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During human infection, Mycobacterium tuberculosis (Mtb) survives the normally bacteriocidal phagosome of macrophages. Mtb and related species may be able to combat this harsh acidic environment which contains reactive oxygen species due to the mycobacterial genomes encoding a large number of dehydrogenases. Typically, dehydrogenase cofactor binding sites are open to solvent, which allows NAD/NADH exchange to support multiple turnover. Interestingly, mycobacterial short chain dehydrogenases/reductases (SDRs) within family TIGR03971 contain an insertion at the NAD binding site. Here we present crystal structures of 9 mycobacterial SDRs in which the insertion buries the NAD cofactor except for a small portion of the nicotinamide ring. Line broadening and STD-NMR experiments did not show NAD or NADH exchange on the NMR timescale. STD-NMR demonstrated binding of the potential substrate carveol, the potential product carvone, the inhibitor tricyclazol, and an external redox partner 2,6-dichloroindophenol (DCIP). Therefore, these SDRs appear to contain a non-exchangeable NAD cofactor and may rely on an external redox partner, rather than cofactor exchange, for multiple turnover. Incidentally, these genes always appear in conjunction with the mftA gene, which encodes the short peptide MftA, and with other genes proposed to convert MftA into the external redox partner mycofactocin.

A convenient synthesis of 2-(2-methyl-1-(4-oxo-3,4-dihydrophthalazin-1-yl)-1H-indol-3-yl)acetic acid derivatives is described using a microwave-promoted multi-step SNAr reaction. The desired products were found to exhibit atropisomerism.

By using a combination of inverse gated (1)H decoupled (13)C-NMR experiments1 with short acquisition times and NMR Cryo-probe technology, the sample requirements and experimental times necessary to accurately measure enantiomeric excess of small chiral molecules has been reduced 16-fold. Quality (13)C-NMR spectra can now be obtained from a 1 to 5 mg sample in 12 minutes. The enantiomeric excess determination achieved from the average integration of all the (13)C-resonances in the spectrum is comparable to enantiomeric excess measured by chiral SFC. The advantage of the NMR method is that enantiomeric excess can rapidly be measured in situ on practical amounts of enantioselective reaction products without the need for chromatographic separation or chemical modification and with substantially less solvent waste.

Potassium can stabilize the formation of chair- or edge-type quadruplex DNA structures and appears to be the only naturally occurring cation that can do so. As quadruplex DNAs may be important in the structure of telomere, centromere, triplet repeat and other DNAs, information about the details of the potassium-quadruplex DNA interactions are of interest. The structures of the 1:1 and the fully saturated, 2:1, potassium-DNA complexes of d(GGTTGGTGTGGTTGG) have been determined using the combination of experimental NMR results and restrained molecular dynamics simulations. The refined structures have been used to model the interactions at the potassium binding sites. Comparison of the 1:1 and 2:1 potassium:DNA structures indicates how potassium binding can determine the folding pattern of the DNA. In each binding site potassium interacts with the carbonyl oxygens of both the loop thymine residues and the guanine residues of the adjacent quartet.

The ability to chemically synthesize biomolecules has opened up the opportunity to observe changes in structure and activity that occur upon single atom substitution. In favorable cases this can provide information about the roles of individual atoms. The substitution of 6-thioguanine (6SG) for guanine is a potentially very useful single atom substitution as 6SG has optical, photocrosslinking, metal ion binding and other properties of potential utility. In addition, 6-mercaptopurine is a clinically important pro-drug that is activated by conversion into 6SG by cells. The results presented here indicate that the presence of 6SG blocks the formation of quadruplex DNA. The presence of 6SG alters the structure and lowers the thermal stability of duplex DNA, but duplex DNA can be formed in the presence of 6SG. These results indicate that some of the cytotoxic activity of 6SG may be due to disruption of the quadruplex structures formed by telomere and other DNAs. This additional mode of action is consistent with the delayed onset of cytotoxicity.

There are DNA sequences which adopt the same quadruplex structural type in the presence of sodium as in the presence of sodium and potassium. There are also sequences that appear to have a requirement for the presence of potassium for the adoption of a particular quadruplex structural type. Information about the basis for these potassium effects has been obtained by examining the structures of a set of DNAs with differing numbers of loop residues and different lengths of runs of dG residues in the presence of sodium alone and in the presence of potassium and sodium. On the basis of the results, obtained primarily via solution-state NMR, it appears that very small loops favor parallel stranded quartet structures which do not require the presence of potassium. DNAs with loops of two to four residues and runs of two dG residues can form quadruplex structures of the "edge" or "chair" type in the presence of potassium but not in the presence of sodium alone. When all of the loops contain four residues, a "crossover" or "basket" type structure can be formed in the presence of sodium as well as in the presence of sodium and potassium. Structures with runs of three or four dG residues and with loops from two to four residues can form basket or crossover type structures in the absence of potassium. The presence of a purine in a loop can block both potassium binding and formation of chair type structures. Modeling of the interactions of cations with these quadruplex structures indicates that the potassium ions required for chair type structures interact with a terminal quartet and residues in the adjacent loop.

The interaction of a DNA quadruplex with thrombin has been studied by first determining the sites of manganese binding to the quadruplex in the absence of thrombin. This has been followed by determining if the interactions with thrombin displace the bound manganese. A different DNA quadruplex has also been studied as a control. The refined solution structures of two DNA quadruplexes have been used to predict the electrostatic potentials of these DNAs. The calculated electrostatic potentials have been used to predict the locations of the binding sites of the paramagnetic ion manganese to these DNAs. The enhanced relaxation of DNA protons due to the binding of the paramagnetic metal ion Mn2+has been used to experimentally determine the locations of the binding sites. The NMR results and the predictions based on the electrostatic potentials both place the binding sites of the manganese in the narrow grooves of these quadruplex DNAs. The predicted locations are spatially close to those experimentally observed, and the predicted and experimental locations also have similar electrostatic potential energy. These results have allowed a validation of the predictions of electrostatic potentials from structure. The 15mer quadruplex has two strong Mn2+binding sites with one in each narrow groove. Both Mn2+are released when the 15mer is complexed with thrombin, indicating that both narrow grooves are involved in the 15mer – thrombin interactions. The dimer quadruplex has a different structural motif than the 15mer and the presence of thrombin does not appreciably affect its interactions with Mn2+.

The hydrolysis of 6-aminopenicillanic acid (APA) at neutral pH, catalyzed by the class C β-lactamase of Enterobacter cloacae P99, was observed from 1H NMR spectra to yield an unexpected product, 8-hydroxypenillic acid, in bicarbonate-containing buffers. This product probably arises from turnover of the carbamate of APA by the enzyme. 1H and 13C NMR spectra of APA in bicarbonate solutions clearly demonstrate the presence of the carbamate. Turnover of APA at saturating concentrations by the enzyme is accelerated by bicarbonate and by methanol. These results suggest that deacylation of the enzyme is rate determining and that the presence of the carbamate affects this step. A mechanism involving rate-determining intramolecular nucleophilic attack by the carbamate on the acyl enzyme is proposed to rationalize these observations and lead to formation of 8-hydroxypenillate. This reaction can be seen as an example of substrate-assisted enzymic catalysis. Quantitative analysis of the data indicated a dissociation ...

The total synthesis of (V) is achieved with the construction of the intermediate (IV) with complete diastereoselectivity as a key step.

An appropriately constructed 2-substituted derivative of L-tryptophane undergoes conversion, in a single step, to a prephalarine structure. The reaction occurs in a diastereoselective fashion, leading shortly thereafter to the naturally occurring version of the alkaloid, phalarine.

The flexibility and curvature of duplex DNAs containing mismatched sites have been monitored as a function of temperature. The diffusion coefficients are dependent on the flexibility and the curvature of the DNA, and these have been determined by NMR-based methods. The diffusion coefficients, D, depend on a Boltzmann term and the viscosity of the solvent, eta, which is also temperature dependent. To analyze the temperature dependence of the diffusion results, the shape function, S(f) = etaD/T, is used. The shape functions do not have the viscosity and temperature dependence of the diffusion coefficients. The presence of mismatched sites significantly enhances the shape function of duplex DNA at all temperatures examined. The observed increases in the shape functions are attributed to the mismatched sites acting as localized flexible joints. The results on the temperature dependence of the shape functions, the optical absorbance, and the proton chemical shifts indicate that local melting at, and adjacent to, mismatched site occurs at a lower temperature than the overall melting of the duplexes. The localized melting gives rise to a considerable increase in the shape function. The contribution of the curvature of the mismatched sites to the enhanced diffusion has been examined. A DNA with mismatches that are in phase with respect to the helical repeat and a DNA which has the mismatches out of phase with respect to the helical repeat have been examined. The results indicate that mismatched sites have modest curvature.

Paramagnetic ions have been used to assist the magnetic alignment of DNA. The anisotropy of the binding sites is sufficient to give rise to significant alignment of the DNA with the observed proton–carbon dipolar couplings spanning a 70-Hz range. The dipolar couplings have been used to determine the positions of the axial and rhombic alignment axes. The positions of the alignment axes relative to the positions of the binding sites of the paramagnetic europium ions have also been determined.