RESUMO
31P NMR spectroscopy was used to investigate a stereochemical course of a nitrite-promoted conversion of phosphoramidate diesters into the corresponding phosphotriesters. It was found that this reaction occurred with almost complete epimerization at the phosphorus center and at the C1 atom in the amine moiety. On the basis of the 31P NMR data, a plausible mechanism for the reaction was proposed. The density functional theory calculation of the key step of the reaction, i.e., breaking of the P-N bond and formation of the P-O bond, suggested a one-step S(N)2(P) process with retention of configuration at the phosphorus center.
Assuntos
Amidas/química , Espectroscopia de Ressonância Magnética , Nitritos/química , Ácidos Fosfóricos/química , Ésteres/química , Modelos Moleculares , Conformação Molecular , Isótopos de Fósforo/química , EstereoisomerismoRESUMO
A number of model structures of the CalA suggested by comparative modeling were tested by site-directed mutagenesis. Enzyme variants were created where amino acids predicted to play key roles for the lipase activity in the different models were replaced by an inert amino acid (alanine). The results from activity measurements of the overproduced and purified mutant enzymes indicate a structure where the active site consists of amino acid residues Ser184, His366, and Asp334 and in which there is no lid. This model can be used for future targeted modifications of the enzyme to obtain new substrate acceptance, better thermostability, and higher enantioselectivity.
Assuntos
Candida , Lipase , Candida/enzimologia , Domínio Catalítico , Lipase/química , Modelos Moleculares , Mutagênese Sítio-DirigidaRESUMO
In this paper a short account of our recent basic studies aiming toward development of new synthetic methods for the preparation of nucleotide analogues using H-phosphonate chemistry is presented.