We have applied a global approach to enzyme active site exploration, where multiple mutations were introduced combinatorially at the active site of Type II R67 dihydrofolate reductase (R67 DHFR), creating numerous new active site environments within a constant framework. By this approach, we combinatorially modified all 16 principal amino acids that constitute the active site of this enzyme. This approach is fundamentally different from active site point mutation in that the native active site context is no longer accounted for. Among the 1536 combinatorially mutated active site variants of R67 DHFR we created, we selected and kinetically characterized three variants with highly altered active site compositions. We determined that they are of high fitness, as defined by a complex function consisting jointly of catalytic activity and resistance to trimethoprim. The k(cat) and K(M) values were similar to those for the native enzyme. The favourable Delta(DeltaG) values obtained (ranging from -0.72 to -1.08 kcal/mol) suggest that, despite their complex mutational pattern, no fundamental change in the catalytic mechanism has occurred. We illustrate that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation.

The development of low molecular weight anionophores is an emerging topic in chemistry, as the need for these compounds increases with the continuous discovery of pathologies involving anomalies in anion transport processes. Development of new concepts to initiate anion imbalance in living cells while fighting multidrug-resistant bacteria is a paramount topic. In this study, three series of compounds including N,N'-diphenylethynylbenzyl benzimidazolium salts (1 and 2), 1,1'-(pyridine-2,6-diyl)bis(3-(4-(phenylethynyl)benzyl)-1H-benzo[d]imidazol-3-ium) salts (3-5), and 1,1'-(pyridine-2,6-diylbis(methylene))bis(3-(4-(phenyl ethynyl)benzyl)-1H-benzo[d]imidazol-3-ium) salts (6-8) displaying high antimicrobial activity and low toxicity against human cells were designed, synthesized, and studied. The most potent compound displayed micromolar minimal inhibitory concentrations in different Gram-negative and Gram-positive bacteria, while its hemolytic activity remained around 10% or less, even after a prolonged period of exposure. The mechanism of action of these benzimidazolium salts on bacterial membrane was assessed by bioanalytical techniques including assays in model membrane liposomes, membrane depolarization studies, and scanning electron microscopy (SEM) in living bacteria.

N,N'-Disubstituted methylenediimidazolium salts allow the formation of flexible inclusion complexes with beta-cyclodextrin, cucurbit[7]uril, tetrapropoxycalix[4]arene, and dibenzo-24-crown-8 ether. Due to the salt nature of the imidazolium guest, the counterion largely determines its solubility in a given solvent. Moreover, by the judicious choice of the imidazolium substituents, inclusion complexes of guest salts were obtained with a variety of macrocyclic hosts, and the binding parameters of the inclusion were determined for each complex.

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