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 Gd­(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-AP₁₀CP₁₀CP₁₀-NH₂ was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP₁₀C­(Gd-PyMTA)­P₁₀C­(Gd-PyMTA)­P₁₀-NH₂, 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 Gd­(III)–Gd­(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 Gd­(III) ion and the C_α of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP₁₀C­(Gd-PyMTA)­P₁₀C­(Gd-PyMTA)­P₁₀-NH₂ 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^III-Based Pulsed Dipolar EPR Spectroscopy (PDS) with Porphyrin–Gd^III 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 m² g^–1, respectively) consisting of two mutually interpenetrating UiO-type frameworks with linkers of the type ^–O₂C­[PE-P­(R¹,R²)-EP]­CO₂^– (P, phenylene; E, ethynylene; R¹ and R², side chains at the central benzene ring with R¹ = R² = OCH₂CCH or R¹ = OCH₂CCH and R² = O­(CH₂CH₂O)₃Me). Their syntheses, using benzoic acid as a modulator, and their characterization are reported herein. The propargyloxy (OCH₂CCH) 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 [Zr₆O₄(OH)₄(O₂C)₁₂] 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 m² g^–1, respectively. PEPEP-PIZOFs, even those with very hydrophilic oligo­(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