ABSTRACT Glycopeptide antibiotics (GPAs) are among the most important therapeutic agents world-wide. The founding member of this natural product family, vancomycin, is used a drug of last resort against infections by methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile. Along with a handful of other antibiotics, vancomycin provides an important weapon against “superbugs”, pathogenic bacteria that have acquired resistance to multiple clinical antibiotics. But as resistance to even this last line of defense spreads, it is ever more important to develop means of chemically tailoring vancomycin and other GPAs to create new derivatives that counter known resistance mechanisms. Synthetic derivatization has proven to be a successful method for creating new antibiotics, but this approach is severely restricted within the GPAs, primarily due to their chemical complexity and size. Key to the structural complexity and biological activity of vancomycin are three aromatic crosslinks, consisting of two aryl ether connections and a biaryl carbon-carbon bond. Research over the past 20 years has shown that a cytochrome P450 enzyme (OxyB) installs the first aryl ether bond. The origin of the remaining two crosslinks, however, remained elusive. We recently showed that OxyA, a second P450 enzyme, introduces the second aryl ether crosslink during vancomycin biogenesis. We further recapitulated the enzymatic activity of OxyC and showed that it installs the final biaryl connection, the first demonstration of this reaction in any GPA. Moreover, we have exploited the reactivities of the native biosynthetic metalloenzymes to implement a chemo-enzymatic route for creating a vancomycin aglycone derivative. The stage is set to fully leverage this chemo-enzymatic approach to chemically derivatize vancomycin in the hopes of generating useful second-generation derivatives. In the current application, we propose to complete the chemo-enzymatic synthesis of not just vancomycin, but also of derivatives known to retain bioactivity, even against resistant pathogens. We further propose to build a library of vancomycin analogs that we refer to as “designer vancomycins”, containing modifications that are inaccessible with current methodologies. We will simultaneously explore the detailed chemical mechanism of OxyB and create an innovative solid-phase approach to enhance the efficiency and scalability of our chemo- enzymatic route. Our studies will shed light onto the biosynthesis of vancomycin and enable the most comprehensive effort yet to create GPA variants with unique structures and possibly new bioactivities via an elegant chemo-enzymatic route.