazolyl).

Teil der Biosynthese von Glycopeptidantibiotika der Vancomycin‐Familie sind mehrere oxidative Phenolkupplungsreaktionen. Eine Oxygenase (OxyB) des Vancomycin‐Produzenten katalysiert die erste dieser Kupplungen nur dann merklich, wenn das vermutliche Hexapeptidsubstrat als Thioester an eine Peptidträgerdomäne (PCD) geknüpft ist, die sich von der nichtribosomalen Peptid‐Synthetase ableitet (siehe Bild).

New classes of thia/oxa-azaspiro[3.4]octanes are synthesized through the implementation of robust and step-economic routes. The targeted spirocycles have been designed to act as novel, multifunctional, and structurally diverse modules for drug discovery. Furthermore, enantioselective approaches to the spirocycles are reported.