Noteworthy Chemistry

January 13, 2014

Milk digestion produces a complex series of organized nanostructures. Dairy milk is a protein-stabilized emulsion of water- and oil-soluble vitamins, salts, proteins, carbohydrates, fat, and other hydrophobic components. Absorption of milk fat by the enterocytes (absorptive cells in the small intestine) relies on lipase-catalyzed hydrolysis into fatty acids and monoglyceride, but little is understood about this process.

B. J. Boyd and coauthors at Monash University (Parkville, Australia) and the Australian Synchrotron (Clayton) discovered that highly ordered geometric nanostructures form as milk undergoes an in vitro digestion process designed to mimic natural digestion. They used a high-intensity synchrotron source and time-resolved small-angle X-ray scattering to track the transition of milk from a normal oil-in-water unstructured emulsion through a series of ordered nanostructures. They displayed the results by using cryogenic transmission electron microscopy.

Adding pancreatin (a mixture of amylase, lipase, and protease) to the milk emulsion at pH 6.5 and 37 ºC converts the milk triglycerides mainly to diglycerides and fatty acids. During digestion, the lipid cores of the milk particles gradually become more hydrophilic as water and hydrophilic molecules move into the lipid phase.

In the absence of bile salt, lipase-catalyzed digestion causes water-filled micelles to form within the oil droplets. As the digestion progresses and more water is incorporated into the system, these micelles self-organize into a cubic lattice and then form water-filled tubes that pack into a hexagonal array that evolves into a bicontinuous cubic array. The high internal surface area of these structures might facilitate lipid digestion in bile-compromised individuals.

Adding bile salt accelerates the digestion of the milk lipids. Within 1 min after adding pancreatin, milk is transformed into a microemulsion that contains multilamellar fragments as long as 400 nm, tubular structures, and unilamellar vesicles—but no higher-ordered liquid crystalline structures. After 30 min, vesicles are the dominant structures. The bile salt is incorporated into the self-assembled structures inside the emulsion particles, creating more hydrophilic interfaces and swelling the internal structures.

Decreasing the pH of the system "back-tunes" the morphological changes developed during digestion and shifts the balance toward the structures formed earlier in the digestive process. (ACS Nano 2014, 8, 10904–10911; Nancy McGuire)

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What is water’s role in anesthesia? Anesthesia modulates nerve activity by binding the anesthetic with proteins in the neuronal membrane. The detailed events at the molecular level, however, are unclear. For example, the role of water in the mechanism of general anesthesia has been the subject of controversy for more than 50 years.

To form a better picture of the anesthesia mechanism, Y. Xu, Y. Wu and coauthors at the University of North Carolina, Chapel Hill, and the University of Pittsburgh School of Medicine identified the effect of water on the binding of anesthetics with proteins. They chose bovine serum albumin (BSA), a protein with known binding pockets for anesthetics, as their model protein. They quantitatively measured the hydration and adsorption processes of anesthetics and BSA by using 1H and 19F NMR spectroscopy, respectively.

The authors found that binding volatile anesthetics, including halothane (1), isoflurane (2), and 1-chloro-1,2,2-trifluorocyclobutane (3), to BSA does not occur unless BSA is hydrated beyond certain thresholds. In contrast, “nonimmobilizers” (e.g., compounds such as 4, 5, and 6 that are structurally similar to anesthetics) do not bind to BSA even after complete hydration.

Structures of anesthetics (1–3) and nonimmobilizers (4–6)

This study provides evidence for the indispensable role of water in anesthetic–protein interactions, even though the anesthetic binding sites are largely hydrophobic. It helps explain the mechanism of general anesthesia and provides guidelines for designing anesthetics with improved potency. (J. Phys. Chem. B 2013, 117, 12007–12012; Xin Su)

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Adding melamine reinforces PVA via hydrogen bonding. P. Song, Z. Xu, and Q. Guo* at Deakin University (Geelong, Australia) and Zhejiang Agriculture and Forestry University (China) used a biomimetic strategy to reinforce poly(vinyl alcohol) (PVA) via hydrogen bonding. In their procedure, melamine, which can form multiple hydrogen bonds, is incorporated at various concentrations within the PVA matrix.

The balance between intermolecular and intramolecular PVA hydrogen bonding within the PVA–melamine structure shifts as the percentage of melamine increases. Rheological measurements confirmed the development of a physically associated network between the PVA chains and melamine as melamine is added that leads to “solid-like” behavior at 1 wt% melamine. Tensile experiments supported these structural changes, which translate to significant enhancements in mechanical properties such as toughness, strength, and extensibility.

At >1 wt% melamine, mechanical and rheological studies showed marked decreases in reinforcement, which the authors attribute to plasticization effects and changes in the associated composite structure. The enhanced hydrogen-bonding structure induced by adding melamine also enhances the thermal stability of PVA. (ACS Macro Lett. 2013, 2, 1100–1104; LaShanda Korley)

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Downsize a protein to a small molecule. The use of proteins in drug discovery, medicine, and industry is restricted because of high manufacturing costs, chemical instability, immunogenicity, and poor bioavailability. D. Fairlie and co-workers at the University of Queensland (Brisbane, Australia) developed a method for making small molecules with proteinlike properties.

The authors studied a 77-residue human inflammatory protein (complement C3a), which plays a role in innate immune responses to infection and tissue injury by interacting with a G protein–coupled receptor called C3aR. C3a rapidly degrades in vivo to a protein that does not bind to C3aR. The authors believe that synthetic C3aR agonists that do not degrade may have immunostimulation and degranulation activities. Stable synthetic antagonists may have anti-inflammatory properties.

The researchers prepared a 3-D homology model for C3aR to identify the interaction residues with C3a. They found a space occupied by three amino acids: leucine, alanine, and arginine. Tripeptides (e.g., 1) composed of these amino acids do not bind C3aR, but hydrogen-bond acceptors (notably oxazole and imidazole) in place of alanine increase C3aR affinity.  Protecting the N-terminus of leucine with acetyl, tert-butoxycarbonyl, or 3-indolecarboxamide further increases potency. 

Leu-Ala-Arg tripeptide and its 3-indolecarboxamide analogue

3-Indolecarboxamide–substituted peptide 2 in the figure is comparable with C3a in terms of agonist response to C3aR, but it has the advantage that 2 remains intact after exposure to plasma. This simple, easy-to-produce compound can be used as a surrogate for human C3a protein. This study may lead to a general method for developing small molecules with the functional potency of proteins. (Nat. Commun. 2013, 4, No. 2802; José C. Barros)

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Use wet-granulation studies to predict agglomeration. M. T. Maloney and co-workers at Pfizer (Groton, CT, and Sandwich, UK) describe the development of laboratory tools to predict agglomeration or attrition upon scale-up in agitated filter dryers.

Agglomeration is undesirable during drying; however, it is desirable for wet granulation. Agglomeration occurs during the early part of the drying process when the filter cake still contains a substantial amount of moisture.

A mixer torque rheometer (MTR) can be used to monitor the rheological properties of wet powders. As the moisture content increases, the torque required to shear the mass increases if agglomeration occurs until enough moisture is present to form a slurry. Fine particulate wet solids that exhibit high viscosities as measured by the MTR are more likely to agglomerate during agitated filter drying. (Org. Process Res. Dev. 2013, 17, 1345–1358; Will Watson)

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Allosteric inhibitor of a microtubule motor protein targets cancer cells. Spindle poles form at the cellular centrosome, the microtubule organizing center that regulates cell cycle progression. Centrosome duplication is highly regulated and occurs alongside DNA replication, ensuring the formation of two functional centrosomes that make up the mitotic spindle poles. Microtubule motor proteins facilitate movement along microtubules and play an integral role in the assembly of functional mitotic spindles.

Supernumerary centrosomes are found in most types of solid tumors and indicate malignancy; they also may drive malignant transformation. Some cells with supernumerary centrosomes form a pseudo-bipolar spindle via centrosome clustering, which prevents multipolar mitosis and cell death. HSET is an essential microtubule motor protein for chromosome clustering and is necessary for the survival of cancer cells.

F. Gergely, D. I. Jodrell, S. V. Ley, and coauthors at the University of Cambridge (UK), the Cambridge Crystallographic Data Center, Cancer Research Technology (Cambridge and London), and the University of Oxford (UK) designed and synthesized a selective allosteric inhibitor of HSET by using chemogenomics-based compound selection. They then biologically analyzed the small-molecule inhibitor CW069 (1) and determined that it causes mitotic spindle defects in cancer cells with supernumerary centrosomes. Treated cancerous cells lacked centrosome clustering, which results in multipolar spindle formation. 

Treatment with the inhibitor has no effect on healthy cells. As a result, CW069 provides a foundation for targeting centrosome clustering as a cancer-treatment strategy. (Chem. Biol. 2013, 20, 1399–1410; Abigail Druck Shudofsky)

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Small-molecule inhibitor CW069