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Chapter 4: A Chemist's Toolbox
To many, the word chemist conjures up images of wafts of steam spiraling out of glass laboratory beakers bubbling with scorching, brightly colored liquids. A vivid scene, perhaps, but a more accurate image of the toolbox of today's chemist might include equipment such as computers, microscopes, and large vats of fermenting bacteria.
The chemistry of yesteryear bears only a slight resemblance to today's endeavor. To be sure, the quest for knowledge—to understand how and why molecules combine and recombine in wondrous ways—remains the same. Yet chemistry, biology, and even physics are all fields of study that are becoming much less self-contained. Biologists use physics trickery with lasers to watch molecules move one at a time. Physicists craft unique radioisotopes that exist for only minutes or hours (as opposed to many that stick around for centuries) and that serve as perfect tools for researchers to track molecules in the bodies of animals and people. Biologists and an increasing number of chemists use living organisms—model systems such as bacteria and yeast—to probe the molecular mysteries of health and disease.
Basic biomedical scientists—chemists, biologists, biochemists, and many others—study health-related problems that may not, at the outset, have a clear connection to a specific disease. Drafting hypotheses and testing them, over and over again, is a lot of hard work. Many experiments "fail" in the sense that the scientist's hunch turns out to be wrong. But every failure is in itself a success: a key tidbit of information pointing the scientist's trained eye in a slightly new direction, where he or she can re-test an idea under a slightly different set of conditions. For most scientists, this ongoing process is addictive!
By using models, researchers can perform all this testing and re-testing with systems (such as microorganisms, plants, animals, or computers) that have a set of defined characteristics that don't change much from day to day, or from experiment to experiment. Remarkably, many creatures that are less highly evolved than humans use similar molecules and pathways to carry out their everyday lives. One example is amylase, an enzyme in your saliva that breaks down starch into smaller sugars. In breadmaking, amylase is "food" for yeast, which use this enzyme to help them produce carbon dioxide, the gas that makes dough rise. In many cases, if a particular enzyme or pathway is very similar in diverse organisms, there is a good chance that it's a very important molecule—often indispensable for life.
A Model Cell
A protective barrier called the plasma membrane (see Form and Function) is one of the most important regulatory locales in a cell, serving as the point of entry and exit for a bounty of large and small molecules that are carried to a cell via the bloodstream. To function properly, cells need a constant supply of nutrients, electrolytes, and structural materials. While some of these necessary components are manufactured in-house, many are obtained from outside the cell. The plasma membrane is also an important communications hub, filtering messages sent by other cells and the outside environment. Membranes are studded with proteins called channels and pores that thread their way from the outside to the inside of the cell, or the other way around.
Scientists often study these proteins because they are a key target for drugs. Scientists have devised molecules "smart" enough to self-assemble into miniature cells or cell parts—such as the plasma membrane. And some researchers are creating teeny, laboratory—made reaction vessels.
M. Reza Ghadiri of the Scripps Research Institute has invented a way to get laboratory—made rings and strings of amino acids to assemble themselves into tube-shaped channels and pores. To create the tubes, Ghadiri constructs rings of eight to ten amino acids. By altering the reaction conditions a little, such as fine-tuning the pH of the test-tube liquid, Ghadiri can get the rings to stack on top of each other, forming a tube. Such artificial versions of naturally occurring molecules might be an extraordinarily useful tool for scientists to use to design—and ultimately deliver—drugs to the right spot in the body. By tweaking the dimensions of the amino acid ring-building blocks, for instance, Ghadiri can design channels to transport substances of varying sizes, ranging from three—atom water molecules to considerably larger molecules of sugars, like glucose. The designer molecules could also be used as antibiotic medicines, by virtue of the artificial channels' ability to poke holes in bacterial membranes—making them too leaky to hold their contents.
Bug Labs
THOMAS EISNER
When it comes to using model organisms, chemist Jerrold Meinwald of Cornell University in Ithaca, New York, goes one step further than many scientists: He looks for critters that run their own chemical laboratories. Insects do virtually all of their communicating—with other insects, plants, and their environment in general—by chemical means, exuding a host of different materials, such as toxic venoms and sexual attractants, in the process. Meinwald and long-time Cornell collaborator Thomas Eisner discovered that one variety of insect, the squash beetle, undertakes the constant production of a bewildering array of chemical compounds—some with enormous molecular rings—starting with just a few small chemical building blocks. Even more amazing, these chemical secretions—which appear as "defensive droplets" on tiny hairs on the surface of the non-moving, immature form of the beetle— continually change themselves (by slight shifts of pliable bonds between connected atoms) to create still more molecules. In general, Meinwald suspects, many such insect-made concoctions serve as defense against predators, but most haven't been analyzed yet in the lab.
This Is Organic Chemistry?
JASON KEIPER
A similar approach, being pursued by Fredric Menger of Emory University in Atlanta, is the design and production of "giant vesicles," which are sort of like artificial cells. Menger can make the vesicles—basically large, membrane-encased bubbles—about the same size as a cell, and with custom-made cellular properties. His innovative work is yet another example of how blurred the classic lines have become between chemistry and biology. Before Menger's graduate students can begin to study cell function—traditionally a biological pursuit—they must pass muster as expert synthetic organic chemists. Menger asserts that such a skill—the mastery of using chemistry to make from scratch a multitude of biological mimics—comes in very handy when asking questions about fundamental cellular processes.
For most of his vesicle experiments, Menger drops into water a greasy material (a lipid called DDAB, didodecyldimethylammonium bromide). Almost instantaneously, the oily liquid balls up, pinching itself off into spheres. Though seemingly tiny, by a chemist's standards they are gargantuan—nearly the size of a cell (something easily seen with a standard light microscope). If you're going to use the vesicles to ask cellular questions, says Menger, it is essential to make the spheres approximately that size (so the angle of curvature of the "membrane" most closely resembles that of a cell). But the real advantage of making the lipid balls so big is that Menger can peer into a microscope and watch what they're doing—in real time—as he dumps other chemicals or hormones on them or subjects them to quick shifts in temperature. Menger hopes these unusual chemistry experiments will shed light on very biological problems, such as how tumor cells stick together, how severely disrupted membranes (such as in burns or wounds) heal themselves, and even how sperm fertilize eggs. And, since cell membranes are a common port of entry for a variety of drugs, the work may also lead to better drug delivery schemes.
Who's Hosting This Party?
Eons of natural selection have arranged perfect fits for biological teammates such as receptors and the proteins they recognize: enzymes and their substrates, for instance, or two twists of matching DNA strands. Starting from scratch in the laboratory, however, chemists must work hard to create an appropriate suitor for a known molecule or for a brand-new one that doesn't even exist in nature. A field of research called host-guest chemistry aims to devise ways to study—and even create—interactions between two molecules (the host and the guest), mimicking or blocking some naturally occurring function. Often, the guests are known biological entities. One example might be a receptor for a hormone. In your body, such receptors are linked to other receptors through biochemical pathways, so activating one sets off an unstoppable cascade of reactions causing some physiological response, such as a change in your blood pressure. To interrupt such pathways—those revved up in heart disease or cancer, for instance—scientists are trying to develop synthetic hosts that can attach themselves tightly to their guest molecule and keep them from talking to other proteins (their real-life hosts). Such a strategy has the potential to trip up an entire signaling pathway. Chemists use computer models along with as much detailed knowledge as they can find about molecules' behaviors and shapes to custom-manufacture new molecular mimics.
A Room Without Much View
REPRODUCED WITH PERMISSION FROM JULIUS REBEK, JAVIER SANTAMARIA, AND MICHAEL PIQUE (SCRIPPS RESEARCH INSTITUTE)
Mimicking cell parts to study chemical reactions in biological places is an important area of chemistry research today. In so doing, scientists can easily make ever-so-slight alterations that may not even exist in nature and then test the physiological outcome of such changes. In addition to manufacturing cell-like components, some chemists have gotten into the business of creating tailor-made, sub-microscopic spaces for reactions to take place. Such spaces aren't very roomy, but they do provide a really snug fit for just a couple of molecules of a defined shape. Julius Rebek of the Scripps Research Institute is an expert at making such molecular cages, which in some cases could make it easier for scientists to study biochemical reactions in the lab.
The clever thing is that Rebek spends little time constructing these molecular cages—they do it themselves. Rebek supplies the scaffolding— enjoined rings of atoms that can be prepared from fairly uncomplicated chemical recipes. Then, chemical attractive forces glue the pieces of the cage together, in much the same way as the seams of a softball are stitched together. The cages are ideal reaction centers. Rebek traps other chemicals inside the cages, so these molecules are forced to touch each other and then react. As the reaction proceeds, the products are also trapped inside, and they can be extracted later. The process is a lot like what happens with enzyme-catalyzed reactions, in which the key job of the enzyme is in positioning the reacting chemicals side by side in close proximity. In addition to acting like enzymes, molecular cages—which can be formed repeatedly and reversibly—may find use as biological sensing devices, designed to detect only molecules that have a certain unique shape.
Pass the Chip, Please
COURTESY OF FRED REGNIER
But don't bother with the sour cream and onion dip. Because this isn't the potato kind, but rather a variety of chip that's made from silicon, plastic, and glass...the kind that fits easily between the tips of two fingers...the kind that contains a laboratory full of instruments! It's true—Purdue University chemist Fred Regnier has succeeded in creating such a "laboratory-on-a-chip" that contains expansive networks of miniature tubes and Lilliputian separation columns, all etched onto a dime-sized silicon wafer, via the same techniques used routinely to manufacture computer chips. Voltage applied to alternate ends of the chip channels a liquid sample throughout the tubing, where it then encounters various mini-instruments, such as dust particle—sized reaction vessels and single, hair-thin chromatography columns (chambers that separate components of a mixture). The tiny chip-labs use far less starting material, requiring a millionth the amount of liquid compared to typically sized instruments-a quantity that equates to just a fraction of a drop. And besides conserving workspace, the chips also save time by permitting several experiments to be conducted at once rather than separately. Such properties could someday also make this technology ideal for measuring multiple components of a solution—cholesterol, sugar, and electrolytes in blood, for instance— in a doctor's office.
Other scientists are developing similar chips to be used as drug delivery devices. Such a "pharmacy- on-a-chip," dreamed up by chemist/engineer Robert Langer of the Massachusetts Institute of Technology is still in the development stages. Langer's chip—also a dime—sized wafer of silicon— has a few dozen tiny reservoirs carved into it. The sample wells, as such reservoirs are called, hold a volume smaller than that of a salt grain. The wells are lined with a gold membrane and filled with various solutions (in the testing phase, the researchers poured in fluorescent compounds that would be easily detectable upon release). The reservoirs are then sealed off with glass. Langer and his co-workers formulate a solution that mimics body fluid (in terms of pH and electrolyte content), submerse the chip in this liquid, and rush electrical current across electrodes laced atop the wells. Then, chemistry happens! Gold particles team up with chloride molecules in the solution and form what chemists call a salt: a duo of alternately charged molecules. Result: The gold membrane well covering collapses and the well's contents—say, a drug—spill out into the solution.
Hard-Working Antibodies
ADAPTED WITH PERMISSION FROM TRENDS IN CELL BIOLOGY 1999, 9, M24-M28. COPYRIGHT 1999 ELSEVIER SCIENCE.
The first thing most people think of when they hear the word antibody is "something that floats around in my body and helps me fend off a cold or the flu." In your body, that's indeed the main task for antibodies. Tailor-made by your immune system, antibodies heal you by first identifying—then supervising the destruction of—the microbes that make you sick. Even better, the next time the same molecule or organism comes around, you have the antibodies on hand, just waiting to do away with it. Perhaps the most remarkable thing about antibodies is how versatile they are. Any foreign molecule, really, can touch off the production of a specific antibody that will match it. Tree pollen. Bacterially produced toxins. DNA. Stop for a minute and think how clever the immune system must be to have the capacity to whip up a perfectly targeted antibody for such a wide variety of different molecules with which we come into contact! Chemists have harnessed the extraordinary power of our antibody-producing immune systems to probe fundamental problems in chemistry. Antibodies, for example, have been prepared against molecular complexes that occur in the middle of a chemical reaction. Such complexes—what chemists call transition states are like a molecular snapshot of what happens during the most important part of a chemical reaction. So-called catalytic antibodies can be prepared that actually influence—by speeding up or even freezing in time—an interesting chemical reaction that a scientist wants to study.
The Many Faces of DNA
DNA—it's not just for heredity anymore. Deoxyribonucleic acid, whose primary function is passing on genes from parents to offspring, is at heart a collection of molecules—a chemical. Scientists are capitalizing on some of the unique features of this versatile substance, and in this way, DNA is acquiring a host of new uses. In the future, electronic devices wired with DNA have the potential to work faster and to fit into a tiny fraction of the space today's larger machines require. DNA based mini-machines also promise to be extremely efficient, consuming less power and producing less heat than the equipment currently in routine use. Many properties of DNA—its size, structure, and the rules that govern how it is copied—may make it superior to current materials for a variety of purposes. Here's a sampling:
DNA Electrical Mini-Wires
COURTESY OF JACQUELINE BARTON
Scientists have known for half a century that the DNA in our bodies—and in microbes, plants, and animals—has a special structure (called a double helix) that looks a lot like an upward spiraling staircase. The two halves of the staircase are complementary— they stick to each other much like the opposite strands of VELCRO®. Each banister of the staircase consists of ringed sugar molecules held together by chemical units called phosphate groups, and the steps in between are also flat— they are stacks of ringed molecules called nucleotides. These steps are the letters in the code of life, and make up each gene's alphabetical sequence. Because of the orderly arrangement of the whole thing, strands of DNA have defined electrical properties. Stacks of ringed molecules exhibit orderly displays of electrons and form what scientists have creatively dubbed "pi-ways." (So-called pi-orbitals are electron-occupied shells that hover above atoms and are particularly common surrounding the types of bonds in ringed molecules.) Scientists have found that electrons can literally hop along these routes. Why would anybody care that DNA can conduct electricity? Damage to DNA, suspect scientists such as Jacqueline Barton of the California Institute of Technology, might be caused—or perhaps even fixed—by electron transfer through DNA. More practically speaking, DNA wires could be very useful components of miniature machines.
DNA Mini-Robots
COURTESY OF NADRIAN SEEMAN
Some of the factories of the future will be far smaller than those of today. That's because such manufacturing plants will have on staff tiny robots, not humans, to perform routine and repetitive tasks. Nadrian Seeman of New York University in New York City has used laboratory-prepared strands of DNA to construct the first DNA-based nanomechanical device. (The prefix nano signifies one-billionth, so that an object that is 1 nanometer is one-billionth of a meter long). Seeman started with a synthetic DNA molecule he calls DX DNA, whose shape is very rigid. This property makes it an ideal robotic arm. Seeman used enzymes that link up the building blocks of DNA to fit together three different DNA pieces, each looped off at the end. The junctions of the mini-machine's parts are twists and turns that naturally occur in DNA. Seeman's tiny nanodevice is much too small to see, even with a microscope. So, as a means to measure the distances between the connecting DNA parts, and to be sure that the entire device is constructed as planned, Seeman labels each of the parts with a fluorescent tag and looks for telltale glows that occur when molecules are close together (or that don't occur when they are farther apart).
DNA Biosensors
In the very near future, DNA will find use as a versatile material from which scientists can craft biosensors, devices that detect the presence of something biological—say, a minute amount of DNA in a virus—and then output a signal. DNA biosensors can theoretically be used for medical diagnostics (for instance, detecting a misspelling in a disease-causing gene), forensic science, agriculture, or even environmental clean-up efforts. A significant advantage of DNA-based sensing devices is that no external monitoring is needed. How do they work? DNA biosensors are complicated mini-machines—consisting of sensing elements ("probes" that match up in sequence with the DNA to be detected), microlasers, and a signal generator, all in one. At the heart of DNA biosensor function is the fact that two strands of DNA stick to each other by virtue of chemical attractive forces. On such a sensor, only an exact fit—that is, two strands that match up at every nucleotide position— gives rise to a fluorescent signal (a glow) that is then transmitted to a signal generator. Ideally, the sensor would be a tiny square of a chip that could be immersed in a test fluid—blood, for instance—to pick up traces of disease-causing bacteria or viruses.
DNA Computers
ADAPTED WITH PERMISSION FROM TOMO NARASHIMA
Scientists crafted the first prototype DNA computer in 1994. These tiny PC-wannabees aren't mainstream yet, but they may present technologists and biologists alike with a powerful tool for solving bewilderingly complicated problems. If you think about it, computers made of DNA make sense. Mother Nature picked DNA as the carrier of life for good reason. DNA is both stable and predictable— intact strands of the genetic material have been unearthed in specimens thousands of years old. A set of mathematical rules defines how DNA is transmitted across generations. And to top it off, DNA is self-replicating-it can copy itself! Take a famously hard-to-solve math problem called the Hamiltonian Path Problem, in which given several points—cities, for instance—the goal is to find the shortest trip from the start city to the end city, but the rules state that you can only travel through each intervening city once. Seems easy, but conventional computers have a miserable time finding the answer, because the only way they know how to solve the problem is to try all the possibilities, one by one. In fact, with 100 or so cities, a supercomputer is needed, and with 1,000 cities, no existing computer can solve this problem! DNA-based computers, on the other hand, would find such a problem a breeze, since they could test all the possibilities at once, in parallel. How might they do that? Scientists made "cities" out of synthetic strands of DNA that they made in the lab (much like a gene, each DNA-city had a different combination of the four different nucleotides). Next, the researchers made connector strands. The lengths of these connectors, which linked the end of each DNA-city with the start of another (several cities could be hooked together in this way), were sized according to the distance between the cities. The scientists then mixed everything together, and all of the matching DNA-cities came together in all possible combinations. Voilà! The shortest string of DNA to come out is the answer to the problem.
Question Box: Life on the Edge
No one in my family was a scientist. I just found it fascinating. I liked biology, but I loved the precision that chemistry can offer.
Life on the edge can be most interesting. To chemist Barbara Imperiali of the Massachusetts Institute of Technology, the borderline between chemistry and biology is indeed an intellectually challenging frontier, and one likely to deliver significant gains in human health. Using a chemistry toolkit, Imperiali is one of many of today's chemists seeking to solve biological mysteries. One problem Imperiali has chosen to tackle is how certain sugar-studded proteins get around compartmentalized cells (those—like human cells—that contain a nucleus and other organelles) which, she describes, "have a need for serious traffic control!" Imperiali focuses on cell pathways responsible for tacking sugars onto proteins, and on those proteins themselves. Using innovative design strategies, she's developing artificial proteins from building blocks not used by Mother Nature. She's also pushing the limits of technology in developing exquisitely sensitive biosensors that can detect trace amounts of metals in biological fluids. The Chemistry of Health asked her what kinds of tools she needs to make these sorts of experiments possible.
CH: What are some of the most exciting technologies at the chemistry-biology interface?
Imperiali: There isn't really any single technique that stands out. Technology has been making enormous leaps and bounds in the last two to three decades. We're in a position now to bring all the tools together to investigate complex biological systems in great detail.
CH: What technologies are indispensable for the kind of work you do?
Imperiali: NMR (nuclear magnetic resonance) spectroscopy, for sure. This is a technique for looking at the structure and movement of molecules in a water-based solution. It's one way we test if the proteins we've designed actually do what we think they do. NMR is also very valuable in medicine these days, where it's known as MRI and is being used to image whole people.
Another great method is fluorescence, a technology that's extremely sensitive in detecting vanishingly small amounts of biological samples and can even be used to follow proteins around inside living cells!
Another fantastic technique is mass spectrometry. With "mass spec," as it's called, we can instantly determine the composition of tiny amounts of sample-with other methods, 10,000 times as much material might be required to analyze some samples.
CH: How are chemistry labs different today from 10 or 20 years ago?
Imperiali: When I was a graduate student, a desk, a bench, and a chemical hood was "my territory." Now, students have a desk, but they move around the lab to different stations to do their experiments. There's also a lot more intermixing of labs. That happens a lot—it's absolutely the thing to do when you're trying to tackle interdisciplinary problems.
CH: What got you interested in science to begin with?
Imperiali: No one in my family was a scientist. I just found it fascinating. I liked biology, but I loved the precision that chemistry can offer. I was excited about looking at individual molecules and understanding biological function, molecule by molecule.
CH: Would you advise a young person interested in science to pursue a chemistry research career?
Imperiali: Absolutely, but I also tell them that they should choose something that they really enjoy. Otherwise, it won't be worth it. I try to tell them to learn how to think rather than to learn a specific skill.
Got It?
How do molecular cages resemble enzymes?
Give three examples of how scientists are using miniature biological chips in research today.
List a few examples of model organisms.
How do researchers use host-guest chemistry to study biology?
Discuss why it is important for chemists to work together with biologists, physicists, and other types of scientists.