Inorganic pyrophosphatases (PPases) catalyze an essential reaction, namely, the hydrolysis of PPi, which is formed in large quantities as a side product of numerous cellular reactions. In the majority of living species, PPi hydrolysis is carried out by soluble cytoplasmic PPase (S-PPases) with the released energy dissipated in the form of heat. In Rhodospirillum rubrum, part of this energy can be conserved by proton-pumping pyrophosphatase (H+-PPaseRru) in the form of a proton electrochemical gradient for further ATP synthesis. Here, the codon-harmonized gene hppaRru encoding H+-PPaseRru was expressed in the Escherichia coli chromosome. We demonstrate, for the first time, that H+-PPaseRru complements the essential native S-PPase in E. coli cells. 13C-MFA confirmed that replacing native PPase to H+-PPaseRru leads to the re-distribution of carbon fluxes; a statistically significant 36% decrease in tricarboxylic acid (TCA) cycle fluxes was found compared with wild-type E. coli MG1655. Such a flux re-distribution can indicate the presence of an additional method for energy generation (e.g., ATP), which can be useful for the microbiological production of a number of compounds, the biosynthesis of which requires the consumption of ATP.
A method is reported to control the stereoselectivity at C(20) in the syntheses of 20-R-21,21,21-trifluorothevinols (12), the opioid ligands incorporating fluorine atoms within the pharmacophore associated with the surroundings of the C(20) carbon atom. The C(20)-alcohols 12 can be prepared either by reaction of 21,21,21-trifluorothevinone (9) with RM (R = alkyl; M = Li, MgX) or by reaction of thevinone (2) and related non-fluorinated ketones with CF3SiMe3. In general, alcohols 12 were formed as mixtures of the C(20)-epimers, with the major epimers of the alcohols obtained from the aforementioned reactions exploiting RLi vs. CF3SiMe3 with opposite absolute configurations at C(20). Some individual C(20)-epimers of the fluorinated alcohols 12 were isolated from the reaction mixtures in pure form by trivial crystallization. The reactions of the ketones with RMgX (R ≠ Me) and RLi (R = tertiary or secondary alkyl) resulted in the reduction of the carbonyl function to produce the secondary alcohols 11a,b rather than the tertiary alcohols 12. The additives of the salts were found to affect the composition of the products in the reactions of 9 with alkyl organomagnesium and organolithium reagents.
