Learning secrets of world's most common organic compound driving research for biofuels

Preliminary research at Kansas State University may make a difference one day at the gas pump.

Many scientists believe that cellulose, the most common organic compound on earth, has enough energy to be the next source for biofuels -- if a procedure to effectively break it down could be devised. Cellulose is a cell wall component that gives plants their rigidity.

Kathrin Schrick, assistant professor in Kansas State University's Division of Biology, has been awarded nearly $900,000 for the next four years from the National Science Foundation to investigate the role sterols, fat-soluble molecules, play in the cell's production of cellulose.

"If we can understand how it is made and how to break it down into simple sugars, then we can generate energy," Schrick said. "We know that sterols are important in making cellulose, but we are not clear how they work. This grant is funding research that should help us with that."

Cellulose is composed of complex fibers made of sugar. Since its strength functions to keep plant tissue sturdy, it also makes it difficult to break down, Schrick said. It requires harsh pretreatment and expensive enzymes, so Schrick hopes her research will provide an understanding of how cellulose is made, which might give insight on how to break it down more easily.

"Not even the structure of cellulose synthase, the enzyme responsible for activating cellulose machinery, is known. We can model it, we can imagine how it looks but we don't really know, and we know even less about how it functions," Schrick said.

Schrick has two hypotheses for sterols' association with the cellulose machinery. She believes that sterols either help to stabilize the construction of cellulose, or they transfer glucose residues to the machinery to make cellulose.

"We know that the machinery that builds cellulose sits in the plasma membrane. Our hypothesis is that the protein complex that makes cellulose actually needs to directly interact with sterols to function properly," she said.

Her hypotheses came from her discovery of a mutation in a dwarf Arabidopsis plant, a common model species used in scientific research. The mutant plant produces about 50 percent less cellulose than normal plants, causing the plant to be smaller and unable to reach maturity in the wild. Schrick went on to discover that mutations in several enzymes, required for the biosynthesis of sterols, affect the amount of cellulose produced.

"The sterol biosynthesis mutants have shown us that sterols are critical for cellulose synthesis, but we still don't understand why. We are using the latest tools to solve the problem at the molecular level, which will potentially lead to advances in the development of biofuels," she said.

Schrick is collaborating with several scientists nationally and internationally. Among them are Seth DeBolt at the University of Kentucky, a co-principle investigator on the grant, and Vincent Bulone at the Division of Glycosciences in the Royal Institute of Technology in Stockholm, Sweden.

Bulone was one of the first scientists to efficiently synthesize cellulose outside of the cell by gathering all the necessary components needed to build cellulose fibers in a test tube. The level of cellulose synthase activity achieved in Bulone's lab represents the highest proportion of cellulose reported from in vitro synthesis to date, Schrick said.

Provided by Kansas State University (news : web)

Scientists reengineer antibiotic to overcome dangerous antibiotic-resistant bacteria

A team of scientists from The Scripps Research Institute have successfully reengineered an important antibiotic to kill the deadliest antibiotic-resistant bacteria. The compound could one day be used clinically to treat patients with life-threatening and highly resistant bacterial infections.

The results were published in an advanced online issue of the Journal of the American Chemical Society.

"[These results] have true clinical significance and chart a path forward for the development of next generation antibiotics for the treatment of the most serious resistant bacterial infections," said Dale L. Boger, who is Richard and Alice Cramer Professor of Chemistry at The Scripps Research Institute and senior author of the new study. "The result could not be predicted. It really required the preparation of the molecule and the establishment of its properties."

The compound synthesized is an analogue of the well-known commercial antibiotic vancomycin.

The new analogue was prepared in an elegant total synthesis, a momentous achievement from a synthetic chemistry point of view. "In addition to the elegantly designed synthesis," said Jian Xie, postdoctoral fellow in Boger's group and first author on the publication, "I am exceedingly gratified that our results could have the potential to be a great service to mankind."

A Single Atom Changes Everything

Vancomycin is an antibiotic of last resort, which is used only after treatment with other antibiotics has failed. Clinically, it is used to treat patients that are either infected with the virulent methicillin-resistant Staphylococcus aureus (MRSA), individuals on dialysis, or those allergic to beta-lactam antibiotics (penicillin, cephalosporins).

The drug was first used clinically in the 1950s, and the first vancomycin-resistant bacterial strains were isolated in the 1980s.

Vancomycin normally works by grabbing hold of and sequestering the bacterial cell-wall making machinery, a peptidoglycan (carbohydrate and peptide containing molecule). Only Gram-positive bacteria have a cell wall, which is a membrane on the cell's outer surface.

The antibiotic binds so tightly to the peptidoglycan that the bacteria can no longer use the machinery to make their cell wall and thus die.

Unfortunately, bacteria have found a way to alter the peptidoglycan in such a way that the antibiotic can no longer grab hold. Think of it as trying to hold a ball without any fingers. Biochemically the bacteria express a mutant form of the peptidoglycan in which properties of a key atom used in the recognition process are changed. This simply means where there once was something attractive there is now something repulsive. Chemically, the bacteria replace an amide (carbonyl, RC=O linked to an amine) with an ester (a carbonyl, RC=O linked to an oxygen, O).

This one atom change changes the entire game and renders vancomycin ineffective. Until now.

Reengineering Vancomycin

Like magnets, molecular interactions can be attractive (oppositely charged) or repulsive (identically charged). What chemists in the Boger lab have done is made this key interaction no longer repulsive, but attractive.

So now the new vancomycin analogue can grab hold of the mutant peptidoglycan, and again prevent the bacteria from making the cell wall and killing the resistant bacteria. But what is so remarkable about the design is that the redesigned antibiotic maintains its ability to bind the wild type peptidoglycan as well.

Changing the properties of a key amide at the core of the natural products structure required a new synthetic strategy that only the most talented chemists could achieve in the lab. The preparation of the entire structure took a great deal of time and a fresh approach.

The new compound has an amidine (an iminium, RC=NH+ linked to a nitrogen, N) instead of an amide at a key position buried in the interior of the natural product. However, to install such a functional group, the chemical properties of the amide carbonyl were not useful, as the natural product has seven of them.

Instead, the group relied on the chemical properties of sulfur (S), oxygen's downstairs neighbor in the periodic table, to install the desired nitrogen. To do this, a second analogue was prepared in which the key amide was chemically altered to a thioamide. "The thioamide allowed us to make any modification at the residue 4 amide that we would like to make, such as the amidine, but we could also make the methylene analogue," said Boger citing work published in another paper (B. Crowley and D. L. Boger, J. Am. Chem. Soc. 128: 2885-2892). "And there are other modifications that we are making at the present time that we haven't disclosed. We are just getting to that work."

The most fundamental finding in the synthesis was that the installation of the amidine could be done in the last step, as a single-step conversion, on the fully unprotected thioamide analogue. Providing an elegant and novel approach to the analogue, which contrasts other published multistep procedures. This chemical behavior was, as Boger said, "an astonishing result as there are no protecting groups and it is a single step reaction… in the end it was the simplest and most straightforward way to prepare the amidine."

Although it is still at its early stages and there is much work ahead. Currently, the only route known to make the new antibiotic is the one published by Boger and his co-workers, which presently provides laboratory amounts of the compound. So Professor Boger now looks forward and will continue to investigate the "host of alternative approaches" for the preparation of the molecule "such as reengineered organisms to produce the material or semi-synthetic approaches to the analogue. That is going to be part of the next stage of the work."

More information: "A Redesigned Vancomycin Engineered for Dual d-Ala-d-Ala and d-Ala-d-Lac Binding Exhibits Potent Antimicrobial Activity Against Vancomycin-Resistant Bacteria," http://pubs.acs.or … 21/ja207142h

Provided by The Scripps Research Institute (news : web)

Molecular scientists develop color-changing stress sensor

It is helpful — even life-saving — to have a warning sign before a structural system fails, but, when the system s only a few nanometers in size, having a sign that's easy to read is a challenge. Now, thanks to a clever bit of molecular design by University of Pennsylvania and Duke University bioengineers and chemists, such warning can come in the form of a simple color change.

The study was conducted by professor Daniel Hammer and graduate students Neha Kamat and Laurel Moses of the Department of Bioengineering in Penn's School of Engineering and Applied Science. They collaborated with associate professor Ivan Dmochowski and graduate student Zhengzheng Liao of the Department of Chemistry in Penn's School of Arts and Sciences, as well as professor Michael Therien and graduate student Jeff Rawson of Duke.

Their work was published in the journal Proceedings of the National Academy of Sciences.

The researchers' work involves two molecules: porphyrins, a class of naturally occurring pigments, and polymersomes, artificially engineered capsules that can carry a molecular payload in their hollow interiors. In this case, Kamat and Liao hypothesized that polymersomes could be used as stress sensors if their membranes were embedded with a certain type of light-emitting porphyrins.

The Penn researchers collaborated with the Therien lab, where the porphyrins were originally developed, to design polymersomes that were studded with the light-emitting molecules. When light is shined on these labeled polymersomes, the porphyrins absorb the light and then release it at a specific wavelength, or color. The Therien lab's porphyrins play a critical role in using the polymersomes as stress sensors, because their configuration and concentration controls the release of light.

"When you package these porphyrins in a confined environment, such as a polymersome membrane, you can modulate the light emission from the molecules," Hammer said. "If you put a stress on the confined environment, you change the porphyrin's configuration, and, because their optical release is tied to their configuration, you can use the optical release as a direct measure of the stress in the environment."

For example, the labeled polymersomes could be injected into the blood stream and serve as a proxy for neighboring red blood cells. As both the cells and polymersomes travel through an arterial blockage, for example, scientists would be able to better understand what happens to the blood cell membranes by making inferences from the stress label measurements.

The researchers calibrated the polymersomes by subjecting them to several kinds of controlled stresses — tension and heat, among others — and measuring their color changes. The changes are gradations of the near infrared spectrum, so measurements must be made by computers, rather than the naked eye. Rapidly advancing body-scanning technology, which uses light rather than magnetism or radiation, is well suited to this approach.

Other advances in medicine could benefit, as well. As cutting-edge pharmaceutical approaches already use similar molecular technology, the researchers' porphyrin labeling system could be integrated into medicine-carrying polymersomes.

"These kinds of tools could be used to monitor drug delivery, for example," Kamat said. "If we have a way to see how stressed the container is over time, we know how much of the drug has come out."

And, though the researchers chose the engineered polymersomes due to the wide range of stress they can endure, the same stress-labeling technique could soon be applied directly to naturally occurring tissues.

"One future application for this is to use dyes like these porphyrins but include them directly in a cellular membranes," Kamat said. "No one has taken a look at the intrinsic stress inside a membrane so these molecules would be perfect for the job."

Provided by University of Pennsylvania (news : web)

Cooling down global warming

Carbon capture has long been identified as a critical technology needed to prevent global warming, but efficient and economical ways to do it have been hard to find.

A new process to capture and convert carbon dioxide, discovered by a chemist in the College of Liberal Arts and Sciences, has just been awarded a patent. It uses cheap catalysts, electricity, and heat to convert CO2 and water into useful chemicals or fuels.

Steve Suib, Board of Trustees Distinguished Professor of Chemistry and the 2011 Connecticut Medal of Science winner, found a way to use metal oxides, such as manganese and zinc oxides, as catalysts in a conversion process that also uses heat and electricity.

The newly patented method can be run as a continuous, rather than a batch, process, to yield large amounts of a product. What is made depends on the catalyst used. Potential products include paraformaldehyde, used in disinfectants and in chemical processes in industry, or ethylene, the largest bulk chemical in the world, used in products such as milk bottles.

The process could also be used to generate hydrogen and a variety of hydrocarbons.

“You can make a significant amount of material at high conversions over a long period of time,” Suib says.

CO2 and H2O are abundant but hard to activate in a chemical process, Suib points out: “It is difficult because both of those molecules are relatively stable.”

The process was developed in Suib’s lab, and Ph.D. candidate Boxun Hu, soon to graduate, was heavily involved in the work, which will be published, Suib says.

The patent was granted in late June. It improves on an earlier process that Suib patented in 2009, which used a more expensive catalyst—platinum—and produced little product. The earlier process was the subject of a 2010 paper in the journal Applied Catalysis Part A.

Both patents are jointly held by UConn, through the Office of Technology Commercialization, and Catelectric Advanced Electrocatalysis, a company that began as part of UConn’s Technology Incubation Program. Catelectric specializes in improved catalysis processes.

Suib has received research grants from Catelectric to work on the process. He has also had extensive Department of Energy grant funding, and is the lead researcher on a $1.8 million DoE grant to make catalysts for a pilot biomass conversion plant that will be built at UConn.

Suib’s latest patent—he has about 50—is a combined process and composition of matter patent. While process patents can be hard to enforce, Suib says—it is difficult to tell whether a possible infringer is varying or using the same process—the composition of matter component of the patent makes infringement easier to pinpoint.

If the newly patented process is used, UConn would get royalties from the use.

The next step would be to take what is a rather crude, laboratory process and scale it up to the pilot plant stage for testing, and eventually to the manufacturing stage, Suib says.

The costs of heat and electricity used in the process would then be considerations. Electricity costs are high in Connecticut compared with other states, Suib notes. The technology of capturing heat, and even excess CO2, from existing manufacturing plants might be considered, allowing factories to use their excess heat in a process that would result in making new chemicals.

The “Buck Rogers” idea now being floated is to capture heat escaping from nuclear power plants and use that as a heat source, he says.

The effort to capture and convert CO2 into useful products, mitigating global warming in the process, is one that has captured his interest: “A lot of people say, ‘you just can’t do this’—but those are the problems that are interesting.”

Provided by University of Connecticut (news : web)

Sweet insight: New discovery could speed drug development

The surface of cells and many biologically active molecules are studded with sugar structures that are not used to store energy, but rather are involved in communication, immunity and inflammation. In a similar manner, sugars attached to drugs can enhance, change or neutralize their effects, says Jon Thorson, a professor of pharmaceutical sciences at the University of Wisconsin-Madison School of Pharmacy.

Thorson, an expert in the attachment and function of these sugars, says that understanding and controlling them has major potential for improving drugs, but that researchers have been stymied because many novel sugars are difficult to create and manipulate. "The chemistry of these sugars is difficult, so we have been working on methods to make it more user friendly," he says.

Now, in a study published online in Nature Chemical Biology on Aug. 21, Thorson, graduate student Richard Gantt and postdoctoral fellow Pauline Peltier-Pain have described a simple process to separate the sugars from a carrier molecule, then attach them to a drug or other chemical. The process also causes a color change only among those molecules that have accepted the sugar. The change in color should support a screening system that would easily select out transformed molecules for further testing. "One can put 1,000 drug varieties on a plate and tell by color how many of them have received the added sugar," Thorson says.

Attached sugars play a key role in pharmacy, says Thorson. Not only can they change the solubility of a compound, but "there are transporters in the body that specifically recognize certain sugars, and pharmaceutical companies have taken advantage of this to direct molecules toward specific tissue or cell types. If we can build a toolbox that allows us to make these molecules on demand, we can ask, 'What will sugar A do when it's attached to drug B?'"

And although the new study was focused more on an improved technique rather than the alteration of drugs, Thorson adds that it does describe the production of some "really interesting sugar-appended drugs: anti-virals, antibiotics, anti-cancer and anti-inflammatory drugs. Follow-up studies are currently under way to explore the potential of these analogs."

The new molecules included 11 variants of vancomycin, a powerful antibiotic, each distinguished by the nature and number of attached sugars.

The essence of the new process is its starting point: a molecule that changes the energy dynamics of the sugar-attachment reaction, Thorson says. "This is one of the first systematic studies of the equilibrium of the reaction, and it shows we can drive it forward or in reverse, depending on the molecule that we start with."

In a single test tube, the new technique is able to detach the sugar from its carrier and reattach it to the biological target molecule, Thorson says. "Sugars are involved a vast range of biology, but there are still many aspects that are not well understood about the impact of attaching and removing sugars, partly because of the difficulty of analyzing and accessing these species."

Making variants of potential and existing drugs is a standard practice for drug-makers, and a recently published study by Peltier-Pain and Thorson revealed that attaching a certain sugar to the anti-coagulant Warfarin destroys its anti-clotting ability. The transformed molecule, however, "suddenly becomes quite cytotoxic — it kills cells," he says. "We don't know the mechanism, but there is some interest in using it to fight cancer because it seems to act specifically on certain cells."

Sugars are also attached to proteins, cell surfaces and many other locations in biology, Thorson says. "By simplifying the attachment, we are improving the pharmacologist's toolbox. This study provides access to new reagents and offers a very convenient screening for new catalysts and/or new drugs, and for other things we haven't yet thought of. We believe this is going to open up a lot of doors."

Provided by University of Wisconsin-Madison (news : web)