In order to accomplish the selective synthesis of
[60]fullerene bisadducts, the reactions of
[60]fullerene
with compounds in which two α,α‘-dibromo-o-xylene
moieties were connected by an oligomethylene chain (n
=
2−5) were investigated. By this method, only two isomers
(cis-2- and cis-3-isomers) were selectively
obtained
when n = 2 and 3, while another isomer
(e-isomer) was obtained when n = 5. When
n = 4, a complex mixture of
bisadducts was formed and has not been separated so far.
cis-2-Bisadducts have been, for the first time,
selectively
obtained in the fullerene chemistry. The structures of bisadducts
were determined on the basis of 1H, 13C NMR,
IR,
UV−vis, and mass spectroscopies. According to the NMR
experiments, the symmetries of cis-2-, cis-3-,
and e-isomers
were concluded to be C
s
,
C
2, and C
1, respectively.
Chiral cis-3- and e-bisadducts were
successfully resolved into
the respective enantiomers on a chiral HPLC column, although
cis-2-bisadducts only gave a single peak. The
UV−vis spectra of cis-2-, cis-3-, and
e-bisadducts were remarkably different from one another.
Specifically, the e-bisadducts
showed a characteristic absorption peak around 420 nm. The
cleavage of the oligomethylene chain produced the
corresponding [60]fullerene derivatives possessing two phenol
moieties. These compounds are applicable to further
functionalization.
SummaryCells adapt to oxidative stress by transcriptional activation of genes encoding antioxidants and proteins of other protective roles. A bZIP transcription factor, Pap1, plays a critical role in this process and overexpression of Pap1 confers resistance to various oxidants and drugs in fission yeast. Pap1 temporarily enters the nucleus upon oxidative stress but returns to the cytoplasm once cells adapt to the stress, suggesting that cellular localization regulates Pap1 function. We report here an additional regulatory mechanism that Ubr1 ubiquitin ligase-dependent degradation lowered the Pap1 protein levels. ubr1 cells were causally resistant to hydrogen peroxide because of the increment of Pap1 levels. Pap1 was preferentially degraded in the nucleus where Ubr1 was consistently enriched. Proteolysis was critical to downregulate Pap1 especially when its activation persisted, as constitutively nuclear Pap1 severely inhibited growth in ubr1 mutants. Inactive mutations in the bZIP DNA binding domain stabilized Pap1 but rescued the lethality caused by constitutively active Pap1 in ubr1 mutants. These findings indicate that either nuclear export or Ubr1-mediated proteolysis must be operative to prevent uncontrolled Pap1 function. Coincidental dysfunction in both inhibitory pathways causes lethality because of prolonged activation of Pap1. Ubr1 is a critical regulator for the homeostasis of oxidative stress response.
Four-base codon strategy was applied to incorporate a fluorophore-quencher pair into specific positions on a single protein; beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap) was employed as a fluorophore and p-nitrophenylalanine (ntrPhe) as a quencher. Their positions were directed by the CGGG/CCCG and GGGC/CCCG four-base codon/anticodon pairs and two doubly mutated streptavidins, i.e., ((52)atnDap, (84)ntrPhe) and ((54)ntrPhe, (84)atnDap) mutants were synthesized through Escherichia coli in vitro protein synthesizing systems. Intramolecular photoinduced electron transfer (ET) was observed as the decrease of intensity in steady-state fluorescence spectroscopy and as the shortening of fluorescence decaytimes. The quenching data indicated that the ET rate reflects the detailed structure of the protein.
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