Noteworthy Chemistry

March 10, 2014

Lycopene effectiveness gets a boost. Lycopene, a carotenoid found in tomatoes, is associated with a decreased risk of cardiovascular disease and certain cancers, and it has been shown to exhibit antioxidant properties. Dried tomato peel and tomato powder can be added as a nutritional supplement to ready-to-eat meat products without the need for chemical extraction of the lycopene component.

All-(E)-Lycopene accounts for 80–97% of the lycopene isomers in tomatoes and related products, but various Z-forms account for 25–70% of the lycopene in human tissues and body fluids. Some Z-isomers show stronger antioxidant activity in vitro than the all-E form.

M. M. Calvo, and coauthors at Complutense University and the Institute of Food Science Technology and Nutrition (both in Madrid) and Friedrich Schiller University Jena (Germany), and Catholic University School of Medicine (Rome) studied the effects of electron-beam (e-beam) irradiation, a common meat sterilization method, on the isomerization and degradation of lycopene additives from dried tomato products. They centrifuged and dried ground fresh tomatoes to obtain the powder, and they freeze-dried the peels. Both products were sealed in low-permeability packaging and irradiated at 10 MeV and 4 kGy at an electron accelerator facility. Temperatures were held to <18 °C, and the samples were protected from light.

The results showed that dried tomato peel has almost twice the lycopene content (0.2 ± 0.02 mM) of the powder (0.1 ± 0.01 mM). Irradiation exposure reduced the lycopene content by ≈50% for both products. HPCL analysis of the peel and powder showed a reduction of all-(E)-lycopene and an increase in (Z)-lycopene isomers during irradiation. This effect was significantly stronger in the peel samples than in the powders.

The authors used RAT-1 fibroblasts to check for antioxidant activity, which they measured as the lycopene's ability to inhibit intracellular reactive oxygen species (ROS), with and without the prooxidant agent H2O2. A weak, but significant, inhibition of ROS production was observed when the fibroblasts were treated with the tomato products in the absence of H2O2. The inhibition effect was stronger for cells incubated with H2O2.

The tomato extracts also inhibited the phosphorylation of mitogen-activated protein kinases, which are associated with stress stimuli, including ROS production. Adding tomato extracts inhibited the expression of the oxidative stress-sensitive transcription factor NF-κB and the oxidative stress–signaling proteins cyclooxygenase-2 and nicotinamide adenine dinucleotide phosphate oxidase.

In all cases, the effect was stronger for peels than for powder extracts and more evident for irradiated samples than for nonirradiated ones. The effects were more pronounced for larger doses of the tomato extracts. All of the effects were observed for a range of carotenoid concentrations that can be reached in vivo after lycopene or tomato supplementation. (J. Agric. Food Chem. 2014, 62, 1557–1563; Nancy McGuire)

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Measure glass-transition temperatures with string resonators. Applications of polymer-based materials are closely related to their properties, especially thermal properties such as the glass-transition temperature (Tg) and melting point. Tg can be determined by using differential scanning calorimetry and dynamic mechanical analysis, but more modern techniques based on beam resonators produce improved sensitivity and accuracy.

S. Bose and co-workers at the Technical University of Denmark (Kongens Lyngby and Roskilde) developed a better analytical system for measuring static and dynamic Tg values simultaneously. Their technique is based on micromechanical string resonators.

The authors used low-stress silicon-rich SiNx to prepare the microstrings, which they attached to a piezo element. The microstrings were heated or cooled with a temperature control system, and their resonance frequencies were detected by a laser-Doppler vibrometer. Samples were loaded by spray-coating polymer solutions uniformly. A schematic drawing of the setup is shown in the figure.

Setup of device for measuring glass-transition temperatures

The change in resonance frequency correlated to the change in static tensile stress (Young’s modulus) of the polymer. The change in quality factor (Q) is directly related to viscoelastic damping. Changes in Young’s modulus and Q as functions of temperature yield the static and dynamic Tg values of the sample. By using this technique, the authors determined the Tg values for an amorphous poly(D,L-lactide) and a semicrystalline poly(L-lactide)  with nanogram quantities of samples. (ACS Macro Lett. 2014, 3, 55–58; Xin Su

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As these dye aggregates grow, they emit stronger red light. Self-organization of building blocks into hierarchical assemblies is an essential part of structural biology. Much research effort is directed toward deciphering the self-organization processes of small molecules, with an emphasis on elucidating the morphologies of the assembled products.

Much less research has been done to evaluate the functional properties of the assembled structures and to explore their practical applications. F. Würthner and co-workers at the University of Würzburg (Germany) monitored the hierarchical growth processes of perylenebisimide derivative 1 and discovered that its self-organized aggregates have uncommon light-emitting behavior.

Perylenebisimide derivative 1

The amphiphilic molecules of 1 spontaneously undergo sequential assembly from nanorods to nanoribbons as their concentration in water increases. As nanorods fuse into nanoribbons, the fluorescence quantum yield (ΦF) and lifetime (τ) increase dramatically. This unusual photophysical phenomenon is in sharp contrast to the common perception that ΦF and τ decrease when concentration increases.

The authors believe that the denser packing of 1 in the nanoribbons disfavors their structural relaxation pathways to less emissive species. This can possibly be attributed to more rigid conformations in the larger dye aggregates caused by more severe 3-D constraints. (Angew. Chem., Int. Ed. 2014, 53, 1270–1274; Ben Zhong Tang)

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Consider all components and byproducts when studying process safety. 3-Amino-4-chloroindazole is prepared by the reaction of 2,6-dihclorobenzonitrile with H2NNH2·H2O in a dipolar aprotic solvent. Z. Wang and co-workers at AbbVie (North Chicago, IL) chose pyridine as the solvent because the boiling point (115 ºC) is close to the reaction temperature (110 ºC).

Even with this temperature match, the reaction had a propensity to run away exothermically. As part of their safety review, the authors studied the reaction to determine the cause of the exotherms. The individual reaction components and intermediates are stable; but the reaction generates HCl, which, when combined with H2NNH2·H2O, generates an exotherm. More importantly, mixing an intermediate with H2NNH2·H2O also creates an exotherm.

Adding base reduces the severity of the exotherm or completely eliminates it, depending on which base is used. Weak bases are preferred; ultimately the authors chose NaOAc. (Org. Process Res. Dev. 2013, 17, 1603–1610; Will Watson)

For additional information on handling H2NNH2 safely, see Niemeyer, J. K.; Kjell, D. P. Org. Process Res. Dev. 2013, 17, 1580–1590.

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Incorporate an azobenzene switch with bioorthogonal ligation. Azobenzenes are classic photoresponsive molecules that change configuration and photophysical properties when they are irradiated. They are prime targets for gaining photocontrol over biological processes. For this process to be effective, it is important to integrate azobenzene units orthogonally into biological systems.

The Staudinger–Bertozzi ligation is a well-known strategy for covalently attaching small-molecule modifiers to biomolecules without disrupting normal biological functions. With this in mind, W. R. Browne, B. L. Feringa, and co-workers at the University of Groningen (The Netherlands) developed a method for modifying biomolecules that incorporates azobenzenes bioorthogonally into azide-containing proteins in aqueous media.

The authors prepared diphenylphosphinoazobenzene derivative 1 by using a method they had previously developed (Szymanski, W.; Wu, B.; Poloni, C.; Janssen, D. B.; Feringa, B. L. Angew. Chem., Int. Ed. 2013, 52, 2068–2072). The ligation of 1 with benzyl azide in a H2O–MeCN–CHCl3 mixture gave product 2 in 95% yield in 38 h. Product 2 can be isomerized to the cis form by visible light irradiation. Its thermal relaxation time scale is similar to those of biological events.

Ligation of azobenzene 1 with a model azide

The authors modified the surface of an azidosilane-functionalized quartz cover slip with 1, but they could not confirm reversible photoisomerization on the surface because the UV–vis spectrum and contact angle did not change significantly upon visible-light irradiation. They were, however, able to attach azobenzene tag 1 to an azide-decorated Sp1-f3 peptide, illustrating the utility of the ligator. (Chem.—Eur. J. 2014, 20, 946–951; Xin Su)

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Integrase polymorphism combined with a resistance mutation increases HIV drug resistance but does not improve viral fitness. The newest class of HIV drugs consists of integrase-strand transfer inhibitors (INSTIs), which target the HIV-1 integrase enzyme. Integrase catalyzes the insertion of proviral DNA into the host chromosome. This is a two-part process: In the first “3′-processing” reaction, a GT dinucleotide is cleaved at the 3′-end of the viral DNA, exposing reactive hydroxyl groups. In the subsequent “strand-transfer” reaction, the hydroxyl groups covalently link the processed viral DNA and the host DNA. INSTIs competitively inhibit this second step.

Currently available INSTIs are effective, tolerable, and minimally toxic; they are recommended for treating newly diagnosed HIV-positive individuals. The INSTI dolutegravir, approved by the US Food and Drug Administration in 2013, has a greater in vivo barrier to the emergence of drug-resistant mutations than do the other two INSTI drugs, raltegravir and elvitegravir.

Thus far, no major resistance mutation has been identified when treatment-naïve patients are treated with dolutegravir. In INSTI-naïve patients who were previously treated with other anti-retroviral drugs, however, dolutegravir treatment fails to work. An R263K integrase mutation has been found in these patients; although it confers low levels of dolutegravir resistance, it decreases viral fitness.

Dolutegravir selection studies in tissue cultures show the emergence of this mutation in HIV subtype B integrase, along with the M50I polymorphism secondary mutation. This polymorphism—at a position that is not highly variable—has been found in 10% of INSTI-naïve patients and in one raltegravir-resistant patient in combination with R263K.

To determine whether M50I complements R263K and further interferes with dolutegravir activity, M. A. Wainberg and colleagues at Jewish General Hospital and McGill University (both in Montreal) biochemically characterized the M50I integrase substitution, alone and with the primary R263K resistance mutation, to determine the significance of the polymorphism. They looked at strand-transfer activity, viral fitness, and INSTI resistance.

On its own, the M50I mutation is associated with low-level resistance to all INSTIs, so, not surprisingly, the M50I–R263K combination moderately increases resistance to dolutegravir. Yet, whereas R263K decreases the activity of the HIV integrase in cell-free assays, the authors found that although M50I is often present with R263K, the polymorphism does not compensate for the reduction in enzymatic activity associated with that mutation. The polymorphic mutant does not affect HIV replication capacity on its own, but when it is combined with R263K, it further decreases viral fitness and diminishes HIV infectivity.

Based on their data, the authors determined that, because M50I increases the resistance of R263K integrase mutants to dolutegravir but does not restore the viral fitness loss that comes with the R263K mutation, it should not lead to a new resistant strain of HIV-1. Accordingly, they believe that dolutegravir may be beneficial as a first-line treatment against HIV (Retrovirology 2014, 11, No. 7; Abigail Druck Shudofsky)

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A simplified synthesis of propofol eliminates neutralization steps. Propofol (2,6-disopropylphenol, 3) is an intravenously administered anesthetic. It is produced commercially by several methods that involve Friedel–Crafts alkylation reactions and purification by vacuum distillation. C. Pramanik and coauthors at Emcure Pharmaceuticals (Pune, India) and the University of Pune developed improved processes for isolating propofol and its intermediates that are suitable for commercial production.

Simplified two-step propofol synthesis

The authors chose a synthetic route to propofol that begins with 4-hydroxybenzoic acid (1; see figure). After alkylating 1 with 2 mol i-PrOH, they optimized the purification step by carefully pouring the reaction mixture over a cooled water–toluene mixture without previously neutralizing the acidic reaction mixture. The HPLC purity of intermediate 2 was >98% after the precipitate was washed with cyclohexane.

In the second step, the authors substituted the traditional ethylene glycol solvent with lower–boiling point 2-ethoxyethanol. After the decarboxylation reaction, they poured the reaction mixture into water and extracted it with toluene, this time without neutralizing the alkaline mixture. After toluene was removed under vacuum, crude propofol was purified by vacuum distillation to give the target compound in >99.8% HPLC purity.

To prepare for profiling the impurities produced in propofol manufacture, the authors synthesized five possible byproducts cited by US and European pharmacopeias. These compounds are expensive or not commercially available. Finally, they validated a multikilogram process for making propofol and are ready to scale up their method to further fulfill market needs. (Org. Process Res. Dev. 2014, 18, 152-156; José C. Barros)

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