A potentially revolutionary new technique for chemical structure determination

I am a big believer in science as a tool-driven rather than an idea-driven revolution, and nowhere do you see this view of science exemplified better than in the development of instrumental techniques in chemistry - most notably NMR and x-ray diffraction. NMR and crystallography were not just better ways to see molecules, but in their scope, their throughput, their cost and their speed, they opened up whole new fields of science like genomics and nanomaterials up to investigation that their developers couldn't even have imagined.

So it is with some interest that I saw a paper from the Nelson, Gonen and Rodriguez labs at UCLA and the Stoltz lab at Caltech that describes a new way to rapidly determine the structures of organic molecules. And I have to say: very few papers in recent times have made me sit up and do a double take, but this one did. At one point in the paper the authors say they were "astounded" by the ease of the technique, and I don't think that word is out of place here at all.

Until now, crystallography has been the gold standard for all kinds of structure determination; it gives you as direct a view of molecules at the atomic level as possible. But the very name "crystallography" implies that you need to get your sample into a crystalline state, and as any chemist who has worked with a headache-inducing list of assorted powders, gels, oils and tars knows, being crystalline is not the natural state of most molecules. In most cases your samples are thus simply not in a convenient form for crystallography.

That's why NMR has been the primary technique for routine organic structure determination. But NMR is still relatively slow and depends on having a machine that's expensive and sometimes breaks down. You also cannot do NMR on a benchtop, and getting the sample in a pure enough condition in the right solvent is also key to good structure determination. Then there are all the problems attendant with shimming, water suppression and other artifacts that NMR presents, although sophisticated software can now take care of most of these. Nonetheless, as powerful as NMR is and will continue to be, it's not exactly a rapid, plug-and-play system.

That's why this recent paper is so promising. It describes a crystallographic technique that uses cryo-EM and micro-electron diffraction (micro ED) for efficiently finding out the structure of organic molecules. Electron diffraction itself is an old technique, pioneered for instance by Linus Pauling in the 1930s, but this is not any old ED, it's micro ED. Cryo-EM already won the Nobel Prize two years ago for determining the structures of complex proteins, but it has never been routinely applied to small molecule structure determination. This new technique could change that landscape in a jiffy. And I mean in a jiffy - the examples they have shown take a few minutes each. The first molecule - progesterone - went from powder to pattern in less than 30 mins, which is quite stunning. And the resolution was 1 Å, and you can't ask for more. Up to twelve samples were investigated in a single experiment.

But what really made me sit up was the variety of starting points that could be investigated. From amorphous powders to samples straight out of flash chromatography to mixtures of compounds, the method made quick work out of everything. As mentioned above, amorphous powders and mixtures are the rule rather than the exception in standard organic synthesis, so one can see this technique being applied to almost every chemical purification and synthetic manipulation done in routine synthesis or structure determination. For me the most amazing application however was the determination of a mixture of four different molecules: no other technique in organic chemistry which I know can do mixtures in a few minutes with such high resolution with such little material.

There are undoubtedly still limitations. For one thing, cryo-electron microscopes are still not cheap, and while sample preparation is getting better, it's also not instantaneous in every case. I also noticed that most of the compounds this study looked at were rather rigid, with lots of fused and other rings; floppy molecules will likely cause some trouble, and although thiostrepton is an impressive-looking molecule, it would be interesting to see how this works for beasts like rapamycin or oligopeptides. In general, as with other promising techniques, we will have to see what the domain of applicability of this method is. 

Nonetheless, this is the kind of technique that promises to take a scientific field in very novel directions. It could accelerate the everyday practice of organic chemistry in multiple fields - natural products, chemical biology, materials science - many fold; and at some point, quantity has a quality of its own. It could allow the investigation of the vast majority of compounds that cannot be easily coaxed into a crystal or an NMR tube. And it could perhaps even allow us to study conformational behavior of floppy compounds, which from first-hand experience I know is pretty hard to do.

If validated, this technique also exemplifies something I have talked about before, which is how scientific tools and discoveries build on each other; in other words, how scientific convergence is a key driving force in science. When cryogenics was invented, nobody foresaw cryo-electron microscopy, and when cryo-EM was invented, nobody foresaw its application to routine organic synthesis. And so it goes on, science and technology piggybacking in ever-expanding spirals.

Science as a messy human endeavor: The origin of the Woodward-Hoffmann rules

A 1973 slide from Roald Hoffmann displaying the 'Woodward
Challenge' - four mysterious reactions which spurred the
Woodward-Hoffmann rules

There is a remarkable and unique article written by my friend and noted historian of chemistry Jeff Seeman that has just come out in the Journal of Organic Chemistry. The paper deals with seven pivotal months in 1964 when Robert Burns Woodward and Roald Hoffmann worked out the basic structure of what we call the Woodward-Hoffmann rules. 

Organic chemists need no introduction to these seminal rules, but for non-chemists it might suffice to say that they opened the door to an entire world of key chemical reactions - both in nature and in the chemist's test tube - whose essential details had hitherto stayed mysterious. These details include the probability of such reactions occurring in the first place and the stereochemistry (geometric disposition) of their molecular constituents. The rules were probably the first significant meld between theoretical and organic chemistry - ten commandments carried down from a mountain by Woodward and Hoffmann, pointing to the discovery of the promised land.The recognition of their importance was relatively quick; In 1980 Hoffmann shared a Nobel Prize for his contributions, and Woodward would have shared it too (it would have been his second) had he not suddenly passed away in 1979.

The first paper on these rules was submitted in November, 1964 and it came out in January, 1965. Jeff's piece essentially traces the conception of the rules in the previous six months or so. The article is very valuable for the light it sheds not just on the human aspect of scientific discovery but on its meandering, haphazard nature. It is one of the best testaments to science as a process of fits and starts that I have recently seen. Even from a strictly historical perspective Jeff's article is wholly unique. He had unprecedented access to Hoffmann in the form of daylong interviews at Cornell as well as unfettered access to Hoffmann's office. He has also interviewed many other important historical figures such as Andrew Streitweiser, George Whitesides and Jack Roberts who were working in physical organic chemistry at the time: insightful and amusing quotes from all these people (such as Whitesides's reference to the demise of a computer at MIT implying that he would now have to perform calculations using an abacus or his toes) litter the account. And there are copious and fascinating images of scores of notebook pages from Hoffmann's research as well as amusing and interesting letters to editors, lists of publications, scribblings in margins and other correspondence between friends and colleagues. Anyone who knows Jeff and has worked with him will be nodding their heads when they see how thorough the job here is.

The story begins when Woodward was already the world's most acclaimed organic chemist and Hoffmann was an upcoming theoretical chemistry postdoc at Harvard. Then as now, Hoffmann was the quintessential fox whose interests knew no bounds and who was eager to apply theoretical knowledge to almost any problem in chemistry that suited his interests. By then he had already developed Extended Hückel Theory (EHT), a method for calculating energies and orbitals of molecules which was the poster child for a model: imprecise, inaccurate, semiquantitative and yet pitched at the right level so that it could explain a variety of facts in chemistry. Woodward had already been interested in theory for a while and had worked on some theoretical constructs like the octant rule. It was a marriage made in heaven.

The most striking thing that emerges from Jeff's exhaustive and meticulous work is how relatively laid back Woodward and Hoffmann's research was in a sense. Hoffmann became aware of what was called the 'Woodward challenge' early in 1964 during an important meeting; this challenge involved the then mysterious stereochemical disposition during some well-known four and six electron reactions, reactions whose jargon ("electrocyclization", conrotatory") has now turned into household banter for organic chemists. The conventional story would then have had both Woodward and Hoffmann burning the midnight oil and persisting doggedly for the next few months until they cracked the puzzle like warriors on a quest. This was far from the case. Both pursued other interests, often ended up traveling and only occasionally touching base. Why they did this is unclear, but then it's no more unclear than why humans do anything else for that matter. Once they realized that could crack the puzzle however they kicked the door open. The paper that emerged in early 1965 was so long and comprehensive that they worried about its suitability for JACS in a cover letter to the editor.

Jeff's story also touches on a tantalizing conundrum whose solution many readers would have loved to know - E. J. Corey's potential role or the lack thereof in the conception of the rules, a role Corey unambiguously acknowledged in his 2004 Priestley Medal address, setting off a firestorm. Unfortunately Corey declined to talk to Jeff about this article (although he does dispute the timing of Woodward and Hoffmann's first meeting). His side of the story may never be known.

There is a lot of good stuff in the 45-page article that is worth reading about which I can only mention in passing here. Many of the actual mechanistic and technical details would be of interest only to organic chemists. But the more general message should not be lost upon more general readers: science is a messy, almost always unheroic, haphazard process. In addition, its real story is often warped by malleable memory, shifting egos, mundane oversights and blind alleys. For a long time science was described in bestselling books and newspaper articles as a determined, heroic march to the truth. These days there is an increasing number of books aimed at uncovering science's massive storehouses of failure and ignorance. But there is a third view of science - that of a journey to the truth which is more mundane, more complex, perpetually puzzling because of its mystery and perpetually comforting because of its human nature.

In this case even Jeff's exhaustive research leaves us with kaleidoscopic questions, questions that may likely remain unanswered. These pertain to Woodward and Hoffmann's occasional indifference to what was clearly a pivotal piece of research, to Corey's claim about the reactions, to the potential cross-fertilization between whatever else Woodward and Hoffmann were doing during this time and the project in question, and to the number of insights they might have imbibed from the community at large. Jeff conjectures answers to these questions, but even his probing mind provides no comforting conclusions, probably because there are none. The quote from Roald Hoffmann with which the piece ends captures the humanity quite well.

"Life is messy. Science is not all straight logic. And all scientists are not always logical. We're just scrabbler for knowledge and understanding."

Here's to the messy scrabblers.

Carl Djerassi (1923-2015): Chemist, writer, polymath, cultural icon

R B Woodward, Vladimir Prelog and Carl Djerassi on the beach at a conference in Riga, Latvia. Photo signed by Djerassi and generously gifted to the author by Prof. Jeffrey Seeman, University of Richmond.

Very few scientists of the 20th century have had as much of both a scientific as well as a cultural impact on the world as Carl Djerassi. It is a measure of how many things Djerassi excelled at that even his amazing purely scientific career seems like a distant horizon. While most scientists are quite happy to become world-renowned in their narrow subfield of science, Djerassi polished off multiple fields of chemistry and then reinvented himself as a notable playwright and writer. And he did all this after significantly contributing to one of the greatest social revolutions of the 20th century, if not of all time - safe, affordable and easily accessible contraception for women. Very few technical inventions in history contributed to giving women control over their lives the way the pill did. Scientists are usually not cultural icons, and the wrong people often are, but Djerassi definitely deserves to be one.

On his 90th birthday, my friend, the noted historian of chemistry Jeff Seeman summarized a few of Djerassi's astonishing contributions and honors:

He has published more than 1,200 scientific papers, 350 in the Journal of the American Chemical Society.

He is a chemical “father of the Pill.”

He has published three autobiographies, one memoir, five novels, two nonfiction books, 11 plays, two collections of poetry, three collections of essays and short stories, and one art book.

He has made significant contributions, in cash and in kind, to charitable causes and artistic endeavors.

He has received the National Medal of Science and the National Medal of Technology.

He has been awarded the Priestley Medal, the first Wolf Prize in Chemistry, and many other awards, as well as 32 honorary doctorates.

He has been recognized by Austria with a postage stamp issued in his honor.

He is in constant demand as a lecturer around the world.

He will turn 90 on Oct. 29.

Yet, Carl Djerassi has never been fully satisfied.

I first encountered Djerassi's work when as an undergraduate I studied the octant rule that he, R B Woodward, Bill Moffit and others pioneered to study the configuration of steroids. Nobody really uses it anymore since more sophisticated methods like NMR spectroscopy have superseded it, but as I came to know more about Djerassi's contributions, it amazed me that the same man who published the octant rule also pioneered the use of mass spectrometry in natural products chemistry, unraveled the biosynthesis of several key steroids and alkaloids and also published some of the first papers on the applications of artificial intelligence in organic chemistry. His 1200 papers span the breadth of the discipline, and among younger chemists only Clark Still comes to my mind as someone who had the same diversity of contributions.

Djerassi's autobiography ("Steroids Made It Possible") which is edited by Jeff is wonderful and a real treat. In it he talks about a variety of scientific and private topics, ranging from the letter to Eleanor Roosevelt that got him a college scholarship to his experiments with mescaline to his accidental grin in the photo showing Richard Nixon awarding him the National Medal of Science (he hated Nixon and in fact was on Nixon's silly "enemies" list, but Nixon said something funny right at the moment the photo was taken). The book also contains painful ruminations such as the one about his daughter's struggle with addiction and her suicide. Djerassi was nothing but upfront about his life, both in this memoir as well as his subsequent two books, the latest of which just came out and which I haven't read yet. 

His writings are also studded with sketches of great chemists like R B Woodward, Gilbert Stork, Bill Johnson and E J Corey, most of whom Djerassi counted among his close friends and colleagues. In some sense, a journey through his science is a journey through the development of postwar organic chemistry in its golden age. And speaking of R B Woodward, Djerassi managed to become the highest cited chemist of the 60s - at a time when his friend had already staked his claim as the the greatest organic chemist of the century and continued to publish seminal papers. That's no small feat.

Djerassi was of course also a noted playwright, reinventing himself during the second half of his life and crafting the play "Oxygen" with his fellow chemist Roald Hoffmann for instance. He was a great example of someone who bridged C P Snow's two cultures, inculcating and displaying a wide storehouse of knowledge ranging from philosophy and art to literature and science. His shares in Syntex Corporation where he researched steroids also made him a wealthy man and allowed him to do this. It also enabled him to retire early, amass an enviable private art collection and spend part of every year in London and other parts of Europe writing and giving talks. His fiction is well worth reading, and his characters are as interesting and honest as the science he pioneered.

Most laymen will of course always know Djerassi as one of the fathers of the contraceptive pill, although those of us who are aware of his chemical contributions appreciate that it was but one part of his prolific career. As many of us also know, Djerassi was a favorite on Nobel Prize lists for a long time, and as they did with many other scientists, the Nobel Prize committee did themselves a disservice by not awarding him one. But Djerassi's career more than that of most others indicates the irrelevance of prizes, as honorable as they may be. In that sense Djerassi is like Gandhi. His work was beyond prizes, and considering the social revolution that The Pill brought about, he will always stand not only as one of the scientific greats of the 20th century but as one of its most important human beings. RIP.

Chemical and Engineering News celebrates 90 years: How chemistry has come a long way

Chemistry is - in the true sense - the central science, reaching inside every aspect of our lives (Image: Marquette University)

Chemical and Engineering News (C&EN) is celebrating 90 years of its existence this year, and I can only imagine how perplexed and awestruck its editors from 1923 would have been had they witnessed the state of pure and applied chemistry in 2013. I still remember devouring the articles published in the magazine during its 75th anniversary, and this anniversary also offers some tasty perspectives on a diverse smattering of topics; catalysis, structural biology and computational chemistry to name a few. 

There's an article in the magazine documenting how the single-most important concept in chemistry - that of the chemical bond - has undergone a transformation; from fuzzy, to rigorously defined, to fuzzy again (although in a very different sense).

Nobel Laureate Roald Hoffmann had something characteristically insightful to say about The Bond:

"My advice is this: Push the concept to its limits. Be aware of the different experimental and theoretical measures out there. Accept that at the limits a bond will be a bond by some criteria, maybe not others. Respect chemical tradition, relax, and instead of wringing your hands about how terrible it is that this concept cannot be unambiguously defined, have fun with the fuzzy richness of the idea.”

In a bigger sense the change in chemistry during these 90 years has been no less than astounding. In 1923 the chemical industry already made up the foundations of a great deal of daily life, but there was little understanding of how to use the concepts and products of chemical science in a rational manner. Since 1923 our knowledge of both the most important aspect of pure chemistry (the chemical bond) and of applied chemistry (synthesis) has grown beyond the wildest dreams of chemistry's founders.

If we had to pinpoint two developments in chemistry during these 90 years that would truly be described as "paradigm shifts", they would be the theoretical understanding of bonding and the revolution in instrumental analysis. As I and others have argued before, chemistry unlike physics is more "Galisonian" than "Kuhnian", relying as much on new instrumental techniques as on conceptual leaps for its signal achievements.

The two most important experimental advances in chemistry - x-ray diffraction and nuclear magnetic resonance - both came from physics, but it was chemists who honed these concepts into a routine laboratory tool for the structure determination of a staggeringly diverse array of substances, from table salt to theribosome. The impact of these two developments on chemistry, biology, medicine and materials science cannot be underestimated; they cut down the painstaking task of molecular structure determination from months to hours, they allowed us to find out the nature of novel drugs, plastics and textiles and they are now used by every graduate student every single day to probe the structure of matter and synthesize new forms of it. Other developments like infrared spectroscopy, electron diffraction, atomic force microscopy and single molecule spectroscopy are taking chemistry in novel directions.

The most important theoretical development in chemistry also derived from physics, but its progress against demonstrates chemists' central role in acting as mediators between concept and application. It also serves to make a key point about reductionism and the drawbacks of trying to reduce chemistry to physics. The chemical bond is an abstract concept going back to "affinities" between atoms (which when illustrated were replete with hooks and eyes). But it was in 1923 that the great American chemist G. N. Lewis propounded the idea in terms of atoms sharing electrons. This was a revolutionary brainwave and illuminated the way for Linus Pauling, John Slater, Robert Mulliken, John Pople and others to use the newly developed machinery of quantum mechanics to fashion the qualitative principle into an accurate, quantitative tool which  - with the development of modern computing - now allows chemists to routinely calculate and predict important properties for any number of chemical substances.

Yet the ramifications of the chemical bond tempt and beguile physicists and constantly escape from their grasp when they try to define them too accurately. The above quote by Roald Hoffmann puts the problem in perspective; quintessentially chemical ideas like aromaticity, the hydrophobic effect, steric effects and polarity "fray at the edges" (in Hoffmann's words) when you try to push them to their limits and try to define them in terms of subatomic physics. Chemistry is a great example of an emergent discipline. It is derived from physics and yet independent of it, relying on fundamental definitions at its own level when progressing.

The chemical bond and other theoretical aspects of chemistry have enabled the rise of the one activity pursued by chemists of which society is an unsurpassed beneficiary - the science, art and commerce of synthesis. Every single molecule that bathes, clothes, feeds, warms, transports and heals us has been either derived from nature using chemical techniques or has been synthetically made in a chemical laboratory. The social impact of these substances is hard to underestimate; even a sampling of a few such as the contraceptive pill, antibiotics or nylon attests to the awesome power of chemistry to completely transform our lives.

In 1923 synthesis was a haphazard process and there was virtually no understanding of how we could do it rationally. All of this changed in the 1950s and 60s when a group of pioneering scientists led by the legendary organic chemist Robert Burns Woodward revolutionized the process and honed synthesis into a precisely rational science which took advantage of the course of chemical reactions, the alignment of orbitals, the development of new chemical reagents and the three-dimensional shape of molecules. Many Nobel Prizes were handed out for these groundbreaking discoveries, but none surpassed the sheer impact that synthesis will continue to have on our way of life.

As is inevitably the case for our embrace of science and technology, with progress also come problems, and chemists have had to deal with their share of issues like environmental pollution, drug side effects and the public perception of chemistry. Suffice it to say that most chemists are well aware of these and are working hard to address them. They recognize that with knowledge comes responsibility, and the responsibility they bear to mitigate the ills of the wrongful application of their science transcends their narrow professional interests and encompasses their duties as citizens.

In the new century chemistry continues to build upon its past and chemists continue to push its boundaries. Another change which the editors of C&EN would not have foreseen in 1923 is the complete integration of chemistry into other disciplines like biology, medicine and engineering and its coming into its own as the true "central science". Today chemistry deeply reaches into every single aspect of our lives. The cardinal problems facing civilization - clean and abundant food and water, healthcare, national security, overpopulation, poverty, climate change and energy - cannot be solved without a knowledge of chemistry. Simply put, a world without chemistry would be a world which we cannot imagine, and we should all welcome and integrate the growth of chemical science into our material and moral worldview.

First published on the Scientific American Blog Network.

Modeling magic methyls

Shamans have their magic mushrooms, we medicinal chemists have our magic methyls. The 'magic methyl effect' refers to the sometimes large and unexpected change in drug potency resulting from the addition of a single methyl group to a molecule (for laymen, a methyl group is a single carbon with three hydrogens, and you don't expect spectacular effects from the addition of such a small modification to a drug). This is part of a broader paradigm in chemistry in which small changes in molecular structure can bring about large changes in properties, especially biological activity.

In this study, researchers from Bill Jorgensen's group at Yale ask what exactly it is that a methyl group can do to a biologically active molecule. They look at more than 2000 cases of purported activity changes induced by methyl groups from two leading medicinal chemistry journals, and their work highlights some unexpected effects of methyls. The techniques used are free-energy perturbation, Monte-Carlo and molecular dynamics simulations to compare methylated and non-methylated versions of published inhibitors in an effort to gain insight into the factors dictating potency.

Firstly, they find that for all their reputation, methyls mostly confer a modest increase in potency. The greatest increase is about 3 kcal/mol in free energy, which considering the exponential relationship between free energy and binding constant, is actually quite substantial. But this happens in a negligible minority of cases; as they find, a 10 fold boost in potency with a methyl is seen in only 8% of the cases, while a 100 fold difference is seen in only 0.4%.

So what does a methyl do? For starters, a methyl is simply a nice, small, lipophilic group so you would expect it to give you some advantage simply by snugly fitting in in an otherwise unoccupied binding pocket. But the real advantage of a methyl is thought to come from kicking out 'unhappy' water molecules; often a small protein pocket is occupied by a highly constrained water molecule that is desperate to join its free brethren in the bulk. A methyl group is usually only too happy to oblige and kick the water out. Now, as the crystallographer Jack Dunitz demonstrated more than a decade back, the maximum free energy gain you could estimate from displacing a water molecule is about 2 kcal/mol. Considered in this light, you would expect a gain of at least that much from a hydrogen to methyl change, but the very rare 3 kcal/mol cases seen seem to call this belief into question. Clearly the common wisdom about methyls displacing waters is not telling us the entire story.

As the authors demonstrate, the common wisdom may indeed point to uncommon cases. They look at five cases where methyls give the greatest potency boost, and in no case do they find evidence for displacement of a water molecule. So where's the potency gain coming from? It turns out that it may be coming from a common but often underappreciated factor; conformational reorganization. When a ligand binds to a protein, it exists in several - often hundreds - of conformations in solution. How tightly the protein can bind the ligand depends on how much energy the protein can expend to twist and turn these conformations into the single bound conformation. You would expect that the more similar a molecule's unbound conformation in solution is to its protein-bound conformation, the easier it would be for the protein to latch on to it.

And indeed, that's what they find. Most of the cases they look at concern potency gains coming from putting methyls at the ortho position of a biaryl ring. Organic chemists are quite familiar with the steric, planarity-disrupting effects of ortho substituents on biaryl rings; in fact it's a tried and tested strategy to improve solubility by disrupting crystal packing effects. It turns out that the bound structures of the molecules present a twisted, non-planar conformation. In the absence of methyls, the rings would prefer to stay almost coplanar (or at least less non-planar) in the unbound conformation. But putting methyl groups on twists the rings in the unbound conformation into a form that's similar to the bound one; basically there's more overlap between the solution and bound conformations in case of the methylated versions compared to the non-methylated ones. Consequently, the protein has to expend less energy to turn an already similar conformation into its bound counterpart. This becomes clear simply by comparing the single dihedral angle bridging the two rings in the bound and unbound conformations of one particular molecule (as shown in the figure above).

The study seems to impart what is part of an important general lesson; when designing ligands or drugs to bind a protein, it is as important to take the solution conformations into account. It's not just how the protein interacts with the drug, but it's what the drug is doing before it meets the protein that's equally important.

Rookie mistakes in molecular modeling: Part 1

Molecular modeling as a general approach is no longer utilized only by experts but has reached the masses. Improved hardware and software capabilities combined with easy-to-use graphical user interfaces have enabled experimental chemists of all kinds to build models of molecules and perform relatively sophisticated calculations on them. Calculations which once required supercomputers can now be routinely done on desktops by organic, inorganic and biological chemists who can use the results to explain, support and predict chemical phenomena. In the coming years we can be confident that we will witness an increasing use of modeling techniques by experimentalists.

An unfortunate (but probably not unexpected) consequence of this ease of use is that it has also become easier to make mistakes while building molecular structures. The main source of errors arises during the translation of 2D chemical structures to their 3D counterparts using some energy minimization protocol. The apparently simple process of drawing a 3D-worthy 2D structure is trickier than it sounds and is therefore quite prone to error. Conformation which was not as important when drawing in 2D is suddenly of overriding importance and it's relatively easy to get it wrong.

As a modeler who has interacted closely with experiment, I have come across a number of rookie mistakes which I have seen myself and others make over the years. Sometimes these mistakes don't matter too much for the final result but sometimes they can completely change it. So I thought I would make a short list of easy-to-avoid errors which may provide checks on modeling structures. In part 1 I will describe mistakes commonly seen during the simple building of structures. Part 2 will deal with interactions with experimentalists.

1. Getting the ionization state wrong: I put this rookie mistake at the top because it's remarkable how many times I have seen even experienced modelers make it. Always remember; amines are protonated at physiological pH while carboxylic acids are deprotonated. The reason why getting this right is important is because it can completely change results from protocols like docking. Just think of the difference a protonated vs unprotonated carboxylate makes for binding to a protein. Also, many modeling algorithms use force fields which are dominated by electrostatic interactions; the wrong protonation state can therefore make a world of difference. A corollary of the ionization state problem results when replacing atoms. For instance you may have a protonated amine which you then want to turn into an alcohol by replacing the N with a O. Unfortunately the atom does change but not the ionization state, and you end up with a weird positively charged doubly bonded oxygen. On a related note, it goes without saying that you shouldn't charge up inappropriate atoms such as those which are conjugated to aromatic systems. The best way to overcome these issues is to simply display charges for all heteroatoms in your final structure.

2. Getting the stereochemistry wrong: The CIP rules were taught to us because they really matter. Here's a typical stereochemical mistake: You construct a structure in 2D and come across a stereocenter. You may even build that stereocenter with the right absolute (R or S) stereochemistry. And then you attach something else to that center and forget to recheck the stereochemistry which may have changed because of the change in CIP priority. The simplest way to make sure about stereochemistry is to always have the program display all absolute stereochemistry for the final structure.

2. Forgetting basic conformational rules: This mistake is most commonly made when converting a 2D structure into a 3D structure. The problem is that when you build a 2D structure, your placement of bonds and angles is somewhat ad hoc based on the rather random way in which you are conveniently rotating and viewing the structure. When you then suddenly convert 2D to 3D, you may end up with axial substituents on six-membered rings, syn-pentane or eclipsing interactions between substituents, funky substructures like non-planar aromatic rings resulting from strain or in the worst cases, even boats for cyclohexanes. Here's another common pitfall: You may try to close a ring by building an unrealistic long bond between two initially separated atoms, thinking that when you then minimize this structure the program will take care of the bond by shortening it to its standard length. This usually happens, but in the process some other parts of your molecule gets messed up. Again, judicious inspection can avoid most of these issues.

3. Cis and trans: The process of building unrealistic bonds between distant atoms and then simply minimizing a structure that I just mentioned can sometimes result in amide bonds becoming cis and this is important enough to be listed as a separate point. This is also a common consequence of importing 2D files in SDF format (which lack hydrogens) and asking a program to add hydrogens. The same thing can happen with double bonds.

4. Forgetting basic chemistry: This mistake has more to do with forgetting basic rules of bonding and chemistry than with modeling. Occasionally you may do things like exceeding the allowed valency of an atom, putting a double bond at a bridgehead carbon (violating Bredt's rule), generating antiaromatic rings, forgetting Baldwin's rules for ring closure, building a vinyl amine or a geminal amino alcohol...and generally creating all sorts of unstable and "impossible" molecules. The problem is that your program won't always raise red flags notifying you about these errors so you need to remember your chemistry and make sure you don't recommend some wacky molecules to make to the synthetic chemist (one of the constant sources of friction between experimentalists and modelers arises from the latter forgetting what's synthetically feasible and stable).

Ultimately, the path to a well-constructed molecule simply depends on being vigilant and judiciously checking your final structure. Remember the well-worn adage; computers don't know any chemistry whatever and they are only as good as the code that goes into them. Nothing can trump a sound knowledge of basic chemical principles.

Image source

Why conduct reactions at low temperature?

The other day I was talking to a synthetic chemist friend about conducting reactions at low temperature and I realized that there is another reason for doing this that is not always appreciated by beginning organic chemistry students. Most students think that the primary purpose of low-temperature reactions is to stop runaway exothermic reactions from getting out of hand and to tame explosive reagents. While this is a perfectly good reason, there is another reason connected to stereochemistry which occasionally necessitates these reactions.

Remember the all important thermodynamic relation ∆G = ∆H - T∆S and recall that life and laboratory chemistry are both games played within a 3 kcal/mol window. In this case the relevant equation would be the Arrhenius equation and the relevant free energy would be the free energy of activation (∆G††). Thus even a 1 kcal/mol energy difference between two transition states can favor the product corresponding to the lower energy TS by a substantial account; for instance you only need a 1.8 kcal/mol energy difference to effect a greater than 95% yield of the more stable species. This principle applies to everything, including conformers, stereoisomers and constitutional isomers. But the important variable for our discussion is the temperature T and it's clear that a lower temperature will affect the free energy favorably.

And that is precisely why it becomes so important in stereoselective reactions. If you are dealing with two diastereometric transition states resulting from attack of a chiral reagent on two enantiomers for instance, you only need a difference of 1.13 kcal/mol to generate a diastereomeric ratio of 95:5. But this phenomenon becomes even more pronounced at -78 degrees celsius which is the temperature of a standard liquid N2 acetone/dry ice bath. For instance, if you conduct a reaction giving you a 95:5 diastereomeric ratio at -78 degrees, the same reaction done at 23 degrees will give you only a 85:15 diastereomeric ratio. And if you look at the energy you need at 23 degrees to overcome that low ratio and bump it to 95:5, it's only 0.58 kcal/mol.

It's incredible to realize how so much of life and chemistry are governed by startlingly small differences in energy between large numbers. But there you have it; a very good reason to lower the temperature of your next aldol condensation to get better stereoselectivity. Make sure to emphasize that to your curious undergrad the next time he/she asks a question about low temperature reactions.

Xtreme C-H functionalization: Natural Edition

Blogging has been swamped lately by that miracle called life but I could not help but be drawn to a paper in this week's Science which describes a most unholy and unexpected stabilizing alliance in a protein's innards.

Proteins are known to form cross-links such as disulfide bonds to stabilize interactions with ligands and substrates. Any reasonable chemist would expect these kinds of interactions to be mediated between polar residues. But nature usurps us low-lifes once again. In this week's Science, a group led by Andrew Karplus reveals a stabilizing covalent cross-link between, hold your breath, a valine and a phenylalanine. Who could have imagined these two otherwise blissfully aloof and stable partners suddenly deciding to...bond?

As chemists know however, there is only one kind of chemical entity that can create such havoc with stable functional groups- a metal. It turns out that the protein is a four-helix bundle diiron protein with two Fe atoms bound in proximity to the Val and Phe. The two irons apparently create their own cofactor by neatly supplying electrons to bond the Val and Phe to each other and molding a cosy bed for themselves. The resolution is 1.2 A so the electron density is unambiguous. The function of the unusual cross-link seems to provide a barrier to protect the iron from potential iron chelators; experiments indicate that the iron is rapidly mopped up by chelators in mutants lacking the cross-link. Intriguingly, the real function of the protein itself remains unknown.

Organometallic chemists who are keeping the midnight oil burning trying to use metals to functionalize unreactive C-H bonds would not be too surprised that a metal is mediating such strange interactions. But the observation demonstrates something that chemists are all too familiar with by now- Nature has been there, and it's done that.

Cooley, R., Rhoads, T., Arp, D., & Karplus, P. (2011). A Diiron Protein Autogenerates a Valine-Phenylalanine Cross-Link Science, 332 (6032), 929-929 DOI: 10.1126/science.1205687

Stimulating quasi-erotic excitement through organic structure determination

Thanks to the graces of the intertubes I came across this rare and fascinating video of R B Woodward put up by some kind soul a couple of months ago. The novelty of the quintessential Bostonian accent, the cigarette and glass of scotch adorning the lectern and the man in blue are only eclipsed by his achievements and what he has to say. He especially saves the coup de grace for the end.

Woodward essentially sheds light on the remarkable developments in organic chemistry until then by providing contrasting examples from his own research. He emphasizes how times had changed between his own work and the state of the art in 1979. One can make similar comparisons right now. Woodward basically attributes the astonishing progress in organic chemistry in the last forty years to two factors- an intense infusion of theoretical concepts in their most general form (MO theory, quantum chemistry etc.), and the path-breaking developments in physical methods, including IR, UV and NMR spectroscopy and x-ray crystallography. He then provides famous examples from his own work to starkly emphasize the contrast.

The first example is from his synthesis of quinine. In this synthesis, one of the steps involved the elimination of a quaternary ammonium ion to form a double bond. The question was whether the double bond formed was a vinyl double bond or an ethylidene double bond; it was the vinyl that was desired.



Nowadays, and even in 1979, a graduate student could settle this question in literally a matter of minutes, but at that point (circa 1945), Harvard did not even have the experimental facilities necessary to chemically investigate this fact. Woodward had to send the sample to the famous chemist Max Tischler at Merck. Tischler got back saying it was an ethylidene. This threw the chemists into a state of despondency for a few days, until Tischler called back to inform them that Merck had made a mistake and it was in fact the vinyl double bond. The tense drama during this situation seems almost comical in the light of modern structure determination methods.

The second example concerned Woodward’s astonishing decade-long synthesis of Vitamin B12. He expressed wonder how an NMR spectrometer had been able to obtain the natural abundance C13 spectrum of 1 mg of the synthetic finished product using 995,000 transient scans. This incredulity would sound almost laughable today. Capillary NMR and 1 GHZ machines have pushed the science and art of structure determination to limits, and doing a million scans on 1 mg of material is almost old hat.

The third example was a nice little anecdote. Woodward had a wager with Linus Pauling in the 1950s whether he could chemically determine the structure of the antibiotic terramycin faster than Pauling could do it with x-ray crystallography. Woodward won the wager, but also admitted that he would probably lose it today because x-ray crystallography had gotten so powerful. Today x-ray crystallography is already at the top of its game, and who knows what breakthroughs in structure determination would be possible with AFM and STM.

The last example cracked everyone up. Woodward talked about the structure determination of cantharidin, the active principle of the Spanish fly. Chemists had isolated up to 500 grams of cantharidin to find out its structure. “Just think of it, 500 grams of cantharidin”, says Woodward. “There are many people who would think it’s an absolute tragedy. Realize that that would be enough to keep the entire population of Spain in a state of quasi-erotic excitement for a period of a full year!”

What would be Woodward’s reaction if he were to suddenly materialize today in a poof of chemical pixie dust and survey the synthesis landscape? My humble guess is that he would not be too impressed. He would undoubtedly be excited by the development of the Sharpless and Grubbs methods and the great success of the palladium-catalyzed reactions (not to mention the general development of organometallic chemistry, in the founding of which he himself played a role). But beyond that, I doubt if he would notice any fundamental change in the science of organic synthesis compared to what he witnessed and orchestrated during his own lifetime. Sure, things have become more efficient, streamlined and automated, but those details, as impressive as they are, are really operational details.

My personal guess is that Woodward would be much more impressed by the application of organic synthesis to biology and materials science. But as for the science itself, it probably still stands very close to where Woodward left it thirty years ago, and the whiz-kid from Quincy would have little trouble bringing himself up to speed on it in no time at all.

The origin of life cannot escape basic organic chemistry

One of the key challenges facing any theories of the molecular origins of life concerns the synthesis, stability polymerization and self-assembly of early life's molecular components. If you cannot explain the chemical origin of these components, you cannot really explain the origin of life. In case of life as we know it, this boils down to explaining the origin of the building blocks of living organisms, namely nucleotides and amino acids.

The simplest principles and quirks of chemistry could have had an influence on how life could have evolved. A neat paper in ACS Chemical Biology offers a potential explanation based on basic organic chemistry for why a certain class of phosphorylated nucleotides formed in preference to others, even though 'conventional' organic chemistry would dictate the opposite.

An anhydroarabinonucleoside has been postulated as an important potential precursor to further nucleotide synthesis. A key step is the phosphorylation of this nucleoside to yield an activated cyclic nucleoside phosphate. Having an activated molecule makes all the difference since activation primes the molecule to be attacked by further nucleophiles, thus triggering polymerization and growth.

However, the phosphorylation of the arabinose nucleoside raises a fundamental question (hopefully) familiar to sophomore organic chemistry students. Why does phosphorylation take place preferentially on the secondary 3'-OH while sterically, as every student of organic chemistry knows, it should be preferred much more on the primary 5'-OH?

To tackle this question, the authors get a crystal structure of the nucleoside in question. This x-ray structure shows an unusually short distance between the 2'-OH oxygen and the C2 carbon (2.7 A, a).


Energy optimization using quantum chemical techniques surprisingly does not get rid of the short distance. Because of this proximity, the 2'-OH can undergo an internal attack on this carbon to generate a reactive intermediate (1), whose ring can be opened in turn by a 3'-OH phosphate to form the activated phosphate product. Now, the 5'-OH also gets phosphorylated; it's just that it cannot attack the C3 carbon of the activated intermediate the way the 2'-OH can because it's not in proximity to this carbon the way the 2'-OH is.


The authors explain the short distance between the 2'-OH and the C3 carbon by postulating an interaction between the lone pair of the 2'-OH oxygen and the pi* orbital of the C2=N bond. This kind of interaction is quite familiar to organic chemists; it is invoked in the famous Burgi-Dunitz trajectory that enables nucleophilic attack on carbonyl carbons. Indeed, the authors perform a theoretical analysis that shows the angle of attack for the 2'-OH to be about a 100 degrees, close enough to the Burgi-Dunitz trajectory.

This is a classic case of there being two competing pathways in chemistry, one of which is preferred to the other because of a subsequent low-energy route that can be traversed. It's a common theme in chemistry and biochemistry and illustrates how otherwise counter-intuitive reactions can be accelerated by putting them at the top of the right energy cliffs. No matter how complex life may be, it still cannot get around the basic laws of organic chemistry. Score one for thermodynamics.

Choudhary, A., Kamer, K., Powner, M., Sutherland, J., & Raines, R. (2010). A Stereoelectronic Effect in Prebiotic Nucleotide Synthesis ACS Chemical Biology DOI: 10.1021/cb100093g