4 research outputs found
Gd(III)-PyMTA Label Is Suitable for In-Cell EPR
Distance measurement in the nanometer
range by electron paramagnetic
resonance spectroscopy (EPR) in combination with site-directed spin
labeling is a very powerful tool to monitor the structure and dynamics
of biomacromolecules in their natural environment. However, in-cell
application is hampered by the short lifetime of the commonly used
nitroxide spin labels in the reducing milieu inside a cell. Here,
we demonstrate that the GdÂ(III) based spin label Gd-PyMTA is suitable
for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was
proven to be inert in in-cell extracts of <i>Xenopus laevis</i> oocytes at 18 °C for more than 24 h. The proline rich peptide
H-AP<sub>10</sub>CP<sub>10</sub>CP<sub>10</sub>-NH<sub>2</sub> was
site-directedly spin labeled with Gd-PyMTA at both cysteine moieties.
The resulting peptide, H-AP<sub>10</sub>CÂ(Gd-PyMTA)ÂP<sub>10</sub>CÂ(Gd-PyMTA)ÂP<sub>10</sub>-NH<sub>2</sub>, as well as the model compound Gd-spacer-Gd,
which consists of a spacer of well-known stiffness, were microinjected
into <i>Xenopus laevis</i> oocytes, and the GdÂ(III)–GdÂ(III)
distances were determined by double electron–electron resonance
(DEER) spectroscopy. To analyze the intracellular peptide conformation,
a rotamer library was set up to take the conformational flexibility
of the tether between the GdÂ(III) ion and the C<sub>α</sub> of
the cysteine moiety into account. The results suggest that the spin
labeled peptide H-AP<sub>10</sub>CÂ(Gd-PyMTA)ÂP<sub>10</sub>CÂ(Gd-PyMTA)ÂP<sub>10</sub>-NH<sub>2</sub> is inserted into cell membranes, coinciding
with a conformational change of the oligoproline from a PPII into
a PPI helix
Increasing the Modulation Depth of Gd<sup>III</sup>-Based Pulsed Dipolar EPR Spectroscopy (PDS) with Porphyrin–Gd<sup>III</sup> Laser-Induced Magnetic Dipole Spectroscopy
Distance determination
with pulsed EPR has become an
important
technique for the structural investigation of biomacromolecules, with
double electron–electron resonance spectroscopy (DEER) as the
most important method. GdIII-based spin labels are one
of the most frequently used spin labels for DEER owing to their stability
against reduction, high magnetic moment, and absence of orientation
selection. A disadvantage of GdIII–GdIII DEER is the low modulation depth due to the broad EPR spectrum of
GdIII. Here, we introduce laser-induced magnetic dipole
spectroscopy (LaserIMD) with a spin pair consisting of GdIII(PymiMTA) and a photoexcited porphyrin as an alternative technique.
We show that the excited state of the porphyrin is not disturbed by
the presence of the GdIII complex and that herewith modulation
depths of almost 40% are possible. This is significantly higher than
the value of 7.2% that was achieved with GdIII–GdIII DEER
Postsynthetic Modification of Metal–Organic Frameworks through Nitrile Oxide–Alkyne Cycloaddition
Postsynthetic modification
of metal–organic frameworks is an important method to tailor
their properties. We report on the nitrile oxide–alkyne cycloaddition
(NOAC) as a modification tool, a reaction requiring neither strained
alkynes nor a catalyst. This is demonstrated with the reaction of
nitrile oxides with PEPEP-PIZOF-15 and -19 at room temperature. PIZOF-15
and -19 are porous Zr-based MOFs (BET surface areas 1740 and 960 m<sup>2</sup> g<sup>–1</sup>, respectively) consisting of two mutually
interpenetrating UiO-type frameworks with linkers of the type <sup>–</sup>O<sub>2</sub>CÂ[PE-PÂ(R<sup>1</sup>,R<sup>2</sup>)-EP]ÂCO<sub>2</sub><sup>–</sup> (P, phenylene; E, ethynylene; R<sup>1</sup> and R<sup>2</sup>, side chains at the central benzene ring with
R<sup>1</sup> = R<sup>2</sup> = OCH<sub>2</sub>Cî—¼CH or R<sup>1</sup> = OCH<sub>2</sub>Cî—¼CH and R<sup>2</sup> = OÂ(CH<sub>2</sub>CH<sub>2</sub>O)<sub>3</sub>Me). Their syntheses, using benzoic
acid as a modulator, and their characterization are reported herein.
The propargyloxy (OCH<sub>2</sub>Cî—¼CH) side chains contain
the ethyne moieties needed for NOAC. Formation of nitrile oxides through
oxidation of oximes in aqueous ethanolic solution in the presence
of PEPEP-PIZOF-15 and -19 resulted in the reaction of 96–100%
of the ethyne moieties to give isoxazoles. Thereby the framework was
preserved. The type of nitrile oxide RCNO was greatly varied with
R being isopentyl, tolyl, 2-pyridyl, and pentafluorophenyl. A detailed
NMR spectroscopic investigation showed the formation of the 3,5-disubstituted
isoxazole to be clearly favored (≥96%) over that of the constitutional
isomeric 3,4-disubstituted isoxazole, except for one example
Expanding the Group of Porous Interpenetrated Zr-Organic Frameworks (PIZOFs) with Linkers of Different Lengths
A Zr-based MOF of
the PIZOF type, which consists of two independent
and mutually interpenetrating UiO-type frameworks with [Zr<sub>6</sub>O<sub>4</sub>(OH)<sub>4</sub>(O<sub>2</sub>C)<sub>12</sub>] nodes,
does not only form with a PEPEP dicarboxylic acid (P = phenylene,
E = ethynylene). Also dicarboxylic acids with the shorter PPPP and
PEPP spacers were found to give PIZOFs, denoted PPPP-PIZOF and PEPP-PIZOF,
respectively. Reducing the spacer length even further to a PEEP segment
caused a switchover to the formation of a UiO framework. The hysteresis
in the Ar sorption curve of PEPP-PIZOF-1 and the slightly too large
amount of combustion residue from PPPP-PIZOF-1 suggest structural
defects. These hint at a mismatch between the requirement of the optimal
linker length for PIZOF formation and the lengths of the PEPP and
PPPP dicarboxylates. Nevertheless, these dicarboxylates prefer the
formation of a PIZOF over the formation of a UiO structure. PEPEP-PIZOF-2,
PPPP-PIZOF-1, and PEPP-PIZOF-1 are stable in air up to 325, 350, and
300 °C, respectively, and have BET surface areas of 2350, 2020,
and 1650 m<sup>2</sup> g<sup>–1</sup>, respectively. PEPEP-PIZOFs,
even those with very hydrophilic oligoÂ(ethylene glycol) side chains
on the linkers, are very stable in water and also during drying from
a water-wetted state. On the contrary, PEPP-PIZOF-1 and PPPP-PIZOF-1
that had been exposed to water required exchange of water for ethanol
before drying to mostly preserve the framework. The results emphasize
the importance of differentiating between framework damage caused
through hydrolysis in water and through drying from a water-wetted
state. The sensitivity of PEPP-PIZOF-1 and PPPP-PIZOF-1 against drying
from a water-wetted state may be the consequence of defects. The drying
stability of water-wetted PEPEP-PIZOFs lets us suggest that reversible
bending of the linkers contributes to the stability of the PEPEP-PIZOFs