Conformational changes caused by specific interactions with protic solvents were studied for 2-(2′-pyridyl)indole and related compounds. Both syn and anti rotameric forms are possible for 2-(2′-pyridyl)-indole. Only the syn conformers are able to form cyclic, doubly hydrogen-bonded complexes with protic solvents. These cyclic solvates undergo efficient fluorescence quenching due to photoinduced double proton transfer and internal conversion. This feature makes it possible to distinguish between the two rotamers and to determine their relative abundance. In aprotic solvents, only the syn form is detected. On the contrary, fluorescence measurements reveal that in alcohols about 80% of the excited-state population are due to the anti conformer. Similar results are obtained for the ground state from NMR NOE experiments, which imply that no interconversion between the two forms occurs in the excited state. Ab initio calculations predict that the syn form should be more stable by about 4.3 kcal/mol. Therefore, the data obtained in alcohol solvents show that the reversal of the syn/anti relative stability is due to hydrogen bonding to the solvent. These conclusions are confirmed by experiments performed for the N-methylated derivative, for bridged 2-(2′-pyridyl)indoles which can only exist in the syn form, and for 2-(4,6-dimethyl-2′-pyrimidyl)indole, where syn and anti conformers are identical. In bulk water solutions no evidence for syn f anti rotamerization was found. However, the process was detected in acetonitrile/water mixtures.

Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.

Spectral, synthetic, and theoretical studies were performed for a family of bifunctional compounds possessing both a hydrogen bond donor (aromatic NH group) and an acceptor (pyridine-type nitrogen atom). The series included 1H-pyrrolo[3,2-h]quinoline, 7,8,9,10-tetrahydropyrido[2,3-a]carbazole, pyrido[2,3-a]carbazole, dipyrido[2,3-a:3‘,2‘-i]carbazole, and 2-(2‘pyridyl)indoles. In alcohol solutions, all these compounds reveal the phenomenon of excited state intermolecular double proton transfer, occurring in complexes of the excited chromophore with an alcohol molecule. This process was identified by comparing the fluorescence of the phototautomeric products with the emission of molecules synthesized to serve as chemical models of the tautomeric structures. Detailed investigations demonstrate that the excited state reaction occurs in solvates that, already in the ground state, have an appropriate stoichiometry and structure. These species correspond to 1:1 cyclic, doubly hydrogen bonded complexes with alcohol. Other types of complexes with alcohol were also found, which, upon excitation, undergo deactivation to the ground state via internal conversion. The relative populations of the two forms of alcohol solvates, characterized by different photophysics, vary strongly across the series. The properties of the presently investigated compounds differ from those of a structurally related 7-azaindole and 1-azacarbazole, in which the phototautomerization involves solvent relaxation around the excited chromophore. Molecular dynamics calculations, performed to predict and compare the ground-state structure of 7-azaindole and 1H-pyrrolo[3,2-h]quinoline alcohol complexes, allow one to rationalize the observed differences in the excited-state reaction mechanisms for the two kinds of systems.