Microbiota of healthy corals are active against fungi in a light-dependent manner.

Coral reefs are intricate ecosystems that harbor diverse organisms, including 25% of all marine fish. Healthy corals exhibit a complex symbiosis between coral polyps, endosymbiotic alga, and an array of microorganisms, called the coral holobiont. Secretion of specialized metabolites by coral microbiota is thought to contribute to the defense of this sessile organism against harmful biotic and abiotic factors. While few causative agents of coral diseases have been unequivocally identified, fungi have been implicated in the massive destruction of some soft corals worldwide. Because corals are nocturnal feeders, they may be more vulnerable to fungal infection at night, and we hypothesized that the coral microbiota would have the capability to enhance their defenses against fungi in the dark. A Pseudoalteromonas sp. isolated from a healthy octocoral displayed light-dependent antifungal properties when grown adjacent to Penicillium citrinum (P. citrinum) isolated from a diseased Gorgonian octocoral. Microbial MALDI-imaging mass spectrometry (IMS) coupled with molecular network analyses revealed that Pseudoalteromonas produced higher levels of antifungal polyketide alteramides in the dark than in the light. The alteramides were inactivated by light through a photoinduced intramolecular cyclization. Further NMR studies led to a revision of the stereochemical structure of the alteramides. Alteramide A exhibited antifungal properties and elicited changes in fungal metabolite distributions of mycotoxin citrinin and citrinadins. These data support the hypothesis that coral microbiota use abiotic factors such as light to regulate the production of metabolites with specialized functions to combat opportunistic pathogens at night.

Two antimycin A analogues from marine-derived actinomycete Streptomyces lusitanus.

Two new antimycin A analogues, antimycin B1 and B2 (1-2), were isolated from a spent broth of a marine-derived bacterium, Streptomyces lusitanus. The structures of 1 and 2 were established on the basis of spectroscopic analyses and chemical methods. The isolated compounds were tested for their anti-bacterial potency. Compound 1 was found to be inactive against the bacteria Bacillus subtilis, Staphyloccocus aureus, and Loktanella hongkongensis. Compound 2 showed antibacterial activities against S. aureus and L. hongkongensis with MIC values of 32.0 and 8.0 µg/mL, respectively.

Metabolism of intravenous methylnaltrexone in mice, rats, dogs, and humans.

Methylnaltrexone (MNTX), a selective mu-opioid receptor antagonist, functions as a peripherally acting receptor antagonist in tissues of the gastrointestinal tract. This report describes the metabolic fate of [(3)H]MNTX or [(14)C]MNTX bromide in mice, rats, dogs, and humans after intravenous administration. Separation and identification of plasma and urinary MNTX metabolites was achieved by high-performance liquid chromatography-radioactivity detection and liquid chromatography/mass spectrometry. The structures of the most abundant human metabolites were confirmed by chemical synthesis and NMR spectroscopic analysis. Analysis of radioactivity in plasma and urine showed that MNTX underwent two major pathways of metabolism in humans: sulfation of the phenolic group to MNTX-3-sulfate (M2) and reduction of the carbonyl group to two epimeric alcohols, methyl-6alpha-naltrexol (M4) and methyl-6beta-naltrexol (M5). Neither naltrexone nor its metabolite 6beta-naltrexol were detected in human plasma after administration of MNTX, confirming an earlier observation that N-demethylation was not a metabolic pathway of MNTX in humans. The urinary metabolite profiles in humans were consistent with plasma profiles. In mice, the circulating and urinary metabolites included M5, MNTX-3-glucuronide (M9), 2-hydroxy-3-O-methyl MNTX (M6), and its glucuronide (M10). M2, M5, M6, and M9 were observed in rats. Dogs produced only one metabolite, M9. In conclusion, MNTX was not extensively metabolized in humans. Conversion to methyl-6-naltrexol isomers (M4 and M5) and M2 were the primary pathways of metabolism in humans. MNTX was metabolized to a higher extent in mice than in rats, dogs, and humans. Glucuronidation was a major metabolic pathway in mice, rats, and dogs, but not in humans. Overall, the data suggested species differences in the metabolism of MNTX.

Discovery of a new series of monoamine reuptake inhibitors, the 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ols.

A novel series of monoamine reuptake inhibitors, the 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ols, have been discovered by combining virtual and focused screening efforts with design techniques. Synthesis of the two diastereomeric isomers of the molecule followed by chiral resolution of each enantiomer revealed the (2R,3S)-isomer to be a potent norepinephrine reuptake inhibitor (IC(50)=28 nM) with excellent selectivity over the dopamine transporter and 13-fold selectivity over the serotonin transporter.

Suitability of tetrahydofuran as a dopant and the comparison to other existing dopants in dopant-assisted atmospheric pressure photoionization mass spectrometry in support of drug discovery.

In this paper, we investigated the suitability of tetrahydofuran (THF) as a dopant and compared it against other common dopants for atmospheric pressure photoionization mass spectrometry (APPI-MS). In a systematic analysis of 37 drug standards and 100 Wyeth proprietary drug candidates, THF was found to increase ionization efficiency as high as 33-fold when introduced through a syringe pump at a flow rate of 20 microL/min, and as high as 114-fold when introduced through the mobile phase at 100 microL/min. As a dopant, THF is as effective as acetone, better than anisole, and slightly less effective than toluene for the majority of the test compounds. The increase in ionization efficiency by THF was found to be compound-dependent. THF was more effective in facilitating the ionization of polar compounds than of non-polar compounds. With THF, toluene and acetone as dopants, a single type of molecular ion ([M+H](+) or M(+*)) is produced for analyte molecules. However, anisole can cause the formation of an ion cluster for polar analytes. The cluster contains [M-2H+H](+), M(+*), and [M+H](+) ions with varied ratios. This complexity may make interpretation of spectra difficult for unknown compounds when complimentary data are not available. Our findings indicate that THF is a suitable dopant in the daily usage for increasing ionization efficiency, especially when THF is used as the mobile phase or as an organic modifier in the mobile phase.

Normal-phase automated mass-directed HPLC purification of a pyrrolobenzodiazepine library with vasopressin agonist activity.

A 23-member library of pyrrolobenzodiazepine derivatives with vasopressin agonist activity was purified on a 100-mg per injection scale using normal-phase (NP) automated mass-directed HPLC. Analytical NP APCI-LC/MS on an experimental monolith silica CN column utilizing gradients of methanol in ethoxynonafluorobutane (hexane-like solvent) was used to provide data on chromatographic purity and ionization of the solutes. The analytical data collected were used to program a preparative LC/MS instrument for "smart" fraction collection based on the protonated molecular ion of the component of interest. Preparative HPLC was carried out on a preparative cyano column with gradients of polar organic solvents in heptane containing n-propylamine as a basic additive. Flow rates twice as high as conventional ones were used for purification of library compounds. Small aliquots of the preparative flow were mixed with makeup solvent and introduced into an APCI source of a quadrupole mass spectrometer, which triggered collection of solutes. Two methods with fixed instrument parameters were used for purification. The system utilized commercially available instrumentation and software, which provided excellent recovery and purity of the library components and appeared to be useful as a fast and efficient alternative to traditional purification technologies based on reversed-phase LC/MS.

Unexpected rearrangement of enantiomerically pure 3-aminoquinuclidine as a simple way of preparing diastereomeric octahydropyrrolo[2,3-c]pyridine derivatives.

Reaction of (S)- or (R)-3-aminoquinuclidine with 2-chloropyrimidine or 2-bromopyrimidine led to an unexpected formation of both cis- and trans-octahydropyrrolo [2,3]pyridine derivatives. A single-step synthesis of two of the four stereoisomers of these octahydropyrrolo[2,3]pyridine derivatives provides a convenient way of generating stereochemically defined isomers. Optimization of reaction conditions was carried out by (1)H NMR monitoring. The relative and absolute stereochemistry of all four stereoisomers was determined by a combination of (1)H, (13)C, and (15)N NMR spectroscopy and vibrational circular dichroism spectroscopy.

In vitro glucuronidation of thyroxine and triiodothyronine by liver microsomes and recombinant human UDP-glucuronosyltransferases.

Glucuronidation, which may take place on the phenolic hydroxyl and carboxyl groups, is a major pathway of metabolism for thyroxine (T4) and triiodothyronine (T3). In this study, a liquid chromatography/mass spectrometry (LC/MS) method was developed to separate phenolic and acyl glucuronides of T4 and T3. The method was used to collect the phenolic glucuronide of T4 for definitive characterization by NMR and to determine effects of incubation pH, species differences, and human UDP-glucuronosyltransferases (UGTs) involved in the formation of the glucuronides. Formation of T4 phenolic glucuronide was favored at pH 7.4, whereas formation of T4 acyl glucuronide was favored at pH 6.8. All the UGTs examined catalyzed the formation of T4 phenolic glucuronide except UGT1A4; the highest activity was detected with UGT1A3, UGT1A8, and UGT1A10, followed by UGT1A1 and UGT2B4. Formation of T3 phenolic glucuronide was observed in the order of UGT1A8 > UGT1A10 > UGT1A3 > UGT1A1; trace activity was observed with UGT1A6 and UGT1A9. UGT1A3 was the major isoform catalyzing the formation of T4 and T3 acyl glucuronides. In liver microsomes, phenolic glucuronidation was the highest in mice for T4 and in rats for T3 and lowest in monkeys for both T4 and T3. Acyl glucuronidation was highest in humans and lowest in mice for T4 and T3. Phenolic glucuronidation was higher than acyl glucuronidation for T4 in humans; in contrast, the acyl glucuronidation was slightly higher than phenolic glucuronidation for T3. UGT activities were lower toward T3 than T4 in all the species. The LC/MS method was a useful tool in studying glucuronidation of T4 and T3.

Rapid screening of enzymes for the enzymatic hydrolysis of chiral esters in drug discovery.

Thirty-five enzymes were rapidly screened for their ability to selectively hydrolyze chiral esters to their corresponding carboxylic acids for the efficient generation of chiral intermediates in drug discovery. Optimization of the enzymatic reactions at various incubation times was performed using a robotic liquid handler. Enantiomeric pairs of chiral esters and carboxylic acids were then analyzed simultaneously by chiral GC/MS in a single analysis. This analytical approach is particularly useful for compounds that do not possess a conjugated chromophore or are volatile and difficult to analyze by chiral HPLC/UV or HPLC/MS. The resulting data was used to determine enantiomeric excesses and percent conversions to the desired enantiomer of the carboxylic acid for the selection of efficient enzymes for bioconversions in drug discovery in a pharmaceutical company.

Enantiomeric separation and determination of absolute stereochemistry of asymmetric molecules in drug discovery: building chiral technology toolboxes.

The application of Chiral Technology, or the (extensive) use of techniques or tools for the determination of absolute stereochemistry and the enantiomeric or chiral separation of racemic small molecule potential lead compounds, has been critical to successfully discovering and developing chiral drugs in the pharmaceutical industry. This has been due to the rapid increase over the past 10-15 years in potential drug candidates containing one or more asymmetric centers. Based on the experiences of one pharmaceutical company, a summary of the establishment of a Chiral Technology toolbox, including the implementation of known tools as well as the design, development, and implementation of new Chiral Technology tools, is provided.

Application of chiral technology in a pharmaceutical company. Enantiomeric separation and spectroscopic studies of key asymmetric intermediates using a combination of techniques. Phenylglycidols.

Phenylglycidols substituted in the 2-, 3-, and 4- positions with fluorine, chlorine, and trifluoromethyl, and with methoxy in the 3- position, were synthesized from the corresponding E-cinnamic acids and separated into their (R,R)- and (S,S)- enantiomers using subcritical fluid chromatography with mixtures of MeOH in CO(2), on either a Chiralpak AD or AS chiral stationary phase. These compounds and commercially-available (R,R)- and (S,S)-phenylglycidol were analyzed for their vibrational circular dichroism (VCD), electronic circular dichroism (ECD), and optical rotation (OR) properties to exemplify a strategy whereby the absolute stereochemistry of common and key chiral intermediates is established early in the structure-activity and structure-property relationship phase of a drug discovery program in a pharmaceutical company. From this study, substituents in the phenyl group of the synthesized molecules were found not to grossly alter spectroscopic features, and therefore, diagnostic absorption bands in the respective VCD spectra, and the sign and shape of the measured ECD curves could be used to determine and track the absolute stereochemistry of analogs without necessarily requiring time-consuming ab initio calculations of all low energy conformers for all compounds. VCD, OR, and ECD calculations for the determination of absolute configuration carried out at the DFT level with the hybrid B3PW91 functional and the TZVP basis set were found to be especially useful in this study.

NMR characterization of an S-linked glucuronide metabolite of the potent, novel, nonsteroidal progesterone agonist tanaproget.

Tanaproget is a first-in-class nonsteroidal progesterone receptor agonist that is being investigated for use in contraception. A major in vitro and in vivo metabolite of tanaproget formed in humans was initially characterized as a glucuronide of tanaproget. However, whether the glucuronide was linked to the nitrogen or sulfur of the benzoxazine-2-thione group in tanaproget could not be determined by liquid chromatography/mass spectrometry (LC/MS) and LC-tandem mass spectrometry analysis. To obtain additional structural details for this metabolite, additional quantities were generated from rat liver microsomal incubations and purified by high-performance liquid chromatography (HPLC) for NMR analysis. The NMR data for the metabolite confirmed that the glucuronide was covalently bound to either the sulfur or the nitrogen of the benzoxazine-2-thione moiety. The lack of key through-bond (scalar) and through-space (dipolar) one-dimensional (1D) and two-dimensional (2D) NMR couplings and correlations in the metabolite spectra (due primarily to low sample concentration) precluded an unambiguous structure elucidation. Subsequent synthesis of the S- and N-glucuronides of tanaproget from tanaproget facilitated the unambiguous regio- and stereochemical assignment of the metabolite by comparison of 1D NMR chemical shifts and scalar coupling constants, 2D NMR correlations, and HPLC and LC/MS characteristics between the synthetic compounds and the metabolite. From extensive comparison of the spectral and chromatographic data of the microsomally derived metabolite and the synthetic compounds, the metabolite has been determined to be the S-(beta)-D-glucuronide of tanaproget.

Enhanced chromatographic resolution of amine enantiomers as carbobenzyloxy derivatives in high-performance liquid chromatography and supercritical fluid chromatography.

The carbobenzyloxy (cbz) protecting group is evaluated for it's potential to enhance the resolution of chiral amine enantiomers using high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC). A series of cbz derivatives of commercially available racemates was prepared and analyzed by enantioselective chromatography using a variety of mobile phases and polysaccharide and Pirkle-type chiral stationary phases (CSPs). The cbz-derivatized product consistently demonstrated enhanced chiral resolution under HPLC and SFC conditions. Improved selectivity and resolution combined with an automated preparative HPLC or SFC system can lead to the rapid generation of highly purified enantiomers of desirable starting materials, intermediates or final products.

Integrity profiling of high throughput screening hits using LC-MS and related techniques.

Integrity profiling of HTS hits is valuable for verification of the hit identity and purity. This provides early discovery researchers with more confident SAR theories. Methodology for integrity profiling of HTS hits must be high throughput, consume little material, and selectively provide structure-based data. Analytical techniques that can be utilized for integrity profiling methods are reviewed for their appropriateness in sample preparation, component separation, detection, purity quantitation, identity confirmation, and follow-up.

Absolute configuration of C2-symmetric spiroselenurane: 3,3,3',3'-tetramethyl-1,1'-spirobi[3 H,2,1]benzoxaselenole.

The enantiomers of 3,3,3',3'-tetramethyl-1,1'-spirobi[3 H,2,1]benzoxaselenole have been separated on a chiral preparative chromatographic column. The experimental vibrational circular dichroism (VCD) spectra have been obtained for both enantiomers in CH(2)Cl(2). The theoretical VCD spectra have been obtained by means of density functional theoretical calculations with the B3 LYP density functional. From a comparison of experimental and theoretical VCD spectra, the absolute configuration of an enantiomer with positive specific rotation in CH(2)Cl(2) at 589 nm is determined to be R. This conclusion has been verified by comparing results of experimental optical rotatory dispersion (ORD) and electronic circular dichroism (ECD) to predictions of the same properties using the B3 LYP functional for the title compound.

Advantages of atmospheric pressure photoionization mass spectrometry in support of drug discovery.

The performance of the atmospheric pressure photoionization (APPI) technique was evaluated against five sets of standards and drug-like compounds and compared to atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The APPI technique was first used to analyze a set of 86 drug standards with diverse structures and polarities with a 100% detection rate. More detailed studies were then performed for another three sets of both drug standards and proprietary drug candidates. All 60 test compounds in these three sets were detected by APPI with an overall higher ionization efficiency than either APCI or ESI. Most of the non-polar compounds in these three sets were not ionized by APCI or ESI. Analysis of a final set of 201 Wyeth proprietary drug candidates by APPI, APCI and ESI provided an additional comparison of the ionization techniques. The detection rates in positive ion mode were 94% for APPI, 84% for APCI, and 84% for ESI. Combining positive and negative ion mode detection, APPI detected 98% of the compounds, while APCI and ESI detected 91%, respectively. This analysis shows that APPI is a valuable tool for day-to-day usage in a pharmaceutical company setting because it is able to successfully ionize more compounds, with greater structural diversity, than the other two ionization techniques. Consequently, APPI could be considered a more universal ionization method, and therefore has great potential in high-throughput drug discovery especially for open access liquid chromatography/mass spectrometry (LC/MS) applications.

Rapid method development for chiral separation in drug discovery using multi-column parallel screening and circular dichroism signal pooling.

A novel strategy for rapid chiral method development has been developed using multi-column parallel screening and circular dichroism (CD) signal pooling. Described is the first use of a customized HPLC system that integrates an HPLC auto-sampler, one pump and five divided channels with five columns and five UV detectors to screen five chiral stationary phases (CSPs) simultaneously in parallel. A high-pressure semi-prep on-line pre-filter, a six-port manifold and five individually adjusted backpressure restrictors were installed in the system which allowed the sample and mobile phase to be evenly distributed over the five columns and UV detectors. The five CSPs, namely Chiralpak AD and AS, Chiralcel OJ and OD and Whelk-O1, were screened. The system guarantees a five-fold increase in speed for chiral column scouting compared with the widely used automated sequential column switching approach, and does not have the limitations of the coupled column screening approach for enantiomers whose elution order could be reversed on CSPs. Furthermore, the five channels after the UV detectors were recombined using a reversed flow splitter into a CD detector. The pooled CD signal from the five channels was recorded to track the elution order of the resolved enantiomers and to determine their sign, positive or negative. The signal pooling allows for the effective use of a single CD detector for multiple columns since unresolved racemate has little CD signal, and observing the sign of CD signal for one of the two enantiomer UV peaks is sufficient for tracking the enantiomeric elution order.

Rapid, automated screening method for enzymatic transformations using a robotic system and supercritical fluid chromatography.

An automated screening method was developed for enzymatic transformations using a robotic system and rapid chiral supercritical fluid chromatography (SFC) analysis with a run time of 1.5 min. The method accelerates the enzyme selection process for screening biocatalysts, where a large number of enzymes are evaluated for activity and enantioselectivity. Kinetic resolution of secondary alcohols by enzymatic transesterification was used as a prototype for method development. The rapid automated method can be used effectively for screening enzymes and optimizing reaction conditions in biocatalysis.