טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentDovgolevsky Ekaterina
SubjectDesign, Synthesis and Evaluation of Mechanism-Based
Inhibitors for the Reaction Catalyzed by the
Enzyme KDO8P Synthase
DepartmentDepartment of Chemistry
Supervisor Professor Timor Baasov


Abstract

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase (KDO8PS) catalyzes the unusual condensation of phosphoenolpyruvate (PEP) with D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate (Pi). This important enzymatic reaction plays a crucial role in the assembly process of lipopolysaccharides of most Gram-negative bacteria, and is therefore an attractive target for the design of novel antibacterial drugs.


Our laboratory is strongly involved with the mechanistic and structure-function studies of KDO8PS-catalyzed reaction. Recently, the most potent in vitro inhibitor, the amino phosphonophosphate 3 was synthesized. However, no antibacterial activity was observed when 3 was tested against a series of different Gram-negative bacteria. The observed lack of antibacterial activity by 3 may be explained either because its low permeability through the membrane of bacteria or the hydrolysis of its crucial phosphate monoester by various phosphatase enzymes. To solve this problem, we have synthesized an isosteric phosphonate analogue of 3, amino bisphosphonate 4 in eight chemical steps from commercially available methyl a-D-mannopyranoside in an overall yield of 15.7%. It was found that 4 serves as a competitive inhibitor of the enzyme with respect to both PEP and A5P binding, with inhibition constants, Ki values, of 49.29 ?5.09 mM and 49.72 ?7.64 mM, respectively. However, no antibacterial activity was observed when the inhibitor 4 was tested in vivo. Therefore, we concluded that the lack of antibacterial activity of these inhibitors, 3 and 4, results from their low permeability through the membrane of bacteria. The overcoming of this problem will help us to develop potent inhibitors that may serve as new antibacterial drugs against specific human pathogens.

In addition, in attempts to test the validity of the proposed acyclic intermediate 1, we have designed its stable phosphonate analogue 5. During this research work, we have developed two alternative synthetic pathways for the synthesis of 5 and succeeded in the construction of its unique b-hydroxy phosphonate moiety. The results that will be attained through examination of these compounds as inhibitors of the enzyme are expected to provide support for the existence of acyclic bisphosphate 2 as a true intermediate.