Noteworthy Chemistry

January 31, 2011

Extract DNA and RNA serially from frozen tissues. Analyzing sets of genes is accomplished better with fresh-frozen tissue samples than with paraformaldehyde-fixed, paraffin-embedded materials because the genes have longer, higher quality nucleic acids. However, a technology for extracting nucleic acids from frozen tissues and cell pellets that is suitable for automation does not exist.

L. Mathot, M. Lindman, and T. Sjöblom* at Uppsala University (Sweden) developed a procedure to extract DNA and RNA from frozen cell pellets or frozen tissues embedded in the gel-like medium “optimal cutting temperature compound”. The frozen tissues are ground in a salt solution and captured in siliceous solid supports.

The authors extracted DNA first because it competes with RNA for binding to solid supports. They screened several magnetic silica-based solid supports with different surface groups to extract the DNA of breast, colon, spleen, tonsil, and bone marrow cells. The use of MagPrep Silica HS beads produced the best results; electrophoresis showed that the DNA fragments have lengths similar to those extracted by the standard procedure (phenol–CHCl3).No RNA contamination was detected.

In the following step, the authors used MagPrep Silica Basic particles to extract RNA from the tissues. The RNA yields were higher than the DNA yields, and the integrity of the RNA was not affected by the procedure. The flowchart illustrates the nucleic acid extraction process; LB, WB, and EB refer to lysis, washing, and elution buffers, respectively.

This is the first method to extract nucleic acids from frozen tissues. It is suitable for automation because all operations can be performed in 96- or 384-well formats; there is little solvent manipulation; binding of the nucleic acids to silica bead is controlled only by pH; and quantification can be evaluated by spectrophotometric methods. (Chem. Commun. 2011, 47, 547–549; JosÉ C. Barros)

Improve MALDI-TOF-MS for determining large molecule masses. Matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is used to determine the mass of large molecules such as biomolecules and polymers. The matrix molecules absorb laser energy, ionize, and deliver some of the energy to the large-molecule analyte. The ionized analyte fragments can neutralize or migrate to the charge detector. The detected signal intensity is proportional to the quantity of analyte ions. When the analyte’s molecular weight is >100 kDa, however, the number of ions is insufficient to guarantee reliable signals for analysis.

To improve this technique, C.-H. Chen and co-workers at Academia Sinica (Taipei, Taiwan) made several improvements to a MALDI-TOF mass spectrometer to amplify the signal intensity. They added an ion trap in front of the detector to capture the generated ions and raised the inert gas pressure in the chamber to increase the number of trapped ions. The separate collection of the ions minimizes neutralization. They also used multiple laser shots to enhance ion accumulation. The improved spectrometer achieved a quantitative analysis of 1000-kDa immunoglobulin M in one scan. (Anal. Chem. 2010, 82, 10125–10128; Sally Peng Li)

Stable deep-blue-light emitters are based on poly(silafluorenes). An important challenge to commercializing polymeric light emitting diodes for flat panel displays is to form efficient blue-light–emitting polymers with good color purity. The large body of research points to 2,7-fluorene–based polymers as the best blue-light emitters. Poly(9,9-dialkylsilafluorene)s, however, are also promising and produce even more stable blue-light emission because the oxidative stability of silicon at the fluorene 9-position is greater than that of carbon.

Y.-q. Mo, X. Chen, H-b. Wu, and coauthors at South China University of Technology (Guangzhou), Canton Oledking Optoelectronic Materials (Guangzhou), and CSIRO Materials Science and Engineering (Clayton, Australia) note that no processible homopolymers of 9,9-diarylsilafluorene appear in the literature. They describe the synthesis and properties of a highly soluble, processible polymer (1) based on 9,9-dialkoxyphenyl-2,7-dihalosilafluorenes.

Treating diiododibromobiphenyl 2 with strong base, then SiCl4, forms key intermediate 3. The reaction of 3 with p-alkoxyphenyllithium 4 creates the desired monomer 5 for the polymerization reaction.

To produce polymer 1, 5 is homopolymerized using the Yamamoto reaction. (COD is cyclooctadiene). At the end of the polymerization, bromobenzene is added to provide phenyl groups at the ends of the polymer chains. This minimizes any green-light emission that might emanate from residual bromo end groups.

After purification, 1 is obtained with an acceptable molecular weight (Mw) of 92 kDa and easy solubility in common organic solvents. The glass-transition temperature of 1 is ~120 °C, and it begins to decompose at 426 °C. The absorption maximum in solution and film form occurs at 391–392 nm, which suggests that the polymer chain conjugation length does not change after spin-coating from solution.

The electroluminescence spectra from single active-layer devices made from 1 are consistent with a deep-blue emission that remains almost unchanged even after annealing at 120 °C for 30 min. These findings contrast with the behavior of poly(dioctylfluorene), which has pronounced emission in the longer wavelength region and unstable blue-light emission after temperature increases and operational stresses.

Polymer 1 has a high external quantum efficiency of 75% and a luminous efficiency of 2.1–3.2 cd/A. (Macromolecules 2011, 44, 17–19; W. Jerry Patterson)

Use a blend of materials to form vascular scaffolds. P. I. Lelkes* and colleagues at Drexel University (Philadelphia) and the Chinese Academy of Science (Changchun) developed blends of various compositions of poly(lactide-co-glycolide) (PLGA), gelatin, and elastin as potential vascular scaffolds that address challenges of mechanical integrity and thrombogenicity. This electrospun blend of synthetic and natural materials yields uniform fibers with a homogeneous distribution of components and elasticity that promotes the influx of migratory cells into the pores.

Hydration of the nanofibers increases the fiber diameter without the loss of mat integrity or the need for post-cross-linking strategies. The mechanical behavior of the biocompatible electrospun scaffold is controlled primarily by the relative amounts of PLGA and gelatin in the blended nanofiber.

The mechanical properties dictate the adhesion and spreading of endothelial cells on the fiber surfaces and the penetration of smooth muscle cells. Swelling is governed by the hydrophobic PLGA content, whereas the introduction of elastin promotes cell adhesion and proliferation. The results show promise for tailored materials for vascular constructs. (Biomacromolecules 2011, 12, Article ASAP DOI: 10.1021/bm101149r; LaShanda Korley)

Prepare Janus nanoparticles by a simple interfacial process. Nanoparticles with two distinct components are often called Janus nanoparticles after the double-faced Roman god of doorways. These nanoparticles may lead to a variety of high-tech applications, from controlled drug delivery to cancer cell therapy.

Most processes for making Janus nanoparticles require special techniques and long procedures. A team led by F. Wang and C. He at the Institute of Materials Research and Engineering (Singapore) and the National University of Singapore developed an easy, rapid process for preparing hybrid Janus nanoparticles.

The researchers used bipolar nanoparticles as model systems for demonstrating the concept of their interfacial process. Negatively charged gold nanoparticles are made compatible with organic solvents with the aid of a surface coating of amphiphilic poly(ethylene glycol)–functionalized polyhedral oligomeric silsesquioxane. When the surface-modified gold nanoparticles suspended in a solvent are brought into contact with an aqueous suspension that contains positively charged silica nanoparticles, interfacial conjugation of the gold and silica nanoparticles is induced by electrostatic attraction at one side of each type of particle. This asymmetric attraction yields Janus nanoparticles with single and multiple small gold nanoparticles (17 nm diam) attached to one large silica nanoparticle (80 nm diam). (Chem. Commun. 2011, 47, 767–769; Ben Zhong Tang)

Separate reaction steps to improve yield, workup, and isolation. G. Guercio and co-workers at GlaxoSmithKline Medicines Research Center (Verona, Italy) wanted to improve the synthesis of GSK356278, a potent PDE4 enzyme inhibitor. They originally converted 1-ethyl-1H-5-aminopyrazole to ethyl 4-chloro-1-ethyl-1H-pyrazolo[3.4-b]pyridine-5-carboxylate in one step by treating it with diethyl (ethoxymethylidene)malonate in 25 volumes of POCl3 at 160 °C to give a 44% yield after chromatography.

They improved the transformation by separating the reaction into two substeps. In the first, condensation of the aminopyrazole with the (ethoxymethylidene)malonate, the temperature was lowered to 100 °C, and EtOH was removed by distillation as it formed. In the second substep, the amount of POCl3 was decreased to 2.5 volumes for the cyclization–chlorination reaction, which was also carried out at a lower temperature (110 °C). Pouring the reaction mixture onto water crystallized the product in 65–70% yield and high purity (98% by HPLC). (Org. Process Res. Dev. 2010, 14, 1153–1161; Will Watson)

Form optically pure ω-trifluoromethylated amino acids via a chiral nickel complex. The bioactivity of fluorine-containing compounds is well documented, particularly in pharmaceuticals. About 40% of marketed drugs and an estimated 80% of drug candidates contain at least one fluorine atom. Thus, researchers are exploring synthetic techniques for introducing fluorine moieties.

The Ni(II) complex of the chiral Schiff base of glycine is an established entrÉe to enantiopure amino acids via several reactions. V. A. Soloshonok, H. Liu, and coauthors at the Chinese Academy of Sciences (Shanghai) and the State University of New York at Stony Brook extended this procedure by the reaction of ω-trifluoromethylalkyl iodides with the Schiff base in a two-step synthesis of amino acids with linear ω-trifluoromethylalkyl groups.

An example of this synthesis begins with chiral ligand 1, which is treated with a nickel salt in the presence of (S)-glycine to form chiral nickel complex 2. The reaction of 2 with the alkylating agent 1,1,1-trifluoropropyl iodide 3 introduces the desired trifluoromethyl substituent to give 4 with high diastereoselectivity.

Disassembly of 4 under acidic conditions releases free amino acid 5 in high yield and almost enantiomerically pure form. Ligand 1 is quantitatively recovered for reuse. The authors note that this reaction proceeds by the alkylation of nucleophilic glycine and alanine to provide new fluorinated amino acids for use in medicinal chemistry. (J. Org. Chem. 2011, 76, 684–687; W. Jerry Patterson)

What do you think of Noteworthy Chemistry? Let us know.