Metal-Promoted Higher-Order Assembly of Disulfide-Stapled Helical Barrels.

Peptide-based helical barrels are a noteworthy building block for hierarchical assembly, with a hydrophobic cavity that can serve as a host for cargo. In this study, disulfide-stapled helical barrels were synthesized containing ligands for metal ions on the hydrophilic face of each amphiphilic peptide helix. The major product of the disulfide-stapling reaction was found to be composed of five amphiphilic peptides, thereby going from a 16-amino-acid peptide to a stapled 80-residue protein in one step. The structure of this pentamer, 5HB1, was optimized in silico, indicating a significant hydrophobic cavity of ~6 Å within a helical barrel. Metal-ion-promoted assembly of the helical barrel building blocks generated higher order assemblies with a three-dimensional (3D) matrix morphology. The matrix was decorated with hydrophobic dyes and His-tagged proteins both before and after assembly, taking advantage of the hydrophobic pocket within the helical barrels and coordination sites within the metal ion-peptide framework. As such, this peptide-based biomaterial has potential for a number of biotechnology applications, including supplying small molecule and protein growth factors during cell and tissue growth within the matrix.

Targeting Enterococcus faecalis HMG-CoA reductase with a non-statin inhibitor.

HMG-CoA reductase (HMGR), a rate-limiting enzyme of the mevalonate pathway in Gram-positive pathogenic bacteria, is an attractive target for development of novel antibiotics. In this study, we report the crystal structures of HMGR from Enterococcus faecalis (efHMGR) in the apo and liganded forms, highlighting several unique features of this enzyme. Statins, which inhibit the human enzyme with nanomolar affinity, perform poorly against the bacterial HMGR homologs. We also report a potent competitive inhibitor (Chembridge2 ID 7828315 or compound 315) of the efHMGR enzyme identified by a high-throughput, in-vitro screening. The X-ray crystal structure of efHMGR in complex with 315 was determined to 1.27 Å resolution revealing that the inhibitor occupies the mevalonate-binding site and interacts with several key active site residues conserved among bacterial homologs. Importantly, 315 does not inhibit the human HMGR. Our identification of a selective, non-statin inhibitor of bacterial HMG-CoA reductases will be instrumental in lead optimization and development of novel antibacterial drug candidates.

An Optimized Preparation of 1,1-Dimethylallyl Esters and Their Application to Solid-Phase Peptide Synthesis.

A one-step preparation of 1,1-dimethylallyl (DMA) esters was optimized for the C-terminal protection of a range of Fmoc-protected amino acids. This preparation is not sensitive to the scale of reaction and affords the corresponding DMA esters in 70-99% yield with high regioselectivity. Additionally, these DMA-protected amino acids were used with the backbone amide linker (BAL) of Albericio and Barany and found to resist diketopiperazine formation during the synthesis of a series of tripeptide esters. C-terminal DMA protection is compatible with the BAL linkage and allows for standard Fmoc-based methods to be used throughout the synthesis.

Death effects of reveromycin A in normal and disease-associated cells of the joint.

Earlier work in our laboratory demonstrated that naturally occurring reveromycin A (Rev A) causes apoptosis in osteoclasts without accompanying necrosis. Rev A death effects in both normal and diseased joint cells were investigated in this study. A dose of 10 µM Rev A did not cause apoptosis nor necrosis in monolayer chondrocytes, even at pH 6.8, a pH mimicking that of an inflamed joint. In contrast, at the acidic pH Rev A did induce significant apoptosis (fourfold increase at 48 h of treatment, P < 0.005) in normal synoviocytes without accompanying necrosis. Western blot of the normal synoviocyte proteins revealed that cytochrome c levels were not significantly changed over the time course of treatment nor did caspase 8 activity increase; therefore, Rev A appears to exert this apoptotic effect through a mechanism independent of the classical intrinsic and extrinsic pathways. Fibroblast-like synoviocytes isolated from rheumatoid arthritis patients (RAFLS) as well as normal human fibroblast-like synoviocytes (NHFLS), cells known to play key roles in arthritic joint pathology, were also subjected to Rev A treatment at both physiologic and acidic pH's. Neither apoptosis nor necrosis was induced in either RAFLS or NHFLS. Parallel mitomycin C treatment of NHFLS induced both apoptosis and necrosis. Comparative structure-activity analyses of Rev A and mitomycin C revealed that Rev A is less likely to cross the cell membrane at near neutral pH. Collectively the data reveal that a physiological dose of Rev A under acidic conditions induces normal synoviocytes to undergo apoptosis while pathologic fibroblast-like synoviocytes are resistant to apoptosis and necrosis.

Rare case of gallbladder agenesis presenting with pancreatitis.

Gallbladder agenesis (GA) is a rare congenital abnormality with an incidence of 0.01-0.09%. Majority of GA exist alone although it can be associated with other systemic malformations involving the gastrointestinal, genitourinary, cardiovascular and skeletal systems. It is thought that biliary and pancreatic pathologies coexist and this is the second case reported in the literature of GA presenting with pancreatitis.

Design, synthesis, biological and structural evaluation of functionalized resveratrol analogues as inhibitors of quinone reductase 2.

Resveratrol (3,5,4'-trihydroxylstilbene) has been proposed to elicit a variety of positive health effects including protection against cancer and cardiovascular disease. The highest affinity target of resveratrol identified so far is the oxidoreductase enzyme quinone reductase 2 (QR2), which is believed to function in metabolic reduction and detoxification processes; however, evidence exists linking QR2 to the metabolic activation of quinones, which can lead to cell toxicity. Therefore, inhibition of QR2 by resveratrol may protect cells against reactive intermediates and eventually cancer. With the aim of identifying novel inhibitors of QR2, we designed, synthesized, and tested two generations of resveratrol analogue libraries for inhibition of QR2. In addition, X-ray crystal structures of six of the resveratrol analogues in the active site of QR2 were determined. Several novel inhibitors of QR2 were successfully identified as well as a compound that inhibits QR2 with a novel binding orientation.

Generation and characterization of a distonic biradical anion formed from an enediynone prodrug in the gas phase.

A negatively charged biradical intermediate was successfully generated in the gas phase via cyclization of the deprotonated bicyclo[8.3.0]trideca-12-ene-2,7-diyn-1-one precursor. The inherent negative charge of this biradical allows its characterization via collision-activated dissociation and reactions with a variety of neutral substrates in an FT-ICR mass spectrometer. Although the biradical is unreactive toward reagents that usually react rapidly with positively charged biradicals, such as dimethyl disulfide, it reacts with the halogen-containing substrates carbon tetrachloride, carbon tetrabromide, and bromotrichloromethane via bromine or chlorine atom abstraction, which supports its biradical structure. The results presented in this study indicate that cyclizations commonly used in solution to form biradical intermediates from enediyne compounds may also occur in the gas phase.

Perceptions of health information exchange in home healthcare.

The aim of this study was to understand home healthcare nurses' current experiences in obtaining outside clinical information at the point of care and the type of clinical information they most desire in their patients' health information exchange profile. A Web-based survey was deployed to home health workers in New York to learn about their experiences retrieving outside clinical data prior to having access to health information exchange, preferred data elements and sources in their patients' health information exchange profiles, and how availability of outside clinical data may affect emergency department referrals. Of the 2383 participants, 566 responded for a 23.8% overall response rate, and 469 of these respondents were RNs. Most RNs, 96.7%, agreed that easier and quicker access to outside information would benefit delivery of care, and 72.6% said the number of emergency department referrals would decrease. When asked about pre-health information exchange access to patient data, 96.3% said it was problematic. Inpatient discharge summaries were chosen most often by the RNs as a top five desired data element 81.5% of the time. Obtaining outside clinical information has been a challenge without health information exchange, but improved access to this information may lead to improved care. Further study is required to assess experiences with the use of health information exchange.

Small molecule inhibitors of anthrax toxin-induced cytotoxicity targeted against protective antigen.

Two molecular scaffolds were designed using the CAVEAT molecular design package to inhibit the oligomerization of protective antigen (PA(63) ), a key protein component of anthrax toxin. The inhibitors were designed to prevent heptamerization of PA(63) by mimicking key residues of PA(63) needed for the intermolecular interactions that stabilize the heptamer. Using the scaffolds identified by CAVEAT, seven candidate inhibitors were synthesized and tested for their ability to inhibit anthrax toxin-induced cytotoxicity, with three of the agents demonstrating modest inhibition in murine J774A.1 macrophage cells.

Synthesis and biological evaluation of a library of resveratrol analogues as inhibitors of COX-1, COX-2 and NF-kappaB.

Resveratrol (4,3',5'-trihydroxystilbene) is a naturally occurring antioxidant that inhibits cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and the transcription factor NF-kappaB. A 78-membered library of resveratrol analogues in which the substituents on the two aryl rings and alkene were varied was synthesized using a solid-phase Wittig olefination reaction. The library contains inhibitors against all three proteins that were more potent than resveratrol itself. Preliminary structure-activity relationships were also obtained from these data that permitted the derivation of pharmacophore models for each of the three targets.

Synthesis and Spectroscopic Correlation of the Diastereoisomers of 2,3-Dihydroxy-2,6,8-trimethyldeca-(4Z, 6E)-dienoic Acid: Implications for the Structures of Papuamides A-D and Mirabamides A-D.

All 4 diastereomeric possibilities for the 2,3-dihydroxy-2,6,8-trimethyldeca-(4Z,6E)-dienoic acid (Dhtda) residue, found in the cyclic depsipeptide natural products papuamides A-D and mirabamides A-D, were stereoselectively synthesized using a Z-selective Wittig reaction of both enantiomers of 2,4-dimethylhex-2-enyl-triphenylphosphonium bromide with all four diastereoisomers of ethyl-3-formyl-2-methyl-1,4-dioxaspiro[4,4]nonane-2-carboxylate. To elucidate the configuration of Dhtda, the 1H- and 13C-NMR spectra of the synthetic isomers were compared to those of the natural residue. On the basis of that comparison, it is suggested that the likely configuration of the diastereomer present in Dhtda residue is either (2R,3S,8S) or (2S,3R,8S) in the papuamides and mirabimides.

Entropy-driven population distributions in a prototypical molecule with two flexible side chains: O-(2-acetamidoethyl)-N-acetyltyramine.

Resonant two-photon ionization (R2PI), resonant ion-dip infrared (RIDIR), and UV-UV hole-burning spectroscopies have been employed to obtain conformation-specific infrared and ultraviolet spectra under supersonic expansion conditions for O-(2-acetamidoethyl)-N-acetyltyramine (OANAT), a doubly substituted aromatic in which amide-containing alkyl and alkoxy side chains are located in para positions on a phenyl ring. For comparison, three single-chain analogs were also studied: (i) N-phenethyl-acetamide (NPEA), (ii) N-(p-methoxyphenethyl-acetamide) (NMPEA), and (iii) N-(2-phenoxyethyl)-acetamide (NPOEA). Six conformations of OANAT have been resolved, with S(0)-S(1) origins ranging from 34,536 to 35,711 cm(-1), denoted A-F, respectively. RIDIR spectra show that conformers A-C each possess an intense, broadened amide NH stretch fundamental shifted below 3400 cm(-1), indicative of the presence of an interchain H bond, while conformers D-F have both amide NH stretch fundamentals in the 3480-3495 cm(-1) region, consistent with independent-chain structures with two free NH groups. NPEA has a single conformer with S(0)-S(1) origin at 37,618 cm(-1). NMPEA has three conformers, two that dominate the R2P1 spectrum, with origin transitions between 35,580 and 35,632 cm(-1). Four conformations, one dominate and three minor, of NPOEA have been resolved with origins between 35,654 and 36,423 cm(-1). To aid the making of conformational assignments, the geometries of low-lying structures of all four molecules have been optimized and the associated harmonic vibrational frequencies calculated using density functional theory (DFT) and RIMP2 methods. The S(0)-S(1) adiabatic excitation energies have been calculated using the RICC2 method and vertical excitation energies using single-point time-dependent DFT. The sensitivity of the S(0)-S(1) energy separation in OANAT and NPOEA primarily arises from different orientations of the chain attached to the phenoxy group. Using the results of the single-chain analogs, tentative assignments have been made for the observed conformers of OANAT. The RIMP2 calculations predict that interchain H-bonded conformers of OANAT are 25-30 kJ/mol more stable than the extended-chain structures. However, the free energies of the interchain H-bonded and extended structures calculated at the preexpansion temperature (450 K) differ by less than 10 kJ/mol, and the number of extended structures far outweighs the number of H-bonded conformers. This entropy-driven effect explains the presence of the independent-chain conformers in the expansion, and cautions future studies that rely solely on relative energies of conformers in considering possible assignments.

Enantio- and diastereoselective synthesis of (R,R)-beta-methoxytyrosine.

The nonproteinogenic amino acid (2R,3R)-beta-methoxytyrosine, a constituent of several cyclic depsipeptide natural products, was synthesized in protected form (8) from a readily available cinnamate ester in four steps and 62% overall yield with a greater than 28:1 er and 19:1 dr. This method provides a highly efficient, enantio- and diastereoselective synthesis of an important amino acid.

Synthesis and cytotoxicity of desmethoxycallipeltin B: lack of a quinone methide for the cytotoxicity of callipeltin B.

The desmethoxy analogue of the cytotoxic, cyclic depsipeptide callipeltin B was synthesized to evaluate the role of its beta-MeOTyr residue. The IC(50) of desmethoxycallipeltin B, in which the beta-MeOTyr residue was replaced by d-Tyr, against HeLa cells was found to be 128+/-10 microM in an MTT assay, compared to 98+/-5 microM for the natural product itself. The roughly comparable cytotoxicities suggest that the cytotoxicity of callipeltin B does not arise through the formation of a quinone methide intermediate.

An improved method for the protection of carboxylic acids as 1,1-dimethylallyl esters.

1,1-Dimethylallyl (DMA) esters of various N-protected amino acids have been synthesized using prenyldimethylsulfonium tetrafluroborate, a reagent that can be readily made and stored, in conjunction with catalytic CuBr. These reactions were complete within several hours and afforded DMA esters in high yields. As has been previously shown in our group, DMA esters represent a palladium-labile proctecting group for carboxylic acids that resists nucleophilic attack as a tert-butyl ester would.

Solid-phase total synthesis and structure proof of callipeltin B.

The cytotoxic, cyclic heptadepsipeptide, natural product callipeltin B was synthesized on a solid-phase support in 15% overall yield. Comparison of the 1H NMR spectra of three synthetic isomers with those of callipeltin B confirmed the configurational reassignment of its threonine residues as d-allothreonine and the assignment of the configuration of its beta-methoxytyrosine residue as (2R,3R).

Ring size and substituent effects in oxyanion-promoted cyclizations of enyne-allenes: observation of a Myers-Saito cycloaromatization at cryogenic temperature.

A series of acetoxy-substituted enyne-allenes, fused to cyclopentene and cyclohexene ring systems, were synthesized and treated with methyllithium to generate the corresponding enolates. It was found that whereas the cyclohexannulated examples underwent either C2-C7 (Myers-Saito) cycloaromatization or C2-C6 (Schmittel) cyclization depending on their terminal subsituents, the cyclopentannulated examples either failed to cyclize altogether or underwent C2-C7 cyclization. Both of these results lie in contrast to the behavior of their benzannulated analogues, which underwent exclusive C2-C6 cyclization independent of substituents. These findings are rationalized on the basis of both ring strain effects and the steric encumbrance of the terminal alkynyl and allenyl subsituents.

Approaches to patient health information exchange and their impact on emergency medicine.

Regional health information organizations and electronic health information exchange may have an important impact on the practice of emergency medicine in the United States. Regional health information organizations are local or regional information-sharing networks that enable electronic data interchange among stakeholders in a given geographic area. These stakeholders may include hospitals, skilled nursing facilities, clinics, private physicians' offices, pharmacies, laboratories, radiology facilities, health departments, payers, and possibly the patients themselves. Regional health information organizations are being formed across the country to improve the safety and efficiency of clinical care; improve public health efforts, biosurveillance, and disaster management response; and potentially create large databases of deidentified aggregate data for research. Because of the unique need for rapid access to information and the acuity of the clinical environment, few areas of the health care delivery system stand to change and benefit more from health information exchange than our nation's emergency departments. This article will explain the motivation for the development of regional health information organizations, identify some of the important issues in their formation, and discuss how their development might affect the practice of emergency medicine.

Solid-phase synthesis and configurational reassigment of callipeltin E. Implications for the structures of callipeltins A and B.

Two possible isomers of the natural product callipeltin E (1, 5) were synthesized by using an Fmoc-based solid-phase strategy in 7 steps, in 20% and 26% overall yields, respectively. The (1)H NMR spectrum of synthetic 5 correlated closely with that of the natural product, whereas that of 1 did not, providing confirmation of the configurational reassignment of the N-terminal residue of callipeltin E as D-allothreonine. This result strongly implies that the corresponding residue in the closely related cyclic depsipeptides callipeltins A and B should also be considered a D-allothreonine residue.

Solid-phase synthesis of callipeltin D. Stereochemical confirmation of the unnatural amino acid AGDHE.

[structure: see text] The lipopeptide callipeltin D (1) was synthesized using an Fmoc-based solid-phase strategy in seven steps and 35% overall yield. The 1H NMR of synthetic 1 correlated closely with that of the natural product, confirming the configurational assignment of the novel amino acid constituent (2R,3R,4S)-4-amino-7-guanidino-2,3-dihydroxyheptanoic acid.