Noteworthy Chemistry

May 19, 2014

An engineered enzyme detoxifies cocaine. The US Food and Drug Administration has no approved medication for treating cocaine abuse; however, C.-G. Zhan and co-workers at the University of Kentucky (Lexington) have engineered an enzyme that acts as a catalyst to detoxify cocaine efficiently.

The authors observed that hydrolyzing cocaine (1) at the benzoyl ester group produces ecgonine methyl ester (2) and benzoic acid, both of which are biologically inactive. They engineered butyrylcholinesterase (BChE), an enzyme similar to acetylcholinesterase (AChE), to function as a cocaine hydrolase. AChE helps regulate the amount of acetylcholine (ACh), the molecule that relays signals between the brain and the muscles. BChE was the enzyme of choice because it is thermally stable. In addition, BChE is highly specific—ACh is the only natural target of BChE and AChE in the human body.

Structures of cocaine and ecgonine methyl ester

The researchers designed an engineered BChE that they named E30-6 by using computational modeling based on the correlation between hydrogen-bonding energy and H–O distance in the first transition state. To confirm the design of E30-6, they performed a sophisticated computational evaluation on E30-6–catalyzed cocaine hydrolysis to determine the detailed reaction coordinate and free-energy changes during the enzymatic process. The evaluation was based on hybrid quantum mechanical, molecular mechanical, and free-energy calculations.

The authors prepared E30-6 by using site-directed mutagenesis, protein expression, purification, active-site titration, and enzyme activity assays. Kinetic evaluation indicated that its catalytic efficiency for cocaine hydrolysis was ≈25-fold higher than for ACh hydrolysis. The catalytic efficiency was comparable with that of AChE for hydrolyzing ACh, which is recognized as the most efficient enzyme hydrolysis.

E30-6 showed no signs of toxicity in mice models. Most importantly, it protected mice against cocaine-induced lethality. This study demonstrates the therapeutic potential of E30-6 as a tool for cocaine detoxification and the potential of computational chemistry for enzyme redesign. (Nat. Comm. 2014, 5, No. 3457; José C. Barros)

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“Click” DNA onto silicon oxide substrates. DNA arrays bound to solid substrates serve as useful probes for examining molecular interactions. A. Maquieiria and colleagues at the Polytechnic University of Valencia (Spain) and the CIBER-BBN/IQAC-CSIC institutes (Barcelona) used thiol–ene chemistry to bind DNA to silicon oxide substrates (see figure). To bind DNA rapidly, the authors used silanization reactions to modify the substrate surfaces with alkyne (strategy A) or thiol (strategy B) functional groups. Complementary thiol- or alkyne-modified oligonucleotides were covalently attached (“clicked”) via UV irradiation. 

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Thiol–ene reaction for immobilizing DNA

Using fluorescence spectroscopy, the authors evaluated variations in immobilization density between strategies A and B. Strategy B yielded higher coverage as the result of covalent immobilization of UV-generated radicals.

The researchers demonstrated the high specificity of the covalent functionalization by photopatterning the oxide surfaces. They used topographical measurements to show that the DNA strand is deposited in a planar configuration onto the substrate. As a proof of concept, they demonstrated that this platform can be used to detect Escherichia coli in DNA at concentrations as low as 50 pM. (Bioconjugate Chem. 2014, 25, 618–627; LaShanda Korley)

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Extract spectroscopic data from digital photographs. Many analytical techniques took a leap forward when it became possible to extract quantitative digital information from them, rather than relying on visual interpretation. Could digital photography be next? Digital photography is widely used in chemical research, mostly to capture visual evidence of experimental setups and results. Photographs are also used to demonstrate changes in fluorophore emissions, but they rarely offer more useful information than direct color comparisons.

Photographic information would be enhanced if pixel data in digital photographs could be correlated with fluorescence spectral data. T. Schwaebel, S. Menning, and U. H. F. Bunz* at Heidelberg University Ruperto Carola (Germany) established a protocol for the precise analysis of fluorescence profiles from digital cameras by mathematically transforming the color information.

The authors first examined the relationship between fluorescence spectroscopic information and digital photographic data by using the digital color analysis technique (Suzuki, H., et al. Anal. Chem. 2002, 74, 5766–5773). After they identified pseudo–color matching functions (PCMFs) as the key factor for converting chromaticity data (color wavelength and saturation) between cameras and fluorimeters, they developed a calibration algorithm that allowed direct comparison between chromaticity values from photographs and emission spectra. The digital camera serves as a three-filter detector for providing readily processible color data simultaneously from a large array of samples.

The authors tested their setup against different light sources, including LEDs, inorganic quantum dot solutions, and organic fluorophore solutions. In all cases, the calculated chromaticity values from normalized emission spectra were consistent with those extracted from photographs.

The ability to obtain and process large amounts of spectral data rapidly and inexpensively makes this method useful for many applications that require fast array detection. (Chem. Sci. 2014, 5, 1422–1428; Xin Su)

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Hydrogen bonds affect the growth of reverse micelles around metal ions. Reverse micelles are used for solvent extraction, nanosynthesis, and drug delivery, among other applications. In reverse micelles, a hydrophobic phase surrounds clusters of amphiphilic molecules that encapsulate a hydrophilic phase. Structural studies of metal ions inside reverse micelles have focused on the inner coordination sphere of the metal ions or on the effect of the ions on the nanoscale structure of the micelles, but these two structural scales have not been linked. M. Olvera de la Cruz, R. J. Ellis, and colleagues at Northwestern University (Evanston, IL) and Argonne National Laboratory (IL) used X-ray analysis and molecular dynamics calculations to explain how acidity affects the ways metal atoms connect to the inner surfaces of reverse micelles, and how this affects their size and shape.

The authors examined the interactions that connect "hard" coordinating metal ions to the "soft" cores of reverse micelle aggregates. Their model system consisted of europium nitrate [Eu(NO3)3] solutions in water or aqueous nitric acid (HNO3) that are encapsulated in micelles made from N,N′-dimethyl-N,N′-dioctylhexylethoxymalonamide and suspended in n-heptane.

Increasing the acidity of the system drives hydrogen-bond (H-bond) donor and acceptor molecules (acid and water) into the organic phase, along with increasing concentrations of Eu3+. The Eu3+ partitioning ratio was 0.6:1 in the acidic system versus 0.4:1 in the neutral-pH system.

Small-angle X-ray scattering (SAXS) analysis indicates that the micelles have a more elongated shape in the acidic system. There is, however, significant variation in shape: Some clusters are globular and others more rod-shaped. In the neutral system, the micelles tend to be small and spherical.

From the SAXS data, the authors calculated the diameters of the polar reverse micelle cores to be ≈11 and ≈15 Å for the neutral and acidic phases, respectively. This result compares well with diameters of 10.4 and 15.6 Å derived from molecular dynamics calculations.

The figure illustrates typical reverse micelle structures in the neutral system (left) and the acidic system. The large Eu3+ ions are shown in pink.

Typical reverse micelle structures

The average number of H-bonds per Eu3+ ion is calculated to be 4.17 in the neutral system and 18.4 in the acidic system. In the latter, water and HNO3 molecules coordinate with the metal ions via H-bonds and dative (coordinate) bonds. Some of these molecules bond directly with malonamide molecules in the outer sphere. Others form H-bonded chain structures with other water and HNO3 molecules. These chains form bridges between the Eu3+ ions and the malonamide molecules and produce the swollen reverse micelles observed under acidic conditions. (J. Phys. Chem. Lett. 2014, 5, 1440–1444; Nancy McGuire)

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Use an amide cosolvent to minimize product overhydrolysis. In many synthetic sequences, nitriles are hydrolyzed to amides with sodium hydroxide (NaOH). The reaction can be difficult to stop at the amide; overhydrolysis to the carboxylic acid is a common problem. One way to prevent this is to provide some species that can mop up the excess NaOH after it has finished its job of converting nitriles to amides.

J. K. Niemeier, R. R. Rothhaar, and co-workers at Eli Lilly (Indianapolis) used kinetic modeling to develop reaction conditions that would minimize carboxylic acid formation but maintain high levels of nitrile conversion. One of their key findings is that using N-methylpyrrolidinone (NMP) as a cosolvent with water helps protect the amide from overhydrolysis.

Nitrile hydrolysis to the amide is catalytic with respect to NaOH concentration, but amide hydrolysis to the carboxylic acid requires stoichiometric amounts of NaOH. Therefore, once the amide is formed, NaOH begins to hydrolyze the amide to the acid. NMP is also an amide, and its concentration is greater than that of the product amide. As a result, NMP hydrolysis helps consume excess NaOH and increase the yield of the target amide. (Org. Process Res. Dev. 2014, 18, 410–416; Will Watson)

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Monitor pH in vivo with no wires or batteries. Sensing pH in living organisms (in vivo) in real time is a sought-after goal for a variety of medical applications because it can provide information about pH changes associated with many physiological processes.  Implantable sensors are ideal candidates for in vivo pH measurements, but current devices are limited in size and accuracy. This is because these devices are based mainly on glass membranes and ion-selective field-effect transistors, and their working mechanisms require reference electrodes.

A. Star and colleagues at the University of Pittsburgh and Ortho-tag Inc. (Pittsburgh) report a device that is made with modified carbon nanotubes. It senses pH values and transmits the data wirelessly and does not require batteries or reference electrodes.

The authors first built a semiconducting network by introducing oxidized single-walled carbon nanotubes (ox-SWNTs, 1) into interdigitated gold electrodes on silicon chips, where the gold electrodes interlock like the fingers on two clasped hands. They electropolymerized 1-aminoanthracene (2) in situ, coating the surfaces of the ox-SWNTs with poly(1-aminoanthracene) (PAA, 3) (see figure). 

Preparation of PAA-coated ox-SWNTs

The device translates pH sensing into conductance measurements. Conductance of the ox-SWNT–PAA matrix depends linearly on the concentration of hydronium ions (H3O+) in its surroundings. The PAA layer is critical to the performance of the pH sensor. It must be thick enough to coat the entire ox-SWNT surface; but excessive coating decreases the device’s responsiveness.

The device accurately measures pH levels in the pH 2–12 range after calibration with potentiometry measurements. It is much more sensitive than devices made from SWNTs or ox-SWNTs alone and retains its sensitivity for at least 120 days. It is selective for H3O+, even in the presence of calcium and sodium ions.

The authors made a prototypical wireless in vivo pH sensor by connecting the ox-SWNT–PAA-based device to an implantable radio frequency identification (RFID) tag. They demonstrated that the sensor can transmit real-time pH data through the passively powered tag across simulated human skin. (Sci. Rep. 2014, 4, No. 4468; Xin Su

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