Emulating Nature's Silicon Skills

Diatoms are wondrous creatures. These single-celled microscopic algae live in fresh or salt water that contains minute amounts of silicic acid. The diatoms extract the soluble silicic acid from the water, concentrate it, and polymerize it to form insoluble silica, which they then deposit in a protective coat that can take on a beautifully ornate architecture. Each of these steps is promoted by enzymes and other proteins that serve not only as catalysts but also as scaffolding components for the final silica structure. Other species, including sponges and some grasses, also use proteins to create silica-protein composites for structural components.

These organisms’ meticulous regulation of silicon chemistry has caught the attention of a small band of chemists who want to adapt such techniques for their own synthetic purposes. Researchers don’t fully understand the mechanisms by which these species wield proteins to process silicon substrates. But by studying the organisms, researchers are learning to use proteins to exert greater control over the production of traditional silicon compounds and also to create new types of silicon compounds, according to Alan R. Bassindale, a professor of organometallic chemistry at Open University, in England.

Bassindale and other chemists in the field discussed their work last month during a Division of Polymer Chemistry symposium on bioinspired silicon chemistry at the ACS national meeting in Washington, D.C.

From a human standpoint, one of the most important classes of silicon compounds is silicones, also known as polysiloxanes. These polymers are used in an enormous array of applications, including caulk, grease, insulation, rubber, hair conditioners, and medical devices. They possess a –Si–O–Si– backbone and have organic side groups attached to the silicon atoms.

Silicones are produced from silanes such as Si(CH₃)₂Cl₂. In the presence of water, hydrolysis converts the silane into a silanol, and condensation subsequently converts the silanol into a polysiloxane. Depending on the particular silicone involved and further chemical steps performed, the resulting polymer structure can be linear, branched, or cross-linked, or shaped like a ring or a cage.

One driving force behind the study of interactions between proteins and silicon compounds is green chemistry “because if you can make silicones and other silicon compounds using biological organisms, you can save a lot of energy, time, and money,” Bassindale said. Current synthetic techniques for producing organosilicon compounds rely on harsh conditions at high temperatures and sometimes high pressures, along with extremes in pH, he noted. “If it were possible to do the same chemistry using enzymes or artificial enzymes,” which can operate under mild conditions, “then the energy costs would decrease dramatically,” he added.

Conventional reaction conditions for producing commercial silicones and other silicon compounds have other drawbacks. They aren’t regio- or enantioselective and can promote uncontrolled side reactions and broad molecular-weight distributions in the products, said Richard A. Gross, a professor in the department of chemical and biological sciences at Polytechnic Institute of New York University. These conditions can also cleave siloxane bonds and lead to decomposition of useful functional groups unless protection and deprotection steps are incorporated into the reaction sequence.

Enzyme-catalyzed reactions, on the other hand, offer much greater control over the structure of products, and their mild reaction conditions protect functional groups. Furthermore, Gross said, natural enzymes can be improved through molecular biology techniques to optimize their behavior and make them “industrially relevant.”

Scientists have been studying natural silica formation “since the Victorians, including Charles Darwin, observed diatoms growing and dividing under their optical microscopes,” according to Stephen J. Clarson, a materials science and engineering professor at the University of Cincinnati. But it wasn’t until the late 1990s that chemists working with sponges and diatoms first isolated and characterized proteins involved in silica production. These researchers, including Daniel E. Morse and Galen D. Stucky of the University of California, Santa Barbara, and Nils Kröger of Georgia Institute of Technology, showed that the isolated proteins could catalyze the formation of silica and silicone polymers under mild conditions in vitro.

A few years later, Bassindale and his Open University colleague organic chemistry professor Peter G. Taylor undertook a hunt for enzymes that they could use to carry out silicon chemistry in the lab. Because the reactivity of silicon compounds is similar to that of carbonyl compounds such as esters, the researchers reasoned that enzymes capable of hydrolyzing esters might also be able to hydrolyze silicon compounds. By restricting the search to cheap and readily available enzymes, they narrowed the field to 112 candidates.

The researchers screened these enzymes for hydrolysis and condensation activity at pH 7 and room temperature with two simple silicon compounds, ethoxytri­methylsilane and trimethylsilanol. Bassindale and Taylor found that most of the enzymes could promote hydrolysis of the silane to some degree. But only five, including trypsin, an enzyme found in the digestive tract, aided condensation of the silanol (J. Inorg. Biochem. 2003, 96, 401).

For the group of enzymes as a whole, activity wasn’t particularly high. “We found out that these enzymes were not very good with silicon compounds, which was not surprising given that they evolved to do specific carbon chemistry, not silicon chemistry,” Bassindale told C&EN. “On the other hand, we did find that under the right circumstances enzymes can effect transformations to make silicon-oxygen bonds. And that was a key finding because now the field is open for people to genetically modify the active site of an enzyme or to design new artificial enzymes for making new organosilicon compounds.”

Paul M. Zelisko, an organic chemistry instructor at Brock University, in Ontario, then attempted a more complex reaction. He found that trypsin and other enzymes can catalyze cross-linking of silicones through hydrolysis and condensation reactions of alkoxy groups. The reaction products are similar to those generated with traditional tin catalysts, which must be avoided in silicones intended for biomedical applications because of potential toxicity, he noted. Enzymes could also serve as “green” replacements for heavy-metal catalysts in the synthesis of silicone products for other applications, according to Zelisko, who has applied for a patent on the technique.

In another green method, Zelisko has shown that enzymes such as trypsin and pepsin can catalyze the production of silica sol-gels from alkoxysilanes without the solvent and acid or base catalyst normally required for this reaction (Chem. Commun. 2008, 5544; Silicon 2009, 1, 47).

He is currently studying the mechanism and kinetics of the enzyme-catalyzed reactions and is also looking into functionalization or the incorporation of additives into the sol-gels for applications such as coatings for metal and wood, optical devices, immobilized catalyst systems, and delivery of bioactive compounds for agricultural or biomedical use.

Zelisko, whose university lies in the heart of Ontario’s wine country, is also experimenting with these sol-gels as a coating for corks. Preliminary testing indicates that the coating can block release of 2,4,6-trichloro­anisol, a stinky contaminant that he said sometimes leaches out of corks and makes a bottle of wine taste like swamp water.

Researchers are looking for other ways to make enzymes useful as well as user-friendly. Kröger, Bassindale, Clarson, Zelisko, and others have shown that some enzymes react with silicic acid or its analogs to form silica-enzyme composites. Choice of enzyme, substrate, and reaction conditions—such as stirring the reaction mixture or passing a stream of nitrogen through it—gives chemists the ability to control the morphology of these composites in a rough approximation of the exquisite control maintained by diatoms and other organisms. Depending on those selections, the synthetic composites take the form of nanosized spheres, short loops, fibers, perforated rectangles, or other shapes, Clarson noted (Chem. Commun. 2003, 238 and 1122).

Silica prefers to form a composite with an enzyme that is covered in positive charges, which attract and help polymerize negatively charged polysilicate ions, Bassindale said. He and Taylor determined that such enzymes have a high pI (the pH at which a protein is neutral). Good candidates include proteinase K, from the fungus Tritirachium album, and trypsin, the researchers report in a paper that will appear in the Journal of Materials Chemistry.

Some of the silica-protein composites can be used to carry out enzyme-catalyzed chemistry. In the case of the nanospheres, for instance, a substrate for the enzyme passes through pores and channels in the sphere’s surface and undergoes a reaction in the presence of the enzyme inside the sphere, and then a reaction product passes back out. Bassindale said he and other researchers have found that enzymes protected in this way maintain anywhere between 40 and 90% of their original activity. The coating thwarts enzymes’ natural tendency to digest themselves at room temperature in a process known as autolysis. “That enables you to store the enzymes at room temperature” without them breaking down, Bassindale said.

Immobilizing an enzyme in a silica or silicone matrix also makes good economic sense because it allows chemists to easily recover and reuse the expensive enzyme for subsequent reactions, noted Clarson, who is collaborating with Polytechnic Institute’s Gross in this area.

In a recent project, the two researchers immobilized the digestive enzyme pepsin in cross-linked polydimethylsiloxane (PDMS). They showed that the unprotected enzyme, which normally operates in the extremely acidic environment of the stomach, denatures and loses its activity at neutral pH. By comparison, the immobilized enzyme retained more than 70% of its activity at pH 7 (Silicon 2009, 1, 37). Materials incorporating such silicone-enzyme composites could be used in biocatalytic membranes or biosensors, Clarson believes.

After the success with pepsin, Clarson turned to fibrinogen and thrombin, two proteins involved in the cross-linking that enables blood to clot in a wound. When he mixed the two proteins into a PDMS emulsion, Clarson found that they strongly adsorbed onto the suspended silicone droplets but maintained their cross-linking ability. This work could lead to materials for targeted drug delivery or novel cardiovascular applications, he told C&EN.

Gross has immobilized lipase B, an enzyme isolated from Candida antarctica yeast, on acrylic beads. Lipase is normally used for hydrolysis, a reaction in which the enzyme breaks apart a substrate by catalyzing its reaction with water. But Gross shifts the reaction equilibrium toward synthesis by removing water from the mixture as the reaction proceeds. In this way, he induces the enzyme to catalyze esterification of silicone substrates to form so-called sweet silicones. These organosilicon-sugar conjugates can be used as surfactants, adhesion promoters, or chiral templates (Org. Lett. 2005, 7, 3857).

The method can also be used to form organosilicon amides, noted Gross, who holds a joint patent for the technique with Dow Corning, a major silicone producer. In one example, Gross reacted a series of silicone diamines with diethyl adipate, a diester, to form polysilicone ester amides “of substantial molecular weight,” he said (Macromolecules 2007, 40, 7919). Gross found that the amount of silicone adipamide present in the polymer product dictated whether it was solid, waxy, or sticky. “Clearly, we can make a series of materials based on polyester amides with silicone blocks, and we can get a range of properties from these that require further investigation,” he said.

Several researchers are studying the interactions between proteins and silicon compounds with the aid of phage-display techniques, Bassindale said. Phages are viruses that can be genetically engineered to express or “display” peptide chains on their surfaces. Peptides consist of a small number of amino acids, the building blocks of proteins. A collection of genetically modified phages is typically mixed with a target molecule. Phages displaying peptides that bind to the target molecule are then collected and their DNA sequence analyzed to identify the peptides responsible for target binding. Clarson used phage-display techniques to show that peptides with histidine groups bind strongly to silica (J. Nanosci. Nanotechnol. 2002, 2, 95).

In their own work with phages, Bassindale and Taylor used silsesquioxanes, silicone analogs that are easier to work with than silicones themselves. The study revealed the amino acid sequences that bind especially well to particular types of silsesquioxanes. A silsesquioxane with polar CF₃ groups, for instance, binds tightly through hydrogen bonds to peptides with serine and threonine side chains (New J. Chem. 2008, 32, 240). As a result of this work, Bassindale told C&EN, “we could rationalize for the first time the way that proteins interact with different silicon compounds.”

Such findings could shed light on the molecular-scale interactions between silicon compounds and proteins in the body, a field of study that is gaining importance because silicones are used in many biomedical devices and personal care products, Bassindale noted. Such information could improve the efficacy and safety of silicone-containing materials.

Meanwhile, Bassindale is studying the proteins that diatoms use to drag silicic acid out of water and into their cells. “Given that the diatoms are so effective at making beautiful patterns, anything we can learn about their mechanisms of action increases our ability to control chemical reactions,” he said. In synthetic polymerizations, he added, “frequently entropy takes over, and you get a huge degree of randomness in the structure of the resulting products. We’re trying to put control into polymerizations so that we get very well-defined structures. We’re learning from nature to design our rather more feeble human efforts at such things.”

Bassindale and Clarson both noted that the field of bioinspired silicon chemistry is benefiting from recent advances in sophisticated techniques such as scanning electron microscopy, genome analysis, and complex genetic engineering. “So the time is right,” Clarson said, “for doing this kind of research on silicon-based, bioinspired materials chemistry.”