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Herein, we establish a method to quantitatively monitor a metal-organic framework (MOF)-catalyzed, biomedically relevant reaction directly in blood plasma, specifically, the generation of nitric oxide (NO) from the endogenous substrate S -nitrosoglutathione (GSNO) catalyzed by H 3 [(Cu 4 Cl) 3 -(BTTri) 8 ] (CuBTTri). The reaction monitoring method uses UV-vis and 1 H NMR spectroscopies along with a nitric oxide analyzer (NOA) to yield the reaction stoichiometry and catalytic rate for GSNO to NO conversion catalyzed by CuBTTri in blood plasma. The results show 100% loss of GSNO within 16 h and production of 1 equiv. of glutathione disulfide (GSSG) per 2 equiv. of GSNO. Only 78 ± 10% recovery of NO(g) was observed, indicating that blood plasma can scavenge the generated NO before it can escape the reaction vessel. Significantly, to best apply and understand reaction systems with biomedical importance, such as NO release catalyzed by CuBTTri, methods to study the reaction directly in biological solvents must be developed.

In bulk aqueous environments, the exchange of protons between labile hydroxyl groups typically occurs easily and quickly. Nanoconfinement can dramatically change this normally facile process. Through exchange spectroscopy (EXSY) NMR measurements, we observe that nanoconfinement of glucose and water within AOT (sodium bis(2-ethylhexyl) sulfosuccinate) reverse micelles raises the energy barrier to labile hydrogen exchange, which suggests a disruption of the hydrogen bond network. Near room temperature, we measure barriers high enough to slow the process by as much as 2 orders of magnitude. Although exchange rates slow with decreasing temperatures in these nanoconfined environments, the barrier we measure below ∼285 K is 3-5 times lower than the barrier measured at room temperature, indicating a change in mechanism for the process. These findings suggest the possibility of hydrogen tunneling at a surprisingly high-temperature threshold. Furthermore, differences in exchange rates depend on the hydroxyl group position on the glucose pyranose ring and suggest a net orientation of glucose at the reverse micelle interface.

Copper containing compounds catalyze decomposition of S-Nitrosoglutathione (GSNO) in the presence of glutathione (GSH) yielding glutathione disulfide (GSSG) and nitric oxide (NO). Extended NO generation from an endogenous source is medically desirable to achieve vasodilation, reduction in biofilms on medical devices, and antibacterial activity. Homogeneous and heterogeneous copper species catalyze release of NO from endogenous GSNO. One heterogeneous catalyst used for GSNO decomposition in blood plasma is the metal-organic framework (MOF), H 3 [(Cu 4 Cl) 3 -(BTTri) 8 , H 3 BTTri = 1,3,5-tris( 1 H-1,2,3-triazol-5-yl) benzene] (CuBTTri). Fundamental questions about these systems remain unanswered, despite their use in biomedical applications, in part because no method previously existed for simultaneous tracking of [GSNO], [GSH], and [GSSG] in water. Tracking these reactions in water is a necessary step towards study in biological media (blood is approximately 80% water) where NO release systems must operate. Even the balanced stoichiometry remains unknown for copper-ion and CuBTTri catalyzed GSNO decomposition. Herein, we report a direct 1 H NMR method which: simultaneously monitors [GSNO], [GSH], and [GSSG] in water; provides the experimentally determined stoichiometry for copper-ion vs CuBTTri catalyzed GSNO decomposition; reveals that the CuBTTri-catalyzed reaction reaches 10% GSNO decomposition (16 h) without added GSH, yet the copper-ion catalyzed reaction reaches 100% GSNO decomposition (16 h) without added GSH; and shows 100% GSNO decomposition upon addition of stoichiometric GSH to the CuBTTri catalyzed reaction. These observations provide evidence that copper-ion and CuBTTri catalyzed GSNO decomposition in water operate through different reaction mechanisms, the details of which can now be probed by 1 H NMR kinetics and other needed studies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The research presented here reports the surprising observation that adding glucose and other carbohydrate osmolytes to the polar phase of water-containing reverse micelles causes the particles to shrink. This apparent change in reverse micelle size is attributed to two factors: an increase in the surface area per surfactant molecule induced by the presence of carbohydrate and changes in the particle shape eccentricity. The studies reported here not only focus on glucose but also explore other carbohydrate osmolytes, specifically ethylene glycol, glycerol, erythritol, xylitol, sorbitol, myo-inositol, and trehalose, in the nanoconfined environments of reverse micelles. Through two-dimensional proton nuclear Overhauser enhancement nuclear magnetic resonance spectroscopy, the osmolytes were determined to reside solvated in the aqueous interior of the reverse micelles. This paper reports the loading limit of carbohydrates into AOT [sodium bis(2-ethylhexyl)sulfosuccinate] reverse micelles, demonstrates the location of the carbohydrates in the reverse micelles, and shows an unexpected effect where the carbohydrates add to the reverse micelle volume without causing an apparent increase in the reverse micelle diameter.

Menaquinones (naphthoquinones, MK) are isoprenoids that play key roles in the respiratory electron transport system of some prokaryotes by shuttling electrons between membrane-bound protein complexes acting as electron acceptors and donors. Menaquinone-2 (MK-2), a truncated MK, was synthesized, and the studies presented herein characterize the conformational and chemical properties of the hydrophobic MK-2 molecule. Using 2D NMR spectroscopy, we established for the first time that MK-2 has a folded conformation defined by the isoprenyl side-chain folding back over the napthoquinone in a U-shape, which depends on the specific environmental conditions found in different solvents. We used molecular mechanics to illustrate conformations found by the NMR experiments. The measured redox potentials of MK-2 differed in three organic solvents, where MK-2 was most easily reduced in DMSO, which may suggest a combination of solvent effect (presumably in part because of differences in dielectric constants) and/or conformational differences of MK-2 in different organic solvents. Furthermore, MK-2 was found to associate with the interface of model membranes represented by Langmuir phospholipid monolayers and Aerosol-OT (AOT) reverse micelles. MK-2 adopts a slightly different U-shaped conformation within reverse micelles compared to within solution, which is in sharp contrast to the extended conformations illustrated in literature for MKs.

Cp X Rh(III)-catalyzed C-H functionalization reactions are a proven method for the efficient assembly of small molecules. However, rationalization of the effects of cyclopentadienyl (Cp X ) ligand structure on reaction rate and selectivity has been viewed as a black box, and a truly systematic study is lacking. Consequently, predicting the outcomes of these reactions is challenging because subtle variations in ligand structure can cause notable changes in reaction behavior. A predictive tool is, nonetheless, of considerable value to the community as it would greatly accelerate reaction development. Designing a data set in which the steric and electronic properties of the Cp X Rh(III) catalysts were systematically varied allowed us to apply multivariate linear regression algorithms to establish correlations between these catalyst-based descriptors and the regio-, diastereoselectivity, and rate of model reactions. This, in turn, led to the development of quantitative predictive models that describe catalyst performance. Our newly described cone angles and Sterimol parameters for Cp X ligands served as highly correlative steric descriptors in the regression models. Through rational design of training and validation sets, key diastereoselectivity outliers were identified. Computations reveal the origins of the outstanding stereoinduction displayed by these outliers. The results are consistent with partial η 5 -η 3 ligand slippage that occurs in the transition state of the selectivity-determining step. In addition to the instructive value of our study, we believe that the insights gained are transposable to other group 9 transition metals and pave the way toward rational design of C-H functionalization catalysts.

Glucose nanoconfined by solubilization in water-containing AOT (sodium bis(2-ethylhexyl) sulfosuccinate) reverse micelles has been investigated using 1 H NMR. NMR spectra reveal well-defined signals for the glucose hydroxyl groups that suggest slow chemical exchange between them and the water hydroxyl groups. Using the EXSY (ZZ-exchange) method, the chemical exchange rate from water to glucose hydroxyl groups was measured for glucose in reverse micelles as a function of size (water pool diameter of ∼1-5 nm) at 25 °C. The chemical exchange rates observed in the nanoconfined interior are dramatically slower (5-20 times) than those observed for glucose in bulk aqueous solution at the same concentration as the micelle interior. Exchange rate constants are calculated via a mechanism that accounts for these observations, and implications of these results are presented and discussed.

Inorganic nanoparticles, stabilized by a passivating layer of organic molecules, form a versatile class of nanostructured materials with potential applications in material chemistry, nanoscale physics, nanomedicine and structural biology. While the structure of the nanoparticle core is often known to atomic precision, gaining precise structural and dynamical information on the organic layer poses a major challenge. Here we report a full assignment of (1)H and (13)C NMR shifts to all ligands of a water-soluble, atomically precise, 102-atom gold nanoparticle stabilized by 44 para-mercaptobenzoic acid ligands in solution, by using a combination of multidimensional NMR methods, density functional theory calculations and molecular dynamics simulations. Molecular dynamics simulations augment the data by giving information about the ligand disorder and visualization of possible distinct ligand conformations of the most dynamic ligands. The method demonstrated here opens a way to controllable strategies for functionalization of ligated nanoparticles for applications.

Monogalactosyldiacylglycerol (MGDG) was identified as a host recognition cue for larvae of the western corn rootworm Diabrotica virgifera virgifera LeConte. An active glycolipid fraction obtained from an extract of germinating maize roots was isolated with thin layer chromatography using a bioassay-driven approach. When analyzed with LC-MS (positive ion scanning), the assay-active spot was found to contain four different MGDG species: 18:3-18:3 (1,2-dilinolenoyl), 18:2-18:3 (1-linoleoyl, 2-linolenoyl), 18:2-18:2 (1,2-dilinoleoyl), and 18:2-16:0 (1-linoleoyl, 2-palmitoyl). A polar fraction was also needed for activity. When combined with a polar fraction containing a blend of sugars (glucose:fructose:sucrose:myoinositol), the isolated MGDG elicited a unique tight-turning behavior by neonate western corn rootworm larvae that is indicative of host recognition. In behavioral bioassays where disks treated with the active blend were exposed to successive sets of rootworm larvae, the activity of MGDG increased over four exposures, suggesting that larvae may be responding to compounds produced after enzymatic breakdown of MGDG. In subsequent tests with synthetic blends composed of theoretical MGDG-breakdown products, larval responses to four synthetic blends were not significantly different (P<0.5) than the response to isolated MGDG. GC-MS analysis showed modest increases in the amounts of the 16:0, 18:0, and 18:3 free fatty acids released from MGDG after a 30-min exposure to rootworm larvae, which is consistent with the enzymatic breakdown hypothesis., (© The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Since membrane penetration is important for drug efficacy, how antimalarial precursor material 1-phenylbiguanide (PBG) interacts with an interface was characterized using a reverse micelle (RM) model system. (1)H NMR studies show that PBG partitions across the membrane interface. Specifically, the (1)H NMR studies showed that the 1-phenylbiguanide compound in an aqueous environment changed when placed near an interface. PBG is known to affect hydrogen bonding in water, and as the size of the RMs changes, the water organization in the water pool is changed. The NOESY spectrum of PBG in AOT RM contains cross-peak signals between the PBG protons and AOT protons, which is consistent with the penetration of the PBG into the interface. At the same time, there is a cross peak between the biguanide moiety and the HOD signal. This shows that these NH protons are near the HOD protons, placing the biguanide functional group in the water pool. Preliminary differential FTIR spectroscopic studies confirmed this location. In summary, we found that PBG interacts with different regions of the interface, with the phenyl group penetrating the hydrophobic interface while the biguanide remains in the water pool.

With the understanding that the laboratory propagated strain of Mycobacterium tuberculosis H37Rv is of modest virulence and is drug susceptible, in the present study, we performed a nuclear magnetic resonance-based metabolomic analysis of lung tissues and serum obtained from guinea pigs infected by low dose aerosol exposure to clinical isolates of Mycobacterium tuberculosis. High Resolution Magic Angle Spinning NMR coupled with multivariate statistical analysis of 159 lung tissues obtained from multiple locations of age-matched naïve and 30 and 60 days of infected guinea pig lungs revealed a wide dispersal of metabolic patterns, but within these, distinct clusters of signatures could be seen that differentiated between naive control and infected animals. Several metabolites were identified that changed in concert with the progression of each infection. Major metabolites that could be interpreted as indicating host glutaminolysis were consistent with activated host immune cells encountering increasingly hypoxic conditions in the necrotic lung lesions. Moreover, glutathione levels were constantly elevated, probably in response to oxygen radical production in these lesions. Additional distinct signatures were also seen in infected serum, with altered levels of several metabolites. Multivariate statistical analysis clearly differentiated the infected from the uninfected sera; in addition, Receiver Operator Characteristic curve generated with principal component 1 scores showed an area under the curve of 0.908. These data raise optimism that discrete metabolomic signatures can be defined that can predict the progression of the tuberculosis disease process, and form the basis of an innovative and rapid diagnostic process.

The dramatic impact of differing environments on proton transfer dynamics of the photoacid HPTS prompted us to investigate these systems with two highly complementary methods: ultrafast time-resolved transient absorption and two-dimensional NMR spectroscopies. Both ultrafast time-resolved transient absorption spectroscopy and time-resolved anisotropy decays demonstrate the proton transfer dynamics depend intimately on the specific reverse micellar system. For w(0) = 10 reverse micelles formed with anionic AOT surfactant, the HPTS proton transfer dynamics are similar to dynamics in bulk aqueous solution, and the corresponding (1)H 2D NOESY NMR spectra display no cross peaks between HPTS and AOT consistent with the HPTS residing well hydrated by water in the interior of the reverse micelle water pool. In contrast, ultrafast transient absorption experiments show no evidence for HPTS photoinduced proton transfer reaction in reverse micelles formed with the cationic CTAB surfactant. In CTAB reverse micelles, clear cross peaks between HPTS and CTAB in the 2D NMR spectra show that HPTS embeds in the interface. These results indicate that the environment strongly impacts the proton transfer reaction and that complementary experimental techniques develop understanding of how location critically affects molecular responses.

A crucial and distinctive feature of tuberculosis infection is that Mycobacterium tuberculosis (Mtb) resides in granulomatous lesion at various stages of disease development and necrosis, an aspect that is little understood. We used a novel approach, applying high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) directly to infected tissues, allowing us to study the development of tuberculosis granulomas in guinea pigs in an untargeted manner. Significant up-regulation of lactate, alanine, acetate, glutamate, oxidized and the reduced form of glutathione, aspartate, creatine, phosphocholine, glycerophosphocholine, betaine, trimethylamine N-oxide, myo-inositol, scyllo-inositol, and dihydroxyacetone was clearly visualized and was identified as the infection progressed. Concomitantly, phosphatidylcholine was down-regulated. Principal component analysis of NMR data revealed clear group separation between infected and uninfected tissues. These metabolites are suggestive of utilization of alternate energy sources by the infiltrating cells that generate much of the metabolites in the increasingly necrotic and hypoxic developing granuloma through the glycolytic, pentose phosphate, and tricarboxylic acid pathways. The most relevant changes seen are, surprisingly, very similar to metabolic changes seen in cancer during tumor development.

Microemulsions form in mixtures of polar, nonpolar, and amphiphilic molecules. Typical microemulsions employ water as the polar phase. However, microemulsions can form with a polar phase other than water, which hold promise to diversify the range of properties, and hence utility, of microemulsions. Here microemulsions formed by using a room-temperature ionic liquid (RTIL) as the polar phase were created and characterized by using multinuclear NMR spectroscopy. (1)H, (11)B, and (19)F NMR spectroscopy was applied to explore differences between microemulsions formed by using 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][BF(4)]) as the polar phase with a cationic surfactant, benzylhexadecyldimethylammonium chloride (BHDC), and a nonionic surfactant, Triton X-100 (TX-100). NMR spectroscopy showed distinct differences in the behavior of the RTIL as the charge of the surfactant head group varies in the different microemulsion environments. Minor changes in the chemical shifts were observed for [bmim](+) and [BF(4)](-) in the presence of TX-100 suggesting that the surfactant and the ionic liquid are separated in the microemulsion. The large changes in spectroscopic parameters observed are consistent with microstructure formation with layering of [bmim](+) and [BF(4)](-) and migration of Cl(-) within the BHDC microemulsions. Comparisons with NMR results for related ionic compounds in organic and aqueous environments as well as literature studies assisted the development of a simple organizational model for these microstructures., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cellular envelope of Mycobacterium tuberculosis is highly distinctive and harbors a wealth of unique lipids possessing diverse structural and biological properties. However, the ability to conduct global analyses on the full complement of M. tuberculosis lipids has been missing from the repertoire of tools applied to the study of this important pathogen. We have established methods to detect and identify lipids from all major M. tuberculosis lipid classes through LC/MS lipid profiling. This methodology is based on efficient chromatographic separation and automated ion identification through accurate mass determination and searching of a newly created database (Mtb LipidDB) that contains 2,512 lipid entities. We demonstrate the sensitive detection of molecules representing all known classes of M. tuberculosis lipids from a single crude extract. We also demonstrate the ability of this methodology to identify changes in lipid content in response to cellular growth phases. This work provides a customizable framework and resource to facilitate future studies on mycobacterial lipid biosynthesis and metabolism.

The possibility that a negatively charged organic molecule penetrates the lipid interface in a reverse micellar system is examined using UV-vis absorption and NMR spectroscopy. The hypothesis that deprotonated forms of dipicolinic acid, H(2)dipic, such as Hdipic(-) and dipic(2-), can penetrate the lipid interface in a microemulsion is based on our previous finding that the insulin-enhancing anionic [VO(2)dipic](-) complex was found to reside in the hydrophobic layer of the reverse micelle (Crans et al. J. Am. Chem. Soc. 2006, 128, 4437-4445). Penetration of a polar and charged compound, namely Hdipic(-) or dipic(2-), into a hydrophobic environment is perhaps unexpected given the established rules regarding the fundamental properties of compound solubility. As such, this work has broad implications in organic chemistry and other disciplines of science. These studies required a comprehensive investigation of the different dipic species and their association in aqueous solutions at varying pH values. Combining the aqueous studies using absorption and NMR spectroscopy with those in microemulsions defines the differences observed in the heterogeneous environment. Despite the expected repulsion between the surfactant head groups and the dianionic probe molecule, these studies demonstrate that dipic resides deep in the hydrophobic portion of the reverse micellar interface. In summary, these results provide evidence that ionic molecules can reside in nonpolar locations in microheterogeneous environments. This suggests that additional factors such as solvation are important to molecule location. Documented ability to penetrate lipid surfaces of similar charge provides a rationale for why specific drugs with less than optimal hydrophobicity are successful even though they violate Lipinski's rules.

Taxa-4(20),11(12)-diene-5α-acetate 5 and Taxa-4(20), 11(12)-diene-5α-acetate, 10β-ol 6, have been identified as early stage intermediates involved in the biosynthesis of Taxol® (paclitaxel). Tritium-labeled 5 and 6 were successfully prepared by Barton deoxygenation using tri-n-butyltintritiide of the C-14-hydroxyl group of a taxoid obtained from Japanese Yew.

The complex cell wall of Mycobacterium tuberculosis is the hallmark of acid fast bacteria and is responsible for much of its physiological characteristics. Hence, much effort has been made to determine its primary structure. Such studies have been hampered by its extreme complexity. Also, its insolubility leads to difficulties determining the presence or absence of base labile groups. We have used an endogenous arabinase to solubilize the arabinan region of the cell wall and have shown using mass spectrometry and NMR that succinyl esters are present on O2 of the inner-branched 1,3,5-alpha-d-arabinofuranosyl residues. In addition, an inner arabinan region of 14 linear alpha-1,5 arabinofuranosyl residues has been identified. These and earlier results now allow the presentation of a model of the entire primary structure of the mycobacterial mycolyl arabinogalactan highlighted by three arabinan chains of 31 residues each.

The location and interactions of solutes in microheterogeneous environments, such as reverse micelles, critically influence understanding of many phenomena that utilize probe molecules to characterize properties in chemical, biological, and physical systems. The information gained in such studies depends substantially on the location of the probe used. Often, intuition leads to the assumption that ionic probe molecules reside in the polar water pool of a system. In this work, the location of a charged polar transition metal coordination complex in a reverse micellar system is determined using NMR spectroscopy. Despite the expected Coulomb repulsion between the surfactant headgroups and the negatively charged complex, the complex spends significant time penetrating into the hydrophobic portion of the reverse micellar interface. These results challenge the assumption that ionic probe molecules reside solvated by water in microheterogeneous environments and suggest that probe molecule location be carefully considered before interpreting data from similar systems.

Ca2+ channel beta subunits regulate cell-surface expression and gating of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, beta subunits are predicted to be modular structures composed of five domains (A-E) that are related to the large family of membrane-associated guanylate kinase proteins. The crystal structure of the beta subunit core B-D domains has been reported recently; however, little is known about the structures of the A and E domains. The N-terminal A domain differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4) primarily as the result of two duplications of an ancestral gene containing multiple alternatively spliced exons. At least nine A domain sequences can be generated by alternative splicing. In this report, we focus on one A domain sequence, the highly conserved beta4a A domain. We solved its three-dimensional structure and show that it is expressed in punctate structures throughout the molecular layer of the cerebellar cortex. We also demonstrate that it does not participate directly in Cav2.1 Ca2+ channel gating but serves as a binding site in protein-protein interactions with synaptotagmin I and the LC2 domain of microtubule-associated protein 1A. With respect to beta4 subunits, the interactions are specific for the beta4a splice variant, because they do not occur with the beta4b A domain. These results have strong bearing on our current understanding of the structure of alternatively spliced Ca2+ channel beta subunits and the cell-specific roles they play in the CNS.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne disease in cattle and other ruminants, is proposed to be at least one of the causes of Crohn disease in humans. MAP and Mycobacterium avium subspecies avium, a closely related opportunistic environmental bacterium, share 95% of their genes and exhibit homologies of more than 99% between these genes. The identification of molecules specific for MAP is essential for understanding its pathogenicity and for development of useful diagnostic tools. The application of gas chromatography, mass spectrometry, and nuclear magnetic resonance led to the structural identification of a major cell wall lipopeptide of MAP, termed Para-LP-01, defined as C20 fatty acyl-D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala methyl ester. Variations of this lipopeptide with different fatty acyl moieties (C16 fatty acyl through C17, C18, C19, C21 to C22) were also identified. Besides the specificity of this lipopeptide for MAP, the presence of an N-Me-L-valine represents the first reported N-methylated amino acid within an immunogenic lipopeptide of mycobacteria. Sera from animals with Johne disease, but not sera from uninfected cattle, reacted with this lipopeptide, indicating potential biological importance.

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.

The fluorescent alkaloid norharmane has been isolated from Reticulitermes termites and characterized by 1H NMR, UV/Vis, mass spectrometry, and gas chromatography/mass spectrometry. Microcoil 1H NMR spectroscopy allowed spectra to be obtained from mass-limited material, facilitating the identification of norharmane, which is the major component in termite fluorescence under UV light. Norharmane was uniformly present at approximately 1 ng/mg in Reticulitermes tibialis Banks workers, soldiers, and alates; Reticulitermes flavipes (Kollar) workers; and Reticulitermes virginicus (Banks) workers. Some termites were observed to fluoresce with less intensity, but no differences in norharmane levels were detected. Mechanisms that may account for fluorescent differences are discussed as are the possible ecological implications of norharmane in termites.

In confined media such as reverse micelles, molecular probes frequently reside at and interact strongly with the interface. If the interface is charged, it is often difficult to separate effects arising from interactions with the charged species from the effect of the interfacial environment. With reverse micelles as a model system, the work reported here explores the interaction of the charged surfactant headgroups at a self-assembled interface with the dipicolinatodioxovanadium(V) coordination complex. The vanadium complex studied in these experiments serves as an excellent probe to investigate how charged metal complexes interact with lipid interfaces. For comparison, measurements were also carried out probing the interaction of the vanadium complex with a model cationic headgroup, tetramethylammonium bromide. The impact of the environment is gauged by changes in the 51V chemical shift, longitudinal relaxation times, and 1H NMR pulsed field gradient measurements. These measurements suggest that while interface component parts, as modeled by the dispersed systems, interact with the vanadium complex, the interfacial environment perturbs the complex substantially more strongly than the sum of the components alone. Coulomb attraction dominates the interaction in all systems probed and surprisingly orients the hydrophobic portion into the bulk water.

Current knowledge on the structure of lipoarabinomannan (LAM) has resulted primarily from detailed studies on a few selected laboratory strains of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium smegmatis. Our previous work was the first to report on the salient structural features of M. tuberculosis clinical isolates and demonstrated significant structural variations. A prime effort is to correlate a particular structural characteristic with observed differences in eliciting an immunobiological response, especially in the context of CD1-restricted presentation of LAM to T cells. T cell clones derived from the cutaneous lesions of leprosy patients have been shown to recognize specifically LAM from Mycobacterium leprae and not from M. tuberculosis Erdman or H37Rv. Herein we provide further fine structural data on LAM from M. leprae (LepLAM) and a tuberculosis clinical isolate, CSU20 (CSU20LAM), which was unexpectedly recognized by the supposedly LepLAM-specific CD1-restricted T cell clones. In comparison with the de facto laboratory LAM standard from M. tuberculosis H37Rv (RvLAM), LepLAM derived from in vivo grown M. leprae is apparently simpler in its arabinan architecture with a high degree of exposed, non-mannose-capped termini. On the other hand, CSU20, an ethambutol-resistant clinical isolate, makes a vastly heterogeneous population of LAM ranging from rather small and non-mannose-capped to full-length and fully capped variants. LepLAM and CSU20LAM contain a higher level of succinylation than RvLAM, which, in the context of truncated or less elaborated arabinan, may contribute to selective recognition by T cells. LAM from all species could be resolved into discrete forms by isoelectric focusing based apparently on their arabinan heterogeneity. In the light of our current and more recent findings, we reason that all immunobiological data should be cautiously interpreted and that the actual LAM variants that may be present in vivo during infection and pathogenesis need to be taken into consideration., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.

Taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol 6 has been identified as an early stage intermediate involved in the biosynthesis of taxol (Paclitaxel). This compound has been efficiently prepared by Barton deoxygenation of the C-2- and C-14-hydroxyl groups on a derivative semisynthetically prepared from taxa-4(20),11(12)-dien-2 alpha,5 alpha,10 beta-triacetoxy-14 beta-(2-methyl)butyrate (7), a major taxoid metabolite isolated from Japanese yew heart wood. The synthetic methodology is amenable for the preparation of isotopically labeled congeners that will be useful to probe further intermediate steps in the biosynthesis of taxol.

The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB(-) mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA(-) and embB(-) mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA(-) and embB(-) mutants. Detailed nuclear magnetic resonance studies coupled with enzyme digestion, chromatography, and mass spectrometry analyses revealed that the disaccharide beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f) extension from the 3-position of the 3,5-linked alpha-d-Ara(f) residue is markedly diminished. As a consequence, a linear terminal beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f) is formed, a motif which is a recognized, nonreducing terminal feature of lipoarabinomannan but not of normal AG. Upon complementation with the embB and embA wild-type genes, the phenotype of the mutants reverted to wild-type, in that normal AG was resynthesized. Our results clearly show that both EmbA and EmbB proteins are involved in the formation of the proper terminal hexaarabinofuranoside motif in AG, thus paving the way for future studies to identify the complete array of arabinosyltransferases involved in the synthesis of mycobacterial cell wall arabinan.

Exposure of DNA to oxidative stress produces a variety of DNA lesions including the formamidopyrimidines, which are derived from the purines. These lesions may play important roles in carcinogenesis. We achieved the first chemical syntheses of a monomeric form of Fapy-dA (1) and oligonucleotides containing this lesion or Fapy-dG at a defined site. Monomeric Fapy-dA readily epimerized at 25 degrees C in phosphate buffer (pH 7.5). The beta-anomer was favored by a ratio of 1.33:1.0, and equilibration was achieved in less than 7 h. Deglycosylation of Fapy-dA in the monomer follows first-order kinetics from 37 to 90 degrees C. The rate constants for deglycosylation of Fapy-dA in the monomeric and oligonucleotide substrates were measured at a common temperature (55 degrees C) and found to be the same within experimental error (t(1/2) = 20.5 h). Implementation of the activation parameters measured for the deglycosylation of 1 indicates that the half-life for deglycosylation of Fapy-dA at 37 degrees C is approximately 103 h. Analysis of the rate constant for deglycosylation of Fapy-dG in an oligonucleotide, revealed that this lesion is approximately 25 times more resistant to hydrolysis than Fapy-dA at 55 degrees C. These results indicate that Fapy-dA and Fapy-dG will be sufficiently long-lived in DNA so as to warrant investigation of their genotoxicity, and both anomers will be present during this time.

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.

The biosynthesis of the diterpenoid antineoplastic drug Taxol in Taxus species involves the cyclization of the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation (with allylic rearrangement) of this olefin precursor to taxa-4(20),11(12)-dien-5 alpha-ol, and further oxygenation and acylation reactions. Based on the abundances of naturally occurring taxoids, the subsequent order of oxygenation of the taxane core is considered to occur at C10, then C2 and C9, followed by C13, and finally C7 and C1. Circumstantial evidence suggests that the acetylation of taxadien-5 alpha-ol may constitute the third specific step of Taxol biosynthesis. To determine whether taxadienol or the corresponding acetate ester serves as the direct precursor of subsequent oxygenation reactions, microsomal preparations isolated from induced Taxus cells and optimized for cytochrome P450 catalysis were incubated with each potential substrate. Both taxadienol and taxadienyl acetate were oxygenated to the level of a diol and to higher polyols at comparable rates by cytochrome P450 enzymes of the microsomal preparation. Preparative-scale incubation allowed the isolation of sufficient quantities of the diol derived from taxadienol to permit the NMR-based structural elucidation of this metabolite as taxa-4(20),11(12)-dien-5 alpha,13 alpha-diol, which may represent an alternate route of taxoid metabolism in induced cells. GC-MS-based structural definition of the diol monoacetate derived in microsomes from taxadienyl acetate confirmed this metabolite as taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol, thereby indicating that acetylation at C5 of taxadienol precedes the cytochrome P450-mediated insertion of the C10-beta-hydroxyl group of Taxol., (Copyright 2001 Academic Press.)

The early steps in the biosynthesis of Taxol involve the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation at C5, acetylation of this intermediate, and a second cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5 alpha-acetoxy-10 beta-ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations. The limited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse transcription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of these enzymes in yeast was monitored by in situ spectrophotometry coupled to in vivo screening of oxygenase activity by feeding taxoid substrates. This strategy yielded a family of taxoid-metabolizing enzymes and revealed the taxane 10 beta-hydroxylase as a 1494-bp cDNA that encodes a 498-residue cytochrome P450 capable of transforming taxadienyl acetate to the 10 beta-hydroxy derivative; the identity of this latter pathway intermediate was confirmed by chromatographic and spectrometric means. The 10 beta-hydroxylase represents the initial cytochrome P450 gene of Taxol biosynthesis to be isolated by an approach that should provide access to the remaining oxygenases of the pathway.

Background: The committed step in the biosynthesis of the anticancer drug taxol in yew (Taxus) species is the cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene. The enzyme taxadiene synthase catalyzes this complex olefin cation cyclization cascade involving the formation of three rings and three stereogenic centers., Results: Recombinant taxadiene synthase was incubated with specifically deuterated substrates, and the mechanism of cyclization was probed using MS and NMR analyses of the products to define the crucial hydrogen migration and terminating deprotonation steps. The electrophilic cyclization involves the ionization of the diphosphate with closure of the A-ring, followed by a unique intramolecular transfer of the C11 proton to the re-face of C7 to promote closure of the B/C-ring juncture, and cascade termination by proton elimination from the beta-face of C5., Conclusions: These findings provide insight into the molecular architecture of the first dedicated step of taxol biosynthesis that creates the taxane carbon skeleton, and they have broad implications for the general mechanistic capability of the large family of terpenoid cyclization enzymes.

A new alkaloid, isothalisopavine, was isolated from Thalictrum minus and shown to be 2,3,8-trimethoxy-7-hydroxyisopavine from spectral evidence. The known thalisopavine, from T. dasycarpum, is 2,3,8-trimethoxy-9-hydroxyisopavine. Isothalisopavine is the first of its class to be substituted unsymmetrically in the two aromatic rings.