Mycofactocin-associated mycobacterial dehydrogenases with non-exchangeable NAD cofactors.

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.

An improved method for determining enantiomeric excess by (13)C-NMR in chiral liquid crystal media.

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.

Total synthesis of enantiopure phalarine via a stereospecific Pictet-Spengler reaction: traceless transfer of chirality from L-tryptophan.

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

Stereochemical identification of (R)- and (S)-ibuprofen using residual dipolar couplings, NMR, and modeling.

In this work, we describe an NMR-based method that utilizes an orientation media composed of the chiral polypeptide liquid crystal poly-gamma-benzyl-L-glutamate (PBLG) dissolved in CDCl(3), to measure the (1)H-(1)H, (1)H-(13)C and (13)C-(13)C residual dipolar couplings (RDCs) of (R) and (S)-ibuprofen. Calculated RDCs, obtained from the lowest energy conformers, are then compared with the experimentally measured RDCs to predict the stereochemistry of each enantiomer. Excellent agreement between calculated and experimental RDCs was found when the lowest energy structure of each enantiomer, obtained in a simulated PBLG/CDCl(3) environment, was used to back-calculate the RDCs. This method is generally useful for small molecular weight molecules that possess either one or two chiral centers, are soluble in low viscosity organic solvents, and will not crystallize (Clegg, Crystal Structure Analysis. Principles and Practice. New York: Oxford University Press; 2002) or cannot be derivatized with a Mosher's reagent (Dale and Mosher, J Am Chem Soc 1973;95:512-519).

Simultaneous determination of 1H-1H and 1H-13C residual dipolar couplings in a chiral liquid crystal solvent using a natural abundance HSQC experiment.

A high-resolution, phase-sensitive, natural abundance F2-coupled 1H-13C HSQC (F2HSQC) NMR experiment was developed to measure simultaneously both (n)D(HH) and 1D(CH) residual dipolar couplings (RDCs) of small molecules present in a chiral polypeptide liquid crystal solvent system composed of poly-gamma-benzyl-L-glutamate (PBLG) in CDCl3. Because this is an indirect-detection NMR experiment, the relatively small amount of sample (7.5 mg in this study) and short acquisition times (5 h) that are required make this HSQC experiment well suited for samples that are either limited in solubility or in quantity or require short analysis times. The F2HSQC experiment can be performed without any specialized equipment or sample modification and can enhance our ability to measure RDCs accurately and rapidly in polypeptide liquid crystal solvents.