Selective 2H-, 13C-, and
17O-isotope labeling of the tyrosine amino acid has been
used to map the unpaired
π-electron spin-density distribution of the UV-generated neutral
l-tyrosine phenoxy radical in alkaline frozen
solution.
The use of 13C and 17O labels allowed
accurate determination of the full spin-density distribution and
provided more
insight in the geometrical structure of the neutral tyrosine radical in
vitro. Simulations of the X-band (9.2 GHz) and
Q-band (34.8 GHz) EPR powder spectra yielded the principal components
of the 1H-, 13C-, and 17O-hyperfine
tensors.
For the two β-methylene hydrogens, a static conformational
distribution of the dihedral angles (90° < θ1 < 60°
and
60° < θ2 < 30°) was taken into account. The
major proton hyperfine interactions and the principal g
values for the
neutral tyrosine radical, obtained from selectively deuterated samples,
are consistent with literature values. The spin
density at the specifically labeled postitions (C1‘, C2‘, C3‘, C4‘,
C5‘, O4‘) was evaluated from the anisotropy of the
13C- and 17O-hyperfine tensors. A
quantitative analysis of the positions C3‘ and C5‘ provided evidence
for a planar
distortion of the aromatic ring at these positions.
17O enrichment of the phenol oxygen O4‘ of the
tyrosine radical
unambiguously showed that the spin density at this oxygen is 0.26 ±
0.01. From the relatively large delocalization
of the spin density over the carbonyl group of the tyrosine aromatic
ring system, it is concluded that the C4‘−O4‘
bond has a double-bond character. The experimentally determined
spin-density distribution is compared with several
computational calculated spin-density distributions found in the
literature. The isotropic 13C-hyperfine
interactions
are discussed in the framework of the Karplus−Fraenkel theory.
This theory proved to be accurate for the
determination of sign and magnitude of the isotropic 13C-
and 17O-hyperfine interactions.
A simple procedure for the preparation of the specifically labelled peptide antibiotic zervamicins IC, IIA and IIB has been developed. The zervamicin molecules are labelled with stable isotopes by culturing the Emericellopsis salmosynnemata on a well-defined synthetic medium containing the highly isotopically enriched amino acid. To obtain the peptide with the specifically and highly enriched amino acid residue, precautions have been taken to prevent any de novo biosynthesis of the particular amino acid from unlabelled precursors. The enrichment of the labelled peptide is determined by mass spectrometric analysis. Following this method we have incorporated [2',4',5',6',7'-2H5]-L-Trp-1, [1'-15N]-L-Trp-1 and [2',3',4',5',6'-2H5]-L-Phl-16 into zervamicins IC, IIA and IIB on the preparative scale and without scrambling of the label. Thus, using the procedures described, isotopically labelled zervamicins can be prepared, allowing them to be studied by solid-state NMR.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.