Artificial molecular clips and tweezers, designed for cofactor and amino acid recognition, are able to inhibit the enzymatic activity of alcohol dehydrogenase (ADH). IC50 values and kinetic investigations point to two different new mechanisms of interference with the NAD(+)-dependent oxidoreductase: While the clip seems to pull the cofactor out of its cleft, the tweezer docks onto lysine residues around the active site. Both modes of action can be reverted to some extent, by appropriate additives. However, while cofactor depletion by clip 1 was in part restored by subsequent NAD(+) addition, the tweezer (2) inhibition requires the competitive action of lysine derivatives. Lineweaver-Burk plots indicate a competitive mechanism for the clip, with respect to both substrate and cofactor, while the tweezer clearly follows a noncompetitive mechanism. Conformational analysis by CD spectroscopy demonstrates significant ADH denaturation in both cases. However, only the latter case (tweezer-lysine) is reversible, in full agreement with the above-detailed enzyme switch experiments. The complexes of ADH with clips or tweezer can be visualized in a nondenaturing gel electrophoresis, where the complexes migrate toward the anode, in contrast to the pure enzyme which approaches the cathode. Supramolecular chemistry has thus been employed as a means to control protein function with the specificity of artificial hosts opening new avenues for this endeavor.

A unified pH scale of absolute values (pHabs scale) enables the comparison of acidities in different solvents. To date, very few different experimental setups have been used for the measurement of values on this scale. The article describes the design and performance of the different symmetric cells used for unified pH measurement by several institutions. Well-established and reliable standard aqueous buffer solutions are the first step of method validation necessary to achieve a robust metrological level for more complex media. The pH of aqueous standard buffers was measured by differential potentiometry, where the potential between two glass electrodes is measured directly. All the tested electrochemical cells prove to be suitable for unified pH measurements. This validation highlights that the method is, to a large extent, independent of the used equipment, including the cell geometry. The inherent symmetry of the cell design helps to reduce the experimental workload and improve the accuracy of obtained results.

[structure: see text] Molecular clips functionalized by phosphonate or phosphate groups bind thiamine diphosphate (TPP) and S-adenosylmethionine (SAM) with high affinity in water; both sulfur-based cofactors transfer organic groups to biomolecules. For TPP, various analytical tools point toward a simultaneous insertion of both heterocyclic rings into the electron-rich clip cavity. Similarly, SAM is also embedded with its sulfonium moiety inside the receptor cavity. This paves the way for enzyme models and direct interference with enzymatic processes.

Harned cell pH T measurements were performed on 2-amino-2-hydroxymethyl-1,3-propanediol (TRIS) buffered artificial seawater solutions in the salinity range 5-20, at three equimolal buffer concentrations (0.01, 0.025, 0.04 mol·kg-H 2 O −1 ), and in the temperature range 278.15-318.15 K. Measurement uncertainties were assigned to the pH T values of the buffer solutions and ranged from 0.002 to 0.004 over the investigated salinity and temperature ranges. The pH T values were combined with previous results from literature covering salinities from 20 to 40. A model function expressing pH T as a function of salinity, temperature and TRIS/TRIS·H + molality was fitted to the combined data set. The results can be used to reliably calibrate pH instruments traceable to primary standards and over the salinity range 5-40, in particular, covering the low salinity range of brackish water for the first time. At salinities 5-20 and 35, the measured dependence of pH T on the TRIS/TRIS·H + molality enables extrapolation of quantities calibrated against the pH T values, e.g., the dissociation constants of pH indicator dyes, to be extrapolated to zero TRIS molality. Extrapolated quantities then refer to pure synthetic seawater conditions and define a true hydrogen ion concentration scale in seawater media.

In die Zange genommen: Eine synthetische molekulare Klammer bindet Nicotinamidadenindinucleotidphosphat (NADP+) (siehe Bild) und besetzt zudem sowohl die Cofaktor‐ als auch die Substratbindestelle in Glucose‐6‐phosphat(G6P)‐Dehydrogenase. Diese Kombination zweier Inhibitionsmechanismen macht die Klammer hoch effektiv und selektiv für dieses Enzym gegenüber anderen Dehydrogenasen.

Inherently chiral molecular clips (MCs), pseudoenantiomeric anti-1 and anti-2, as well as mesoid syn-3, were synthesized by diastereodifferentiating repetitive Diels-Alder reactions of the achiral bisdienophile 6 with chiral diene 5 generated in situ from (-)-menthyl 3,4-bis(dibromomethyl)benzoate 4. These MCs were successfully separated by chiral HPLC to give optically active anti-1 and anti-2 and almost optically inactive syn-3. The structures of anti-1, anti-2, and syn-3 were assigned by high-resolution NMR and the absolute configurations of anti-1 and anti-2 were determined by the exciton-chirality method. Optically active anti-2 can serve as a chiral host. It binds the HCl adduct of D-tryptophan methyl ester (D-TrpOMeHCl) 3.5 times stronger than the L-enantiomer (KD/KL=3.5).

Abstract. The acidities of any given solvent or mixtures thereof can be compared by pH measurements on a unified scale, so-called pHabsH2O measurements. The method is quite new and has not been characterized with respect to metrological criteria to date. Metal solid-contact glass electrode half-cells, three commercial, conventional glass electrode half-cells with inner liquid filling and one pair of combined electrodes were used to investigate the stability of the measurement and the reproducibility of pHabsH2O results of ethanol mixtures with water. All electrodes are suitable for unified acidity measurements in standard aqueous buffers. In ethanol mixtures, the combined electrodes were found to be unsuitable. The half-cell electrodes can be reasonably used only in buffered solutions.

The highly selective recognition process of NAD R and NADH (as important cofactors of many redox enzymes) by molecular clips in aqueous solution is studied systematically by a combined experimental and quantum-chemical approach. The strongly pH-dependent complexation-induced 1 H NMR shifts of the guest molecule indicate that in buffered aqueous solution at pH ¼ 7.2 the nicotinamide ring, the active site of NAD R , is preferentially bound inside the cavity of the molecular clip, whereas in pure water under slightly acidic conditions both units (the nicotinamide ring as well as the adenine moiety) are located outside the cavity. The latter finding is explained by a competing selfaggregation of NAD R which prohibits the recognition process. In addition, the investigation of the NAD R fragments NMNA, NMN, and AMP as well as the comparison of measured and computed chemical shieldings provides information on possible binding modes. Under equal conditions the binding of NADH to the molecular clip is significantly weaker than that of NAD R , so that the oxidation states (NAD R /NADH) can be distinguished by the molecular clips. The nucleotides NMN and AMP are bound less strongly by the molecular clips than NAD R . The weaker binding indicates that multiple aromatic pSp and cationSp host-guest interactions only possible in NAD R have a synergetic effect on the complex stability. In addition to the inhibition of the cofactor NAD R , a further implication is the development of sensors since a quenching of fluorescence is observed for specific molecular clips by the addition of NAD R . a Calculated structure (as shown in Figure 4 or Supporting Information: Table QC-1 -QC-9; NAD-Nic ¼ NADþ5, NAD-Ade ¼ NADþ9, NAD 0 -Nic ¼ NADþ3, NAD 0 -Ade ¼ NADþ7, compared to G3; NADH8 is compared to free guest G2 and NADH3 to G3).