A Conformationally Stable π-Expanded X-Type Double Helicene Comprising Dihydropyracylene Units with Multistage Redox Behavior.

A π-expanded X-type double [5]helicene comprising dihydropyracylene moieties was synthesized from commercially available acenaphthene. X-ray crystallographic analysis revealed the unique highly twisted structure of the compound resulting in the occurrence of two enantiomers which were separated by chiral HPLC, owing to their high conformational stability. The compound shows strongly bathochromically shifted UV/vis absorption and emission bands with small Stokes shift and considerable photoluminescence quantum yield and circular polarized luminescence response. The electrochemical studies revealed five facilitated reversible redox events, including three reductions and two oxidations, thus qualifying the compound as chiral multistage redox amphoter. The experimental findings are in line with the computational studies based on density functional theory pointing towards increased spatial extension of the frontier molecular orbitals over the polycyclic framework and a considerably narrowed HOMO-LUMO gap.

Toward a Formyl-to-Phenyl Conversion: An Unexpected Photochemical Fulvene Rearrangement.

Toward a conversion of aldehydes into arenes, we designed a sequence involving the initial reaction of an aldehyde to give a fulvene, followed by photochemical and platinum-catalyzed rearrangements into a Dewar benzene derivative, which finally isomerizes into the targeted arene. While computational studies support the plausibility of this route, we found that fulvene irradiation resulted in an unexpected isomerization into a spiro[2.4]heptadiene. This unusual photorearrangement has been investigated mechanistically and provides access to a variety of spiro[2.4]heptadienes with different substituents.

Synthesis and Characterization of Phenylalanine Amides Active against Mycobacterium abscessus and Other Mycobacteria.

Nα-2-thiophenoyl-d-phenylalanine-2-morpholinoanilide [MMV688845, Pathogen Box; Medicines for Malaria Venture; IUPAC: (2R)-N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide)] is a hit compound, which shows activity against Mycobacterium abscessus (MIC90 6.25-12.5 µM) and other mycobacteria. This work describes derivatization of MMV688845 by introducing a thiomorpholine moiety and the preparation of the corresponding sulfones and sulfoxides. The molecular structures of three analogs are confirmed by X-ray crystallography. Conservation of the essential R configuration during synthesis is proven by chiral HPLC for an exemplary compound. All analogs were characterized in a MIC assay against M. abscessus, Mycobacterium intracellulare, Mycobacterium smegmatis, and Mycobacterium tuberculosis. The sulfone derivatives exhibit lower MIC90 values (M. abscessus: 0.78 µM), and the sulfoxides show higher aqueous solubility than the hit compound. The most potent derivatives possess bactericidal activity (99% inactivation of M. abscessus at 12.5 µM), while they are not cytotoxic against mammalian cell lines.

Racemization-free synthesis of Nα-2-thiophenoyl-phenylalanine-2-morpholinoanilide enantiomers and their antimycobacterial activity.

Nα-2-thiophenoyl-D-phenylalanine-2-morpholinoanilide (MMV688845, IUPAC: N-(1-((2-morpholinophenyl)amino)-1-oxo-3-phenylpropan-2-yl)thiophene-2-carboxamide) from the Pathogen Box® library (Medicines for Malaria Ventures, MMV) is a promising lead compound for antimycobacterial drug development. Two straightforward synthetic routes to the title compound starting from phenylalanine or its Boc-protected derivative are reported. Employing Boc-phenylalanine as starting material and the T3P® and PyBOP® amide coupling reagents enables racemization-free synthesis, avoiding the need for subsequent separation of the enantiomers. The crystal structure of the racemic counterpart gives insight into the molecular structure and hydrogen bonding interactions in the solid state. The R-enantiomer of the title compound (derived from D-phenylalanine) exhibits activity against non-pathogenic and pathogenic mycobacterial strains, whereas the S-enantiomer is inactive. Neither of the enantiomers and the racemate of the title compound shows cytotoxicity against various mammalian cells.

Type of Dietary Fiber Is Associated with Changes in Ileal and Hindgut Microbial Communities in Growing Pigs and Influences In Vitro Ileal and Hindgut Fermentation.

BACKGROUND: The degree of ileal organic matter (OM) fermentation appears to be comparable to hindgut fermentation in growing pigs. OBJECTIVES: This study aimed to determine if dietary fiber sources with known different total gastrointestinal tract (GIT) fermentability in humans affect ileal and hindgut microbial communities and ileal fermentation in growing pigs used as an animal model for human adults. METHODS: Male pigs (21 kg bodyweight; 9 wk old; PIC Camborough 46 × PIC boar 356L; n = 8/diet) were fed for 42 d a diet containing cellulose (CEL, low fermentability) as the sole fiber source (4.5%) or diets in which half of the CEL was replaced by moderately fermentable fiber, psyllium (PSY), or kiwifruit (KF) fiber. For each diet, terminal jejunal (substrate) and ileal (inoculum) digesta were collected from euthanized animals for in vitro ileal fermentation (2 h). Terminal ileal (substrate) and cecal (inoculum) digesta were used for in vitro hindgut fermentation (24 h). After in vitro fermentations, OM fermentation and short-chain fatty acid (SCFA) production were determined. Ileal digesta and feces were collected for microbial analysis. Data were analyzed by 2-factor ANOVA (diet × GIT region). RESULTS: In vitro ileal OM fermentation was on average 22% and comparable to hindgut OM fermentation. Ileal and hindgut OM fermentation, SCFA production, and microbial community composition changed (P < 0.05) when CEL was partially replaced by KF or PSY. For instance, pigs fed the PSY diet had 3-fold higher (P ≤ 0.05) number of ileal and fecal bacteria than pigs fed the CEL and KF diets. Pigs fed the CEL diet had 4-fold higher (P ≤ 0.05) hindgut valeric acid production than pigs fed the other diets. CONCLUSIONS: Ileal fermentation is quantitatively significant. Partial substitution of CEL with more fermentable fibers influences both ileal and hindgut microbial communities and the fermentation in growing pigs.

Reduction of Muscle-Invasive Tumors by Photodynamic Therapy with Tetrahydroporphyrin-Tetratosylat in an Orthotopic Rat Bladder Cancer Model.

Photodynamic therapy (PDT) is a promising option for minimal-invasive treatment of bladder cancer. Efficacy of PDT in muscle-invasive urothelial cancer is still hampered by low tissue penetration of most photosensitizers due to short excitation wavelength. The novel light reactive agent tetrahydroporphyrin-tetratosylat (THPTS) is excitable at near-infrared (760 nm), allowing tissue penetration of up to 15 mm. Here, we established an orthotopic rat bladder cancer model and examined the effects of THPTS-PDT on tumor growth in vivo, and analyzed molecular mechanisms in vitro We examined pharmacokinetics and subcellular localization, and evoked cell death mode in cultured rat urothelial carcinoma cells (AY-27). We used female F344 Fischer rats for in vivo studies. Ten rats each were used for THPTS-PDT and light-only control. Bladders were evaluated by macroscopy and histology. Temperature-dependent THPTS uptake resulted in endosomal/lysosomal localization. PDT (0-50 µmol/L THPTS; 10 J/cm2) induced early onset of apoptosis leading to dose-dependent cytotoxicity in AY-27 cells. Single-time transurethral THPTS-PDT (100 µmol/L THPTS; 10 J/cm2) in F344 rats led to significant reduction of muscle-invasive tumor number (2/10 vs. 7/10 in controls) and total tumor volume (60% reduction) 2 weeks after PDT, while sparing healthy tissue. Here, we report for the first time effective tumor growth control by PDT in vivo THPTS is a promising new photosensitizer with the advantage of higher therapeutic depth and the potential of high-selective therapy in muscle-invasive urothelial cancer. This approach possibly allows minimal-invasive bladder preserving treatment of bladder cancer without systemic side effects.

Quantitative prediction of long-term molecular response in TKI-treated CML - Lessons from an imatinib versus dasatinib comparison.

Longitudinal monitoring of BCR-ABL transcript levels in peripheral blood of CML patients treated with tyrosine kinase inhibitors (TKI) revealed a typical biphasic response. Although second generation TKIs like dasatinib proved more efficient in achieving molecular remission compared to first generation TKI imatinib, it is unclear how individual responses differ between the drugs and whether mechanisms of drug action can be deduced from the dynamic data. We use time courses from the DASISION trial to address statistical differences in the dynamic response between first line imatinib vs. dasatinib treatment cohorts and we analyze differences between the cohorts by fitting an established mathematical model of functional CML treatment to individual time courses. On average, dasatinib-treated patients show a steeper initial response, while the long-term response only marginally differed between the treatments. Supplementing each patient time course with a corresponding confidence region, we illustrate the consequences of the uncertainty estimate for the underlying mechanisms of CML remission. Our model suggests that the observed BCR-ABL dynamics may result from different, underlying stem cell dynamics. These results illustrate that the perception and description of CML treatment response as a dynamic process on the level of individual patients is a prerequisite for reliable patient-specific response predictions and treatment optimizations.

Cromolyn/gelatin mixtures as aqueous alignment media and utilization of their mechanical stability for a layering technique.

In this study, aqueous blends of cromolyn and gelatin ("cromogels") are introduced as anisotropic media. The addition of gelatin enables an advantageous adjustability of the strength, the homogeneity, and the stability of the cromolyn alignment. The mechanical stability of these polymer-dispersed liquid crystals is further utilized by stacking layers of D2 O/cromolyn/gelatin with varying component ratio. The resulting distinct phases with correspondingly different degrees of alignment can be targeted by spatially resolved NMR techniques. As a case study, we investigated sucrose in a two-phase system with neat D2 O and analyte layered over the anisotropic medium. A recently presented spatially selective coupled-type HSQC experiment allows the determination of one-bond C-H splitting in both phases.

Front-line imatinib treatment in children and adolescents with chronic myeloid leukemia: results from a phase III trial.

A total of 156 patients (age range 1.3-18.0 years, median 13.2 years; 91 (58.3%) male) with newly diagnosed CML (N = 146 chronic phase (CML-CP), N = 3 accelerated phase (CML-AP), N = 7 blastic phase (CML-BP)) received imatinib up-front (300, 400, 500 mg/m2, respectively) within a prospective phase III trial. Therapy response, progression-free survival, causes of treatment failure, and side effects were analyzed in 148 children and adolescents with complete data. Event-free survival rate by 18 months for patients in CML-CP (median follow-up time 25 months, range: 1-120) was 97% (95% CI, 94.2-99.9%). According to the 2006 ELN-criteria complete hematologic response by month 3, complete cytogenetic response (CCyR) by month 12, and major molecular response (MMR) by month 18 were achieved in 98, 63, and 59% of the patients, respectively. By month 36, 86% of the patients achieved CCyR and 74% achieved MMR. Thirty-eight patients (27%) experienced imatinib failure because of unsatisfactory response or intolerance (N = 9). In all, 28/148 patients (19%) underwent stem cell transplantation (SCT). In the SCT sub-cohort 2/23 patients diagnosed in CML-CP, 0/1 in CML-AP, and 2/4 in CML-BP, respectively, died of relapse (N = 3) or SCT-related complications (N = 2). This large pediatric trial extends and confirms data from smaller series that first-line imatinib in children is highly effective.

Configurationally Stable Chiral Dithia-Bridged Hetero[4]helicene Radical Cation: Electronic Structure and Absolute Configuration.

A stable chiral hetero[4]helicene radical cation was synthesized and characterized by UV/Vis absorption and EPR spectroscopy, as well as X-ray crystallography. For the first time, a combination of chiroptical methods involving ECD, ORD, and VCD, supported by quantum mechanical predictions, enabled the elucidation of the absolute configuration of such open-shell helical species.

Surface modification of PDMS microfluidic devices by controlled sulfuric acid treatment and the application in chip electrophoresis.

Herein, we present a straightforward surface modification technique for PDMS-based microfluidic devices. The method takes advantage of the high reactivity of concentrated sulfuric acid to enhance the surface properties of PDMS bulk material. This results in alteration of the surface morphology and chemical composition that is in-depth characterized by ATR-FTIR, EDX, SEM, and XPS. In comparison to untreated PDMS, modified substrates exhibit a significantly reduced diffusive uptake of small organic molecules while retaining its low electroosmotic properties. This was demonstrated by exposing the channels of a microfluidic device to concentrated rhodamine B solution followed by fluorescence microscopy. The surface modification procedure was used to improve chip-based electrophoretic separations. Separation efficiencies of FITC-labeled amines/amino acids obtained in treated and untreated PDMS-devices as well as in glass chips were compared. We obtained higher efficiencies in H2 SO4 treated PDMS chips compared to untreated ones but lower efficiencies than those obtained in commercial microfluidic glass devices.

Microfluidic chips for chirality exploration.

Microfluidic chips applied to the investigation of chirality allow reaction, separation and analysis of minuscule amounts of enantiomeric molecules. Chiral chip technology is employed in fields as diverse as pharmaceutical high throughput screening and deep space exploration missions.

Chip electrophoresis of active banana ingredients with label-free detection utilizing deep UV native fluorescence and mass spectrometry.

In the present work, we report on a rapid and straightforward approach for the determination of biologically active compounds in bananas applying microchip electrophoresis (MCE). For this purpose, we applied label-free detection utilizing deep UV fluorescence detection with excitation at 266 nm. Using this approach, we could identify dopamine and serotonin, their precursors tryptophan and tyrosine and also the isoquinoline alkaloid salsolinol in less than 1 min. In bananas, after 10 days of ripening, we additionally found the compound levodopa which is a metabolite of the tyrosine pathway. Quantitative analysis of extracts by external calibration revealed concentrations of serotonin, tryptophan, and tyrosine from 2.7 to 7.6 µg/mL with relative standard deviations of less than 3.5%. The corresponding calibration plots showed good linearity with correlation coefficients higher than 0.985. For reliable peak assignment, the compounds were also analyzed by coupling chip electrophoresis with mass spectrometry. This paper demonstrates exemplarily the applicability of MCE with native fluorescence detection for rapid analysis of natural compounds in fruits and reveals the potential of chip-based separation systems for the analysis of complex mixtures.

A new weakly basic amino-reactive fluorescent label for use in isoelectric focusing and chip electrophoresis.

In this study, we present a novel amino-reactive fluorescence marker (referred to as UR-431), which is well suited for electrophoretic techniques. A main feature of this marker is its weakly basic behavior when conjugated to analytes. Labeled primary amines exhibit a positive net charge and accordingly a cathodic mobility below a pH of 2.4. The label features a pH-independent fluorescence emission and is thus very interesting for electrophoretic applications such as IEF. The absorption maximum of this yellow daylight chromophore is at 431 nm, whereas fluorescence emission peaks at 537 nm (quantum yield approximately 0.1). The label was successfully conjugated to amines, peptides and proteins and separated via CE and MCE. The on-chip detection limit of labeled lysine using a mercury-lamp-based fluorescence microscope was determined as 12 nM. An important feature of the new label is that it effects only a subtle change of the pI of proteins compared with common anionic labels, e.g. FITC. pI values of proteins were investigated by comparing native proteins and labeled proteins in CIEF. UR-431 was also applied to sensitive detection of amines and peptides in MCE.

Rapid replication of master structures by double casting with PDMS.

We present a simple method for fast and precise replication of microfluidic master structures for moulding or soft embossing by double casting of microstructured masters with polydimethylsiloxane (PDMS). The significant achievement is a simple approach to inverse a given microstructure multiple times by means of PDMS-based soft lithography utilising hydroxypropylmethylcellulose (HPMC) as non texturing release agent. A series of PDMS copies have been generated from different silicon layouts with excellent reproducibility and precision, even submicron structures were well reproduced. The replicas were successfully applied in hot embossing and soft lithography of microfluic devices. Hence, we believe this technique is ideally suited for the economic replication of precious master structures (master sharing) commonly used in soft lithography and hot embossing.

Progress in microchip enantioseparations.

Advances in microfluidic chips for chiral separations from 2003 to early 2009 are discussed. Microchip-based separation techniques promise higher speed, throughput, portability, less sample and reagent consumption, better environmental compatibility, reduced cost and the prospect of system integration. Microchip electrophoresis is the most promising technique for miniaturized enantioseparations and has been performed with a variety of designs and analytes, however, other formats such as microchip electrochromatography are also gaining in popularity. Microchip fabrication, chemistry and detection issues are critically discussed and highlighted. Integration of enantioseparation techniques into multifunctional microchips are currently a rapidly advancing area of research and methods are discussed that may eventually enable enantioseparations to be the part of a holistic chemical microchip.

Label-free fluorescence detection in capillary and microchip electrophoresis.

Herein, we summarize the current status of native fluorescence detection in microchannel electrophoresis, with a strong focus on chip-based systems. Fluorescence detection is a powerful technique with unsurpassed sensitivity down to the single-molecule level. Accordingly fluorescence detection is attractive in combination with miniaturised separation techniques. A drawback is, however, the need to derivatize most analytes prior to analysis. This can often be circumvented by utilising excitation light in the UV spectral range in order to excite intrinsic fluorescence. As sensitive absorbance detection is challenging in chip-based systems, deep-UV fluorescence detection is currently one of the most general optical detection techniques in microchip electrophoresis, which is especially attractive for the detection of unlabelled proteins. This review gives an overview of research on native fluorescence detection in capillary (CE) and microchip electrophoresis (MCE) between 1998 and 2008. It discusses material aspects of native fluorescence detection and the instrumentation used, with particular focus on the detector design. Newer developments, featured techniques, and their prospects in the future are also included. In the last section, applications in bioanalysis, drug determination, and environmental analysis are reviewed with regard to limits of detection.

Impact of laser excitation intensity on deep UV fluorescence detection in microchip electrophoresis.

A high intensity 266 nm continuous wave (cw-) laser developed for material processing was utilised as an excitation source for sensitive native fluorescence detection of unlabelled compounds in MCE. This 120 mW laser was attached via an optical fibre into a commercial epifluorescence microscope. With this MCE set-up we evaluated the impact of laser power on the S/N of aromatic compounds as well as of proteins. Compared with a previous work which used a 4 mW pulsed laser for excitation, improved S/N for small aromatics and to a lesser extent for proteins could be attained. The LOD of the system was determined down to 24 ng/mL for serotonin (113 nM), 24 ng/mL for propranolol (81 nM), 80 ng/mL for tryptophan (392 nM) and 80 ng/mL for an aromatic diol (475 nM). Sensitive protein detection was obtained at concentrations of 5 microg/mL for lysocyme, trypsinogen and chymotrypsinogen (340, 208 and 195 nM, respectively). Finally, a comparison of the cw- with a pulsed 266 nm laser, operating at the same average power, showed a higher attainable sensitivity of the cw-laser. This can be attributed to fluorescence saturation and photobleaching effects of the pulsed laser at high pulse energies.

Two-photon excited fluorescence detection at 420 nm for label-free detection of small aromatics and proteins in microchip electrophoresis.

Two photon excited (TPE) fluorescence detection was applied to native fluorescence detection of aromatics in microchip electrophoresis (MCE). This technique was evaluated as an alternative to common one photon excitation in the deep UV spectral range. TPE enables fluorescence detection of unlabeled aromatic compounds, even in non-deep UV-transparent microfluidic chips. In this study, we demonstrate the proof of concept of native TPE fluorescence detection of small aromatics in commercial microfluidic glass chips. Label-free TPE fluorescence detection of native proteins and small aromatics in MCE was achieved within the micromolar concentration range, utilising 420 nm excitation light.