Synthesis of stable isotope-labeled chloroquine and amodiaquine and their metabolites.

Anti-malaria drugs chloroquine and amodiaquine and their metabolites were synthesized to incorporate 13 C and 15 N starting from U-13 C-labeled benzene to give M + 7 isotopomers. Chloroquine and its metabolites were prepared from 7-chloro-1,2,3,4-tetrahydroquinolin-4-one through an aryl substitution with the corresponding amines; and the amodiaquine and its metabolites were prepared from 4,7-dichloroquinoline in a similar fashion.

Gram-scale synthesis and efficient purification of 13C-labeled levoglucosan from 13C glucose.

(13)C-Labeled levoglucosan has been synthesized and purified in good yield, and on the gram scale in one step from commercially available (13)C glucose. This one-step protocol uses 2-chloro-1,3-dimethylimidazolinium chloride that serves to selectively activate the anomeric carbon toward substitution reactions. The labeled glucose is then smoothly converted to the anhydroglucose. Purification is efficiently achieved on large scale by chromatography on silica gel.

A general route for 13C-labeled fluorenols and phenanthrenols via palladium-catalyzed cross-coupling and one-carbon homologation.

A series of (13)C-labeled polyaromatic hydrocarbons (PAHs), fluorenols and phenanthrenols were synthesized from commercially available (13)C-labeled starting material giving rise to M + 6 isotopomers. This was accomplished using key palladium-catalyzed cross-coupling and one-carbon homologation strategies. The conditions for these reactions were optimized, and the new chemical routes are efficient in the number of chemical steps, can be scaled to afford gram quantities and occur in good yields based on the (13)C label. These labeled compounds as precursors for more complex PAHs and are useful as internal standards in mass spectrometry and NMR spectroscopy studies for monitoring environmental contamination and biological exposure to PAHs and their metabolites.

Aerobic oxidation reactions catalyzed by vanadium complexes of bis(phenolate) ligands.

Vanadium(V) complexes of the tridentate bis(phenolate)pyridine ligand H(2)BPP (H(2)BPP = 2,6-(HOC(6)H(2)-2,4-(t)Bu(2))(2)NC(5)H(3)) and the bis(phenolate)amine ligand H(2)BPA (H(2)BPA = N,N-bis(2-hydroxy-4,5-dimethylbenzyl)propylamine) have been synthesized and characterized. The ability of the complexes to mediate the oxidative C-C bond cleavage of pinacol was tested. Reaction of the complex (BPP)V(V)(O)(O(i)Pr) (4) with pinacol afforded the monomeric vanadium(IV) product (BPP)V(IV)(O)(HO(i)Pr) (6) and acetone. Vanadium(IV) complex 6 was oxidized rapidly by air at room temperature in the presence of NEt(3), yielding the vanadium(V) cis-dioxo complex [(BPP)V(V)(O)(2)]HNEt(3). Complex (BPA)V(V)(O)(O(i)Pr) (5) reacted with pinacol at room temperature, to afford acetone and the vanadium(IV) dimer [(BPA)V(IV)(O)(HO(i)Pr)](2). Complexes 4 and 5 were evaluated as catalysts for the aerobic oxidation of 4-methoxybenzyl alcohol and arylglycerol ß-aryl ether lignin model compounds. Although both 4 and 5 catalyzed the aerobic oxidation of 4-methoxybenzyl alcohol, complex 4 was found to be a more active and robust catalyst for oxidation of the lignin model compounds. The catalytic activities and selectivities of the bis(phenolate) complexes are compared to previously reported catalysts.

Mild and selective vanadium-catalyzed oxidation of benzylic, allylic, and propargylic alcohols using air.

Transition metal-catalyzed aerobic alcohol oxidation is an attractive method for the synthesis of carbonyl compounds, but most catalytic systems feature precious metals and require pure oxygen. The vanadium complex (HQ)(2)V(V)(O)(O(i)Pr) (2 mol %, HQ = 8-quinolinate) and NEt(3) (10 mol %) catalyze the oxidation of benzylic, allylic, and propargylic alcohols with air. The catalyst can be easily prepared under air using commercially available reagents and is effective for a wide range of primary and secondary alcohols.

Mechanism of alcohol oxidation by dipicolinate vanadium(V): unexpected role of pyridine.

Dipicolinate vanadium(V) alkoxide complexes (dipic)V(V)(O)(OR) (OR = isopropoxide (1), n-butanoxide (2), cyclobutanoxide (3), and α-tert-butylbenzylalkoxide (4)) react with pyridine to afford vanadium(IV) and 0.5 equiv of an aldehyde or ketone product. The role of pyridine in the reaction has been investigated. Both NMR and X-ray crystallography experiments indicate that pyridine coordinates to 1, which is in equilibrium with (dipic)V(V)(O)(O(i)Pr)(pyr) (1-Pyr). Kinetic studies of the alcohol oxidation suggest a pathway where the rate-limiting step is bimolecular and involves attack of pyridine on the C-H bond of the isopropoxide ligand of 1 or 1-Pyr. The oxidations of mechanistic probes cyclobutanol and α-tert-butylbenzylalcohol support a two-electron pathway proceeding through a vanadium(III) intermediate. The alcohol oxidation reaction is promoted by more basic pyridines and facilitated by electron-withdrawing substituents on the dipicolinate ligand. The involvement of base in the elementary alcohol oxidation step observed for the dipicolinate system is an unprecedented mechanism for vanadium-mediated alcohol oxidation and suggests new ways to tune reactivity and selectivity of vanadium catalysts.

Synthesis of chiral 13C,77Se-labeled selones.

Stable isotope-labeled N-acyl selones have been constructed in fewer than four steps from readily available starting materials. Site-specific labeling was achieved using the following synthons: bromo[2-(13)C]acetic acid, [(13)C]formic acid, and elemental (77)Se. These labeled selones have been found to provide unique insights into enolate structure and may be useful in the detection and quantitation of remotely disposed chiral centers in compounds in short supply.

A rapid, sensitive method for the quantitation of specific metabolites of sulfur mustard in human urine using isotope-dilution gas chromatography-tandem mass spectrometry.

Sulfur mustard agent (HD) (2,2'-dichloroethyl sulfide), a Schedule I compound on the Chemical Weapons Convention Schedule of Chemicals, remains a public health concern because it is simple to synthesize and it is in the chemical weapon stockpiles of several countries. A sensitive, rapid, accurate, and precise method was developed to quantitate trace levels of 1,1'-sulfonylbis [2-(methylthio) ethane] (SBMTE) in human urine as a means of assessing exposure to HD. The method used immobilized liquid-liquid extraction with diatomaceous earth, followed by the analysis of the urine extract using isotope-dilution gas chromatography-tandem mass spectrometry. Relative standard deviations were less than 8.6% at 1 ng/mL and 3.6% at 20 ng/mL. The limit of detection for SBMTE was 0.038 ng/mL in 0.5 mL of urine.

Quantitation of the sulfur mustard metabolites 1,1'-sulfonylbis[2-(methylthio)ethane] and thiodiglycol in urine using isotope-dilution Gas chromatography-tandem mass spectrometry.

Sulfur mustard (HD), or bis(2-chloroethyl)sulfide, has several urinary metabolites that can be measured to assess human exposure. These metabolites include the simple hydrolysis product thiodiglycol (TDG) and its oxidative analogue, TDG-sulfoxide, as well as metabolites of the glutathione/b-lyase pathway 1,1'-sulfonylbis[2-(methyl-sulfinyl)ethane] (SBMSE) and 1-methyl-sulfinyl-2-[(methylthio)ethyl-sulfonyl]ethane (MSMTESE). Current methods focus on either the TDG or the b-lyase metabolites. We have developed a single method that measures products of both metabolic branches, with the reduced compound of SBMSE and MSMTESE, 1,1'-sulfonylbis [2(methylthio)ethane] (SBMTE), as the definitive analyte and TDG as a confirmation analyte. Sample preparation included b-glucuronidase hydrolysis for TDG-glucuronide conjugates, titanium trichloride reduction of sulfoxides to SBMTE and TDG, solid-phase extraction, and a chemical derivatization. We analyzed samples using gas chromatography-tandem mass spectrometry with quantitation using isotope-dilution calibration. The method limits of detection for TDG and SBMTE were 0.5 ng/mL and 0.25 ng/mL, respectively, with relative standard deviations of less than 10%. Urine samples from individuals with no known exposure to mustard agent HD had measurable concentrations of TDG, but no SBMTE was detected. The geometric mean concentration of TDG was 3.43 ng/mL, with concentrations ranging from < 0.5 ng/mL to 20 ng/mL.

A comprehensive approach for accurate measurement of proton-proton coupling constants in the sugar ring of DNA.

Stereo-selectivedeuteration has been explored as an approach for improving the accuracy of NMR-derived, three-bond vicinal proton-proton coupling constants in the 12-base-pair DNA Dickerson sequence [d(CGCGAATTCGCG)(2)]. The coupling constants are useful for DNA structure determination in restrained molecular dynamics calculations. Specifically, the A5 and A6 residues were prepared with the H2" proton stereo-selectively replaced with a deuteron. Deuteration of the H2" leads to a 42-fold reduction in the transverse cross-relaxation rate of the H2' spin, effectively negating the contribution of transverse cross relaxation to the cross peak frequencies and phases. Calculated linewidth and polarization transfer functions indicated that the reduced dipolar interaction is also expected to result in a significant increase in intensity for all cross peaks involving the H1', H2', or H3' spin. The spectral complexity is also reduced by selective deuteration. Time-shared homonuclear decoupling of passive spins during acquisition was implemented, reducing the spin system, in some cases, to an effectively isolated two-spin system. This enables the use of a 90 degrees mixing pulse instead of the 35 degrees pulse commonly used in standard P.E.COSY experiments, leading to an additional 75% increase in signal intensity. Selective excitation pulses were used to reduce the number of increments required in the indirect dimension by as much as a factor of 4. The cumulative improvement in sensitivity is striking, approaching three orders of magnitude per unit time. Separate experiments, referred to as Stripe-COSY and Superstripe-COSY, were optimized for each coupling constant measured. Finally, J-doubling was used to obtain the most accurate peak separations. This comprehensive approach shows promise as an effective method for extracting highly accurate homonuclear vicinal coupling constants in DNA.

The high-resolution structure of the triplex formed by the GAA/TTC triplet repeat associated with Friedreich's ataxia.

Expansions of the triplet repeat, GAA/TTC, inside the first intron of the frataxin gene causes Friedreich's ataxia (FRDA). It was of interest to us to examine whether the FRDA repeat forms an unusual DNA structure, since formation of such structure during replication may cause its expansion. Here, we show that the FRDA repeat forms a triplex in which the TTC strand folds on either side of the same GAA strand. We have determined the high-resolution NMR structures of two intramolecularly folded FRDA triplexes, (GAA)2T4(TTC)2T4(CTT)2 and (GAA)2T4(TTC)2T2CT2(CTT)2 with T.A.T and C+.G.C triads. T4 represents a synthetic loop sequence, whereas T2CT2 is the natural loop-folding sequence of the TTC strand. We have also made use of site-specific 15N-labeling of the cytosine residues to investigate their protonation status and their interaction with other protons. We show that the cytosine residues of the Hoogsteen C+.G pairs in this triplex are protonated close to physiological pH. Therefore, it appears that the triplex formation offers a plausible explanation for the expansion of the GAA/TTC repeats in FRDA.

Fragile X DNA triplet repeats, (GCC)n, form hairpins with single hydrogen-bonded cytosine.cytosine mispairs at the CpG sites: isotope-edited nuclear magnetic resonance spectroscopy on (GCC)n with selective 15N4-labeled cytosine bases.

Here, we provide a direct proof that the formation of hairpins by (GCC)n at the 5'-UTR of the FMR-1 gene offers a mechanism for CpG hypermethylation associated with the fragile X syndrome. For this, we have performed hetero-nuclear (15N-1H) magnetic resonance spectroscopy to probe the structure of the CpG sites in the (GCC)n hairpins that are 15N-labeled at the amino (N4) groups of specific cytosine bases. Analyses of chemical shift, pH-induced chemical exchange, and NOE pattern of the (15N-labeled) amino protons of cytosine bases reveal that the cytosine bases at the CpG sites are intrahelical and well-stacked with the neighboring G.C base-pairs in the stem of these hairpins and probably form single hydrogen-bonded C.C mispairs. Measurements of pH-dependent 1H line-width also demonstrate that the C.C mispairs are more susceptible to open-closure than the G.C base-pairs. Thus, the Cs at the CpG sites of the (GCC)n hairpin are "flipped out" more easily to the activated state than those in the corresponding Watson-Crick duplex, (GCC)n. (GGC)n and this makes the hairpin a better target for methylation by the human methyltransferase, the enzyme that methylates the Cs at the CpG sites.

Probing site-specific interactions in protein-DNA complexes using heteronuclear NMR spectroscopy and molecular modeling: binding of Cro repressor to OR3.

In this paper, a general method is developed to study site-specific interactions in DNA-protein complexes using heteronuclear NMR spectroscopy and molecular modeling. This method involves two steps: (a) homonuclear 1H NMR and molecular modeling are used to develop a low resolution model and (b) 15N7-guanosine containing oligonucleotides are employed to probe the specific intermolecular interactions predicted in (a). This method is applied to Cro-operator complex due to its small size and extensive prior characterization. Non-exchangeable and exchangeable base protons have been assigned by nuclear Overhauser effect spectroscopy (NOESY) and chemical shift correlation spectroscopy. Extensive line-broadening has been observed in the 1H NMR spectra of the operator DNA in the presence of protein. Differential line-broadening observed in the imino proton region and the comparison of NOESY spectra in the presence and absence of Cro protein show that guanosine-12 and guanosine-14 are involved in the Cro-DNA interaction, while the three A.T base-pairs at the 3'- and 5'-termini play only a minor role in the binding. A model of the Cro-operator DNA complex has been constructed by docking helix-3 of the Cro protein in the major groove and it predicted specific hydrogen bonds between N7 of guanosines-12 and -14 and the side-chain of Lys-32 and Ser-28, respectively. The appearance of a new resonance in the temperature dependent proton detected heteronuclear multiple quantum coherence (HMQC) spectra of the Cro-DNA complex also demonstrates a specific interaction of Cro with guanosine-14 of the operator DNA.

Solution structures of the Huntington's disease DNA triplets, (CAG)n.

Highly polymorphic DNA triplet repeats, (CAG)n, are located inside the first exon of the Huntington's disease gene. Inordinate expansion of this repeat is correlated with the onset and progression of the disease. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific P1 enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)n expansion. Nondenaturing gel electrophoresis and 1D 1H NMR studies of (CAG)5 and (CAG)6 reveal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations (i.e., 1.0-2.0 mM that is typically required for 2D NMR experiments) both (CAG)5 and (CAG)6 exist predominantly in mismatched duplex forms. Mismatched duplex structures of (CAG)5 and (CAG)6 are useful, because they adequately model the stem of the biologically relevant hairpins formed by (CAG)n. We, therefore, performed detailed NMR spectroscopic studies on the duplexes of (CAG)5 and (CAG)6. We also studied a model duplex, (CGCAGCG)2 that contains the underlined building block of the duplex. This duplex shows the following structural characteristics: (i) all the nucleotides are in (C2'-endo, anti) conformations, (ii) mismatched A x A base pairs are flanked by two Watson-Crick G x C base pairs and (iii) A x A base pairs are stably stacked (and intra-helical) and are formed by a single N6-H--N1 hydrogen bond. The nature of A x A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CA*G)5]2 duplex where the adenines are 15N-labeled at N6. Temperature- and pH-dependent imino proton spectra, nondenaturing electrophoresis, and P1 digestion data demonstrate that under a wide range of solution conditions longer (CAG)n repeats (n> or =10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)8,21 inserts in the M13 single-stranded DNA templates shows a replication bypass for the (CAG)21 insert but not for the (CAG)8 insert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)n even in presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)n may cause slippage during replication and thus may explain the observed length polymorphism.

Selected pharmacodynamic and biochemical properties of selenonium choline.

1. Five minutes after intravenous administration of 50 mg/kg of the novel choline analogue selenonium choline [(CH3)2Se + CH2CH2OH, SeCh], only 8% of the administered dose was accounted for in blood, brain, liver, heart, and kidney tissues. 2. SeCh was acetylated in vivo to acetylselenonium choline (ASeCh) in all of the tissues examined. 3. During postmortem incubation, brain concentrations of SeCh and ASeCh increased to 535% and to 425%, respectively. 4. K(m) and Vmax values for the phosphorylation of SeCh by choline kinase were higher and lower, respectively, compared to the phosphorylation of choline. 5. Acetylation of SeCh was described with K(m) and Vmax values that were both higher than the values for Ch. 6. The data suggest that SeCh is phosphorylated and incorporated into various lipids in brain tissue, and is acetylated to ASeCh by both nonneural and neural tissues.

(R,R)-N,N'-dimethylcyclohexyl-1,2-diazaseleno-phospholidine as a chiral derivatizing agent for the evaluation of chiral alcohols.

Previously, a diazaphospholidine has been synthesized and evaluated as a chiral derivatizing reagent for the determination of the optical purity of chiral alcohols via 31P NMR spectroscopy (Alexakis et al., J. Org. Chem. 57:1224-1237, 1992). Our laboratory is interested in the advantageous and practical applications of 77Se NMR spectroscopic studies in many facets of chemistry and biochemistry. To this end we have used this diazaphospholidine as a starting point and have investigated chiral alcohols coupled to an optically pure diazaselenophospholidine. The diastereomers formed were then evaluated by 77Se NMR spectroscopy, and these results were compared to the 31P NMR results published by Alexakis and co-workers. It was found that addition of the Se atom produced diastereomers that were air stable and, in many cases, the individual diastereomers could be distinguished by 77Se NMR spectroscopy. Preliminary results indicate that the 77Se nucleus is somewhat more sensitive to remotely disposed chiral centers than is the 31P nucleus. Furthermore, because of their stability, these compounds do not readily decompose and can, therefore, be studied by a variety of chromatographic and spectroscopic techniques.

Synthesis of novel tellurium containing analogues of choline and acetylcholine and their quantitation by pyrolysis-gas chromatography-mass spectrometry.

Methods for the synthesis and quantitation of the novel choline analogues, telluronium choline and acetyltelluronium choline, are described. An assay procedure utilizing pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS) with cold trapping was developed with [2H4]telluronium choline and [2H4]acetyltelluronium choline as internal standards. The telluronium compounds were ion-pair extracted from tissue with dipicrylamine, washed with 2-butanone, and pyrolyzed prior to GC-MS analysis. The compounds were monitored using selected ion monitoring at m/z 232 and m/z 190 for acetyltelluronium and telluronium choline, respectively, and at m/z 236 and m/z 194 for the analogous deuterated internal standards. The assay was linear over a range of 20 pmol-20 nmol of compound taken through the assay.

Further biological activities of the novel choline analogs selenonium choline and acetylselenonium choline.

The pharmacological actions of the novel choline analog, selenonium choline [(CH3)2Se+CH2CH2OH] and its acetyl ester acetylselenonium choline (ASeCh) were studied in vivo and in vitro. ASeCh produced a dose-related decrease in mean arterial pressure in the rat similar to acetylcholine (ACh) but was 1% to 2% as potent. ASeCh demonstrated agonist activity on the rat isolated ileum and was approximately 2% as active as ACh. Selenonium chlorine (SeCh) was taken up and acetylated in brain tissue slices in a time- and concentration-dependent manner. The use of KCl as a loading stimulus did not increase the uptake of SeCh but increased tissue levels of ASeCh 1.5-fold over the control concentrations. The uptake of SeCh was described by a single low-affinity uptake component (Km = 167 microM) that was not blocked by hemicholinium-3. In contrast, hemicholinium significantly blocked the acetylation of SeCh. Compared with basal release, depolarization with KCl caused a significant release of ASeCh into the incubation medium. A neural specificity was suggested for the in vitro uptake of SeCh. Acetylation of SeCh in vivo in the rat after intraventricular administration was similar to the extent of acetylation of [2H4]-choline. ASeCh bound to both M1 and M2 cholinergic receptors with 2% to 3% of the affinity observed for ACh. These data suggest that SeCh may satisfy criteria for a false neurotransmitter precursor.