The hydrazone modulation of a penetrating peptide carrier is reported as a suitable and straightforward strategy for the delivery of Cas9 inside living cells.
Enzyme-loaded polymeric vesicles, or polymersomes, can be regarded as nanoreactors, which, for example, can be applied as artificial organelles. We implemented a naturally occurring enzymatic cascade reaction in two types of polymersomes, which are known to possess different permeability properties. The selected cascade reaction involved the antioxidant enzymes superoxide dismutase (SOD1) and catalase (CAT) for combating oxidative stress. The activities of both enzymes were investigated by spectrophotometric and electrochemical assays. Whereas the SOD1 substrate (the radical anion superoxide, O2•-) was able to penetrate both membranes equally well, the CAT substrate (H2O2) showed different rates of diffusion. When O2•- was generated inside polymersomes filled with both SOD1 and CAT, the activities of the two systems were comparable again.
The determination of the three-dimensional structure of organic and biomolecular compounds by NMR spectroscopy usually involves the measurement of 3 J coupling constants, [1] NOEs, [2] and cross-correlated relaxation [3] to obtain information about dihedral angles, distances, and projection angles, respectively. If interconversion of conformers takes place and is fast on the NMR time scale, NMR spectroscopic parameters for flexible parts of the molecule are motionally averaged. This effect is one of the main complications in the structure determination of nonrigid molecules and often prohibits the determination of the relative configuration of organic compounds.It has been shown that residual dipolar couplings (RDCs) can yield information complementary to that obtained from 3 J coupling constants and NOE parameters also for organic compounds [4][5][6][7][8][9][10] and enable the assignment of relative configurations even in the presence of a limited degree of motion. [11][12][13][14][15] The problem of the joint treatment of an unknown configuration and conformational averaging when residual dipolar couplings are used in structure determination has scarcely been tackled. Herein two approaches are discussed, and it is shown that even conformer populations can be obtained from experimentally determined RDCs.The example chosen for illustration is a five-memberedring compound investigated recently by some of us. The amethylene-g-butyrolactone had been synthesized as single diastereoisomer, [16] the relative configuration of which (trans, denoted 1, or cis, denoted 2) was unknown and could not be determined by using conventional NMR spectroscopic parameters.[11] By using RDCs, however, it was possible to assign the relative configuration as trans (Scheme 1).[11]The number of ring conformers of 1 is restricted to two, denoted A and B in the following. These conformers are envelope conformations, one with C2 below (A) and one with C2 above (B) the almost planar arrangement of the remaining ring atoms (see also Figure SI1 in the Supporting Information).[17] In the previous study, [11] we used the transition structure between A and B as a crude approximation of the average ring conformation of 1. Additionally, we fitted the structures of the two rigid conformers to the RDC data by using one order tensor [18,19] each. By using this method we were able to assign the relative configuration as trans.[20]However, we did not attempt to extract information concerning the populations of the two conformers (p A and p B ), which was therefore one subject of the current investigation.The direct (residual) dipolar coupling D IS between spins I and S, with magnetogyric ratios g I and g S , is given by Equation (1): [9,21] ISis the dipole-dipole coupling constant (in Hz), V IS is the angle between the interspin vector and the external magnetic field, and r IS is the interspin distance (which corresponds to the bond length for directly bound nuclei). The angular brackets indicate that the RDCs are averaged over both molecular tumbling ...
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