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Di- and triphosphorylated small molecules represent key intermediates in a wide range of biological and chemical processes. The importance of polyphosphorylated species in biology and medicine underscores the need to develop methods for the detection and characterization of this compound class. We have reported two-dimensional HPP-COSY spectroscopy techniques to identify diphosphate-containing metabolic intermediates at submillimolar concentrations in the methylerythritol phosphate (MEP) isoprenoid biosynthetic pathway. (1) In this work, we explore the scope of HPP-COSY-based techniques to characterize a diverse group of small organic molecules bearing di- and triphosphorylated moieties. These include molecules containing P-O-P and P-C-P connectivities, multivalent P(III)-O-P(V) phosphorus nuclei with widely separated chemical shifts, as well as virtually overlapping (31)P resonances exhibiting strong coupling effects. We also demonstrate the utility of these experiments to rapidly distinguish between mono- and diphosphates. A detailed protocol for optimizing these experiments to achieve best performance is presented.

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The biogenesis of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) is accomplished by the methylerythritol phosphate (MEP) pathway in plants, bacteria and parasites, making it a potential target for the development of anti-infective agents and herbicides. The biosynthetic enzymes comprising this pathway catalyze intriguing chemical transformations on diphosphate scaffolds, offering an opportunity to generate novel analogs in this synthetically challenging compound class. Such a biosynthetic approach to generating new diphosphate analogs may involve transformation through discrete diphosphate species, presenting unique challenges in structure determination and characterization of unnatural enzyme-generated diphosphate products produced in tandem. We have developed (1)H-(31)P-(31)P correlation NMR spectroscopy techniques for the direct characterization of crude MEP pathway enzyme products at low concentrations (200 microM to 5 mM) on a room temperature (non-cryogenic) NMR probe. Coupling the 100% natural abundance of the (31)P nucleus with the high intrinsic sensitivity of proton NMR, (1)H-(31)P-(31)P correlation spectroscopy is particularly useful for characterization of unnatural diphosphate enzyme products in the MEP pathway. As proof of principle, we demonstrate the rapid characterization of natural enzyme products of the enzymes IspD, E and F in tandem enzyme incubations. In addition, we have characterized several unnatural enzyme products using this technique, including new products of cytidyltransferase IspD bearing erythritol, glycerol and ribose components. The results of this study indicate that IspD may be a useful biocatalyst and highlight (1)H-(31)P-(31)P correlation spectroscopy as a valuable tool for the characterization of other unnatural products in non-mammalian isoprenoid biosynthesis.

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Prostate Specific Antigen (PSA) is a biomarker used in the diagnosis of prostate cancer and to monitor therapeutic response. However, its precise role in prostate carcinogenesis and metastasis remains largely unknown. A number of studies arguing in the favor of an active role of PSA in prostate cancer development and progression have implicated this serine protease in the release and activation of growth factors such as insulin-like growth factor 1 (IGF1) through cleavage of insulin like growth factor binding protein 3 and Transforming Growth Factor beta (TGF-beta) through cleavage of Latent TGF-beta. In contrast, other studies suggest that PSA activity might hinder tumor development and progression. In light of these contradictory findings, efficient inhibitors of PSA are needed for exploring its biological role in tumor development and metastasis. Towards the goal of developing potent inhibitors of PSA, we have explored the molecular mechanism of a series of beta-lactam based compounds on binding to PSA using activity assays, matrix assisted laser desorption ionization with a time-of-flight mass spectrometry, and GOLD docking methodology. The mass spectrometry experiments and the activity assays confirmed the time-dependent and covalent nature of beta-lactam binding. To gain insights on the reaction intermediates at the molecular level, we docked beta-lactam inhibitors to a homology modeled PSA using the GOLD docking program in noncovalent and covalent binding modes. The docking studies elucidated the molecular details of the early noncovalent Michaelis complex, the acyl-enzyme covalent complex, and the nature of conformational reorganization required for the long term stability of the covalent complex. Additionally, the molecular basis for the effect of stereochemistry of the lactam ring on the inhibitory potency was elucidated through docking of beta-lactam enantiomers. As a validation of our docking methodology, two novel enantiomers were synthesized and evaluated for their inhibitory potency using fluorogenic substrate based activity assays. Additionally, cis enantiomers of eight beta-lactam compounds reported in a previous study were docked and their GOLD scores and binding modes were analyzed in order to assess the general applicability of our docking results. The close agreement of our docking results with the experimental data validates the mechanistic insights revealed through the docking studies and paves the way for the design and development of potent and specific inhibitors of PSA.

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BACKGROUND: The mucosal lymphocyte population is the largest in the body, and the gastrointestinal compartment has been well characterized in HIV infection. Much less is known about the effects of HIV on the genital tract. OBJECTIVE: : To examine the T-lymphocyte phenotype and receptor repertoire as well as total and virus-specific immunoglobulin concentrations in the endocervix of HIV-infected women at different stages of infection as compared with uninfected women. PATIENTS AND METHODS: Participants were 12 seronegative women, 10 HIV-infected "slow progressors" not taking antiretroviral therapy, and 9 HIV-infected women whose antiretroviral therapy was failing. We used multiparameter flow cytometry to enumerate T-cell populations on cytobrush-obtained cervical specimens, the immunoscope technique to determine the T-cell receptor (TCR) repertoire, and quantitative enzyme-linked immunosorbent assays for antibody determinations on cervical secretions absorbed onto ophthalmic sponges. Nonparametric statistical analyses were performed. RESULTS: We found marked depletion of leukocytes and CD4 T lymphocytes in the endocervix of HIV-infected women as compared with uninfected women; this was significant at more advanced disease stages. Naive T cells were rare in the endocervix of all groups. Activation marker expression was higher in endocervical T lymphocytes than in peripheral blood among control and slow-progressing HIV-infected women but not in women failing therapy. Endocervical T lymphocytes showed highly restricted utilization of Vbeta TCR families. Unlike other mucosal sites, the cervix contained IgG as the predominant immunoglobulin isotype. HIV-IgG was detected in the cervix of most HIV-infected women and in blood of all infected women. CONCLUSIONS: HIV infection induces substantial changes in the immune profile of the female genital tract. Further study of the implications of these findings for HIV acquisition and transmission is needed.

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We report catalytic, enantioselective [4 + 2]-cycloadditions of o-quinones with ketene enolates (derived from readily available acid chlorides) using cinchona alkaloid derivatives as catalysts to produce products in high enantiomeric excess (ee) and good to excellent yields. The thermodynamic driving force for these reactions is due in part to the restoration of aromaticity to the products. The resulting chiral, bicycloadducts can be synthetically manipulated in a variety of useful ways, for example to provide a flexible synthesis of alpha-oxygenated carboxylic acid derivatives.

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We report a mechanistically based study of bifunctional catalyst systems in which chiral nucleophiles work in conjunction with Lewis acids to produce beta-lactams in high chemical yield, diastereoselectivity, and enantioselectivity. Chiral cinchona alkaloid derivatives work best when paired with Lewis acids based on Al(III), Zn(II), Sc(III), and, most notably, In(III). Homogeneous bifunctional catalysts, in which the catalyst contains both Lewis acidic and Lewis basic sites, were also studied in detail. Mechanistic evidence allows us to conclude that the chiral nucleophiles form zwitterionic enolates that react with metal-coordinated imines. Alternative scenarios, which postulated metal-bound enolates, were disfavored on the basis of our observations.

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Cervical cytobrushes provide a tool to sample endocervical T cells for assessment of local immunity. However, most previous studies in HIV-seropositive women have excluded samples containing blood and hence have analyzed selected populations of patients. As determined by multiple-parameter flow cytometric analysis of T lymphocytes from two sequential cytobrushes and concurrently collected blood samples, this study found a minimal effect of blood contamination on cervical T cell phenotypic parameters in normal women. The consequences of blood in endocervical samples will ultimately depend on the design and objective of each study, but these data suggest studies could be more inclusive and should not automatically discard samples that contain red blood cells.

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We present a full account of a tandem catalytic, asymmetric chlorination/esterification process that produces highly optically enriched alpha-chloroesters from inexpensive, commercially available acid halides using cinchona alkaloid derivatives as catalysts and polychlorinated quinones as halogenating agents. We have performed kinetics and control experiments to investigate the reaction mechanism and establish conditions under which the reactions can be best performed. We have developed NaH and NaHCO3 shuttle base systems as the easiest and most cost-effective ways of conducting the reactions, rendering the methodology economically competitive with known chiral halogenation procedures. We have also demonstrated the utility of our reactions by converting the products to synthetically useful derivatives.

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A catalytic asymmetric procedure for the preparation of beta-amino acids (specifically beta-substituted aspartic acid derivatives) is reported. The cinchona alkaloid catalyst benzoylquinine (BQ) mediates up to five distinct steps of a reaction pathway, all in one reaction vessel. The products of this reaction, highly optically enriched beta-substituted aspartic acid derivatives, were prepared from N-acyl-alpha-chloroglycine esters and acid chlorides in the presence of the catalyst. This approach was also amenable to the synthesis of small polypeptides containing beta-substituted aspartic acid units, including a non-natural fragment of the antibiotic lysobactin. The addition of Lewis acids to this system was found to accelerate the rate of specific steps in the reaction pathway. Mechanistic aspects of this reaction, such as imine formation and Lewis acid chelation to the beta-lactam intermediate, were investigated through comparison of IR, NMR, and other physical data.

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