Performance Analysis of Orthogonal Pairs Designed for an Expanded Eukaryotic Genetic Code

Background: The suppression of amber stop codons with non-canonical amino acids (ncAAs) is used for the site-specific introduction of many unusual functions into proteins. Specific orthogonal aminoacyl-tRNA synthetase (o-aaRS)/amber suppressor tRNA CUA pairs (o-pairs) for the incorporation of ncAAs in S. cerevisiae were previously selected from an E. coli tyrosyl-tRNA synthetase/tRNACUA mutant library. Incorporation fidelity relies on the specificity of the o-aaRSs for their ncAAs and the ability to effectively discriminate against their natural substrate Tyr or any other canonical amino acid. Methodology/Principal Findings: We used o-pairs previously developed for ncAAs carrying reactive alkyne-, azido-, or photocrosslinker side chains to suppress an amber mutant of human superoxide dismutase 1 in S. cerevisiae. We found worse incorporation efficiencies of the alkyne- and the photocrosslinker ncAAs than reported earlier. In our hands, amber suppression with the ncAA containing the azido group did not occur at all. In addition to the incorporation experiments in S. cerevisiae, we analyzed the catalytic properties of the o-aaRSs in vitro. Surprisingly, all o-aaRSs showed much higher preference for their natural substrate Tyr than for any of the tested ncAAs. While it is unclear why efficiently recognized Tyr is not inserted at amber codons, we speculate that metabolically inert ncAAs accumulate in the cell, and for this reason they are incorporated despite being weak substrates for the o-aaRSs. Conclusions/Significance: O-pairs have been developed for a whole plethora of ncAAs. However, a systematic and detaile

Evidence for specific solvation of two halocarbene amides.

Laser flash photolysis (LFP, 308 nm) of endo-10-halo-10’-N,N-dimethylcarboxamidetricyclo[4.3.1.0]-deca-2,4-diene (1Cl and 1F) releases indan and halocarbene amide (2Cl and 2F). Although the carbenes are not UV-vis active, they react rapidly with pyridine to form ylides (4Cl, 4F), which are readily detected in LFP experiments (lambda(max) = 450 nm). Dioxane decreases the observed rate of carbene reaction with pyridine in CF(2)ClCFCl(2). Small amounts of THF decrease the observed rate of reaction of carbene 2F with pyridine but increase the rate of reaction of carbene 2Cl with pyridine. LFP (266 nm) of dienes 1Cl and 1F in CF(2)ClCFCl(2) with IR detection produces carbenes 2Cl and 2F with carbonyl vibrations at 1635 and 1650 cm(-1), respectively. In dioxane or THF solvent, LFP produces the corresponding ether ylides (5Cl, 5F) by capture of carbenes 2Cl and 2F. The ylides have broad carbonyl vibrations between 1560 and 1610 cm(-1). The addition of a small amount of dioxane in CFCl(2)CF(2)Cl extends the lifetime of the carbene. This observation, together with the ether-induced retardation of the rates of carbene capture by tetramethylethylene and pyridine, is evidence for solvation of the carbene by dioxane

Evidence for specific solvation of two halocarbene amides.

Laser flash photolysis (LFP, 308 nm) of endo-10-halo-10’-N,N-dimethylcarboxamidetricyclo[4.3.1.0]-deca-2,4-diene (1Cl and 1F) releases indan and halocarbene amide (2Cl and 2F). Although the carbenes are not UV-vis active, they react rapidly with pyridine to form ylides (4Cl, 4F), which are readily detected in LFP experiments (lambda(max) = 450 nm). Dioxane decreases the observed rate of carbene reaction with pyridine in CF(2)ClCFCl(2). Small amounts of THF decrease the observed rate of reaction of carbene 2F with pyridine but increase the rate of reaction of carbene 2Cl with pyridine. LFP (266 nm) of dienes 1Cl and 1F in CF(2)ClCFCl(2) with IR detection produces carbenes 2Cl and 2F with carbonyl vibrations at 1635 and 1650 cm(-1), respectively. In dioxane or THF solvent, LFP produces the corresponding ether ylides (5Cl, 5F) by capture of carbenes 2Cl and 2F. The ylides have broad carbonyl vibrations between 1560 and 1610 cm(-1). The addition of a small amount of dioxane in CFCl(2)CF(2)Cl extends the lifetime of the carbene. This observation, together with the ether-induced retardation of the rates of carbene capture by tetramethylethylene and pyridine, is evidence for solvation of the carbene by dioxane