@article{THESIS,
      recid = {617},
      author = {Zhang, Chen},
      title = {DESIGN, CONSTRUCTION, AND DIRECTED EVOLUTION OF ARTIFICIAL  METALLOENZYMES},
      publisher = {University of Chicago},
      school = {Ph.D.},
      address = {2015-12},
      number = {THESIS},
      pages = {126},
      abstract = {This dissertation describes the design, construction, and  directed evolution of artificial metalloenzymes. Transition  metal catalysts and enzymes possess unique and often  complementary properties that have made them important  tools for chemical synthesis. In hopes of combining the  selectivity and adaptability of enzymes with the reactivity  of transition metal catalysts, researchers have explored  artificial metalloenzymes (ArMs) in which secondary  coordination sphere effects impart selectivity to metal  catalysts, accelerate chemical reactions, and are  systematically optimized via directed evolution. Our  ultimate goal is to achieve non-directed and selective C-H  functionalization.

Chapter one describes unnatural amino  acid (UAAs) synthesis. UAA incorporation into protein  scaffolds could provide a facile method to construct ArMs  and metallopeptides. However, neither the synthesis nor the  incorporation of catalytically active UAAs have been  reported. We developed a general approach for the synthesis  of UAAs using 9-borabicyclononanyl group which enables  simultaneous protection of both the amine and carboxylic  acid functionalities. Both bidentate and tridentate Pd  complexes were synthesized and confirmed by HRMS-ESI and  NMR characterization. These UAAs catalyzed imidate  rearrangements, but peptides containing these UAAs did not  impart enantioselectivity to this reaction, and we were  unable to achieve incorporation of the UAAs into proteins  using codon suppression methods. We next pursued  incorporation of UAAs with catalytically active organic  side chains. Inspired by secondary amine catalyzed  reactions, we designed and prepared two UAAs, prolyl-lysine  and imidazolidinone-lysine, with strong resemblance to  pyrrolysine. Both UAAs catalyzed aldol and Michael  reactions under physiological conditions. This work was  conducted in collaboration with Dr. Zhihui Zhang and Hao  Yang, and was published in Organometallics, 2012, 31,  7328-7331.

Chapter two describes the construction of Mn  terpyridine ArMs for oxygenation. Mn terpyridine is a  robust catalyst for benzylic C-H oxygenation and olefin  epoxidation. We designed and synthesized a  maleimide-substituted Mn-terpyridine cofactor and  demonstrated that this cofactor could be incorporated into  two different scaffold proteins to generate the desired  ArMs. The structure and reactivity of one of these ArMs was  explored, and the broad oxygenation capability of the  Mn-terpyridine catalyst was maintained, providing a robust  platform for optimization of ArMs for selective hydrocarbon  functionalization. This work was conducted in collaboration  with Dr. Poonam Srivastava and Ken Ellis-Guardiola, and was  published in Tetrahedron, 2014, 70, 4245-4249.

Chapter  three describes the construction and directed evolution of  dirhodium ArMs. We first demonstrated that strain-promoted  azide–alkyne cycloaddition could be used for rapid and  general ArM formation. This approach was used to  incorporate an alkyne-substituted dirhodium complex into a  prolyl oligopeptidase (POP), and the resulting ArM  catalyzed carbenoid N-H insertion with low  enantioselectivity. Scaffold mutagenesis was then employed  to improve the enantioselectivity of N-H insertion  reactions involving a range of aryl amines and  donor-acceptor carbene precursors. Despite great success in  evolving natural enzymes, no example of directed evolution  applied to engineering ArMs has been reported. We  established a streamlined, high-throughput protocol for  design, expression, purification and screening of ArM  libraries. After the process was sufficiently optimized,  the first round of directed evolution from an engineered  parent protein, F99H328, produced the mutant G1P1_D5 which  achieved 54% ee. This work was published in Chembiochem,  2014, 15, 223-227. This work was conducted in collaboration  with Dr. Poonam Srivastava, Hao Yang, Hyun June Park and  Ken Ellis-Guardiola.},
      url = {http://knowledge.uchicago.edu/record/617},
}