Field of Science

A coterie of pyridines

Mohammad Movassaghi of MIT has come up with a versatile new pyridine synthesis using the coupling of amides and pi-electron donors such as acetylenes. Better than the formaldehyde guzzling Hantszch synthesis I guess. The variety of molecules produced is impressive indeed. I love em all, not their smell though. In fact I have not come across any other solvent whose smell I hated this much. Also, smelling them is apparently not very good in terms purposes. At least that's what I hear.

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Carbene 'decarbenized'

An interesting exchange has taken place in the pages of Angewandte Chemie. Barbel Shulze and his group at the University of Leipzig claimed that they had isolated the first isothiazolium carbenoid (DOI: 10.1002/anie.200702746)

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Other carbenoids such as imidalolium, thiazolium etc. were already well known. The group characterised this elusive species by solid state NMR and cited the C13 resonance of the carbenoid carbon as proof that they had it in hand. However, they had no crystal structure

One of their "proofs" for the existence of this species was its reaction with morpholine, in which they suggested that the carbene inserts into the morpholino NH in a classic carbene insertion reaction.

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However, Guy Bertrand and his group at UC Riverside challenged this assumption and pretty clearly negated it as far as I can see.(DOI: 10.1002/anie.200702272) They showed that the purported carbene actually forms a different species.

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Very nicely, they got a hint for this process from an old 1966 Woodward publication dealing with ring opening of isoxazolium salts. I love such insights from history!

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Interestingly, their calculations, done at a pretty decent level of quantum chemical theory, suggested the TS for this rearrangement was only 1 kcal/mol more than the purported carbene, this making its existence almost impossible. And the rearranged derivative was several kcal/mol more stable than the purported carbene. Thus their rebuttual clearly says that not only do they think the carbene is not "stable" but they don't observe it at all.

The group also proposed an alternative explanation for the formation of the morpholino derivative, a simple nucleophilic addition to an electrophilic carbon (6S below). Not only that, but they also obtained a crystal structure for product (7s below) of the reaction of the morpholine with their new rearranged derivative.

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In my opinion, Bertrand's group cinched it as well as it can be cinched. I wouldn't have liked to be Shulze in this whole episode. Now anybody can make a mistake, although one would prefer that he or she does not make it in the pages of AC. But at the same time, what is most troublesome is the fact that Bertrand and his colleagues could not observe the NMR resonance that Shulz and his colleagues observed. Not at all.

Now one thing to Schulze's credit is that he and his co-authors graciously accepted Bertrand's alternative explanation which was pretty solid and were generous in their praise of his group's work. However, they also challenge Bertrand's alternative explanation of the formation of the morpholino derivative; after all, both nucleophilic addition and carbene insertion could lead to the same product.

But what's happening here? What exactly did the earlier group see? Only time will tell I guess. Maybe some even more interesting chemistry will be unearthed. I guess an elusive species has to pass some pretty rigorous tests in order to be called "stable".

P.S. Nothing like mulling over and reading an interesting chemistry debate while listening to Mozart's 40th Symphony

Numbers worth knowing

For every scientist, there's this bunch of numbers worth knowing, that are a good guide for understanding and predicting stuff. Here's a short list of numbers (apart from the usual ones like Avogadro's, Planck's, Boltzmann's, pKa values etc.) which are quite useful for chemists and biochemists. It's really a personal list, but it can be generally useful.

1. 3 kcal/mol: the maximum difference that can exist between two or more conformers in solution to be able to observe the minor one by NMR. Because remembering that ∆G=-RTlnK, this means that with a difference of 3 kcal between two conformers, the major one will be present to the extent of 99.96%. Naturally for practical purposes, the difference usually cannot exceed about 2 kcal/mol (98% or so) to detect the minor conformer, as NMR's detection limit is about 2%...below that it's hard to see the stuff. This is an important issue, because for example in the binding of a drug to a receptor, a conformer that exists to the extent of 3% can be the bioactive one.

2. 1.54 A, 1.34 A, 1.22 A Lengths of C-C single, double, triple bonds

3. Milliseconds: NMR time scale

4. Energy at room temperature: 18-20 kcal/mol. Important because that means that if energy differences are below these numbers, interconversion at R.T. can take place. Note that this is the kinetic barrier at RT. Thermal energy at RT is 0.6 kcal/mol only. For reference, energetic input needed for cis-trans interconversion for a typical double bond is about 20 kca/mol.

5. 2-10 kcal/mol: Strength of hydrogen bond. This can widely vary though...2-5 kcal/mol is a better range to remember.

6. 1.2, 1.5, 1.7, 1.5 A, 1.35: Van der Waals radii for H, O, C, N, F from Bondi (JPC, 1966). Useful for noticing "short" contacts in crystals and molecular and protein structures

7. 1 kcal/mol ~ 350 /cm: One of those useful conversion factors, in this case between wave numbers and energies. For quantum chemists, another useful relationship is 1 hartree= 627.5 kcal/mol

8. Relevant dielectric constants: water 78, DMSO 47, ethanol 24, acetonitrile 37

9. Ionic radii of essential ions: Na+ 0.95 A, K+ 1.33 A, Mg2+ 0.65 A, Ca2+ 0.99 A

More when they strike me

Daniel Koshland

Daniel Koshland of induced fit fame has passed away (or should I say passed on...for a chemist, it's all atoms). Koshland was the chief editor of Science from 1985-95

One of his quotes which is one of my favourites is:
"Always tackle the important problems...that's because you are probably going to spend equal time tackling the less important ones anyway"

As a graduate student, I say Amen to that.

Weird scis-strans observation

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Not something I expected...the C1-C2 cis form is more stable than the C1-C2 trans form. Both ab initio methods and MMFF say this, and also indicate the geometry to be perfectly planar. The problem with the perfectly planar geometry is that the C4 hydrogen-carbonyl oxygen distance is short in the cis form (2.35 A) and the C4 hydrogen- sp3 ester oxygen distance is almost the same (2.39 A) in the trans. Both these distances are short. A little non planarity could preserve the conjugation, but get rid of the distances. However, both methods don't seem to be doing this. Electronics seems to overwhelm sterics.

Interestingly, a look through the Cambridge Structural Database also indicates several similar C1-C2 cis esters, with short distances (although longer than the ones above by perhaps 0.1 A or so). So this is not a purely theoretical observation.

The simple things that defy us...I am trying to figure the reasons for this one out. Maybe it's the electrostatic attraction/HB between the C4 hydrogen and carbonyl oxygen.

The BI guys should have used Glide XP Dock

In the last post, I was wondering why the BI guys did not use any docking to conjecture the binding mode of their new p38 inhibs. While docking is not a foolproof predictor, it can shed light on possible anomalous modes. Particularly interesting was this observation about the benzothiophene sulfur reversing its trans preference to become cis to the adjacent amide oxygen. This counterintuitive observation was later explained by alluding to the glutamate that would have had an unfavourable electrostatic interaction with the sulfur had it been trans to the oxygen. The observation was revealed by the crystal structure. However, before the crystal structure was obtained, they went through the design and synthesis of two compounds based on the reasonable hypothesis that the sulfur would be trans. It was only after their puzzlement with the failure of these designed compounds that the situation became clear through crystallography.

Well, the observation is also revealed by Glide XP docking, and I think this could have saved them some time. I first duplicated the X-ray pose of the earlier BIRB compound in p38 (1KV2 PDB id), and then docked the new compound in. The result; the binding score was still good, if not as good as for BIRB. But the important fact was that all the top 5 docking poses from Glide indicated the sulfur to be cis to the oxygen and away from the Glu, just as in the crystal. In fact, even the fragment docked in the same position as the crystal structure indicated, with the sulfur cis. Not totally surprising if electrostatic interactions are well parametrized and recognised by the scoring function. A further advancement where polarization effects are taken into account during docking would help a lot (This is in the works)

This was one case in which docking could have said basically the same thing that the crystal structure did. In this case, docking could have saved them the design and synthesis of two extra compounds based on a misleading hypothesis, and perhaps additional head-scratching validated by crystallography. On a related note, Glide is pretty well-parametrized on a couple of kinases, including Lck, CDK2, and p38.

BI p38 inhibitors: breaking from the urea

Boehringer Ingelheim has a new series of p38 kinase inhibitors (DOI: 10.1021/jm070415w), where they seek to do something different from their famous diaryl urea inhibitors. These diaryl urea inhibitors have the single oxygen of a morpholino ring bonding to the NH group of the backbone residue (Met in this case). BI decided to use a computer program called LigBuilder to find a suitable link between the newly envisaged backbone-amide bonding pyridine and a sulfonamide which bound to the Phe DFG out residue, thus blocking one conformation of the kinase.

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LigBuilder helped them find a 5,6 fused heterocyle ring which would substitute in part for the urea moiety; the best activity came from a benzothiophene ring. An interesting physical-organic issue came up at this point. They had assumed that the sulfur of the benzothiophene would prefer to be trans to the oxygen of the succeeding amide. However, in the protein, things changed and the sulfur prefered to be cis to the oxygen, because the trans form had electrostatic repulsion with a Glu bound to the amide NH. This was confirmed by the crystal structure. It's important to note that it was essentially through trial and error that they discovered this; they designed and synthesized two compounds based on the intuitive hypothesis, and when there was a radical drop in potency, surmised that perhaps something funny was going on.

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Why they chose to add an ortho -NH2 to the backbone binding pyridine at this late stage is something that puzzles me. The ortho amino pyridine/pyrimidine moiety is well-known to form a set of correlated hydrogen bonds to the backbone carbonyl oxygen and the NH and is present in hundreds of inhibitors, and it is not surprising that this moiety increased potency. Standard stuff like inhibition of LPS-induced TNF-alpha production was noted. In the end, they had a good viable inhibitor.

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X=S, R=NH2 etc.

Favourite author among 2006 medalists

I am happy that the author of my all-time favourite biochemistry textbook is being awarded the National Medal of Science. Others include Dervan (Caltech bioorganic), Caruthers (Colorado, DNA etc.) and Langer (MIT materials, biology). All great scientists.

Is there cause for optimism?

I am not a big fan of unfounded optimism based on raw hope, especially in the kind of world that we seem to be living in, where reason is routinely given short shrift, and where decisions seem mandated by politics, emotions, and most importantly, by the loud voice of people wallowing in faith. However, the more I look at history, the more optimistic I seem to get...

Read the rest of the entry on Desipundit...

Book review: Pharmacology for Chemists

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Pharmacology for Chemists: Joseph G. Cannon, Oxford University Press, 2007

This book is a one stop shop for chemists wanting to be familiar with the essentials of pharmacology. Cannon also adopts the same perspective, always giving stress on the chemical basis of the action of drugs, as well as neurotransmitters, hormones and intracellular signalling molecules. The first part of the book deals with general principles, which Cannon beautifully explains in a nutshell. Topics include the blood-brain barrier, drug receptors, pharmacological assays, metabolic inactivation and modifications of drugs by organs, basic pharmacokinetics, and properties of membranes and cells. The latter part is focused on particular systems. It is especially in discussing the central nervous system that the book really shines. Cannon talks in detail about the neurochemistry and physiological action of several important neurotransmitters and their agonists & antagonists. He also discusses the most important diseases arising from malfunctioning of the CNS. The chemistry of addiction also receives due exaplanation. Later chapters include discussions of cardiovascular drugs and drugs which are used to treat asthma and allergies.
Pharmacology is a vast science, and it not possible for a utilitarian chemist to work through the grand tomes on the subject to get what he wants. Cannon's book largely remedies this situation, and provides him with the essentials, without overloading him with information. This slim volume is a must on the shelves of medicinal chemists, biochemists and any professional working in, or interested in drug design.

Killing polyavians with a unistone: designing promiscuous drugs

Decapitate multiple avians by means of a single projectile (Kill many birds with a single stone)

There is an interesting review (doi:10.1016/ on the "rational" design of promiscuous ligands and drugs which target multiple receptors. The review tries to break with the current paradigm of drug discovery, that of highly selective and potent ligands targeting a single receptor. According to the authors, there is an addition to this paradigm of selective ligand design; instead, design promiscuous ligands that would modulate multiple targets and bring about the intended action. This may look like a somewhat suprising strategy, because we know that promiscuous ligands usually carry the risk of toxicity and off-target side effects. But the paradigm seems to be more subtle than that. Here are some points from the review that I found interesting:

1. Many drugs which were initially thought to be effective because of their selectivity are now recognised to be promiscuous, and perhaps effective because they are promiscuous. A most striking example is Gleevec. This way of thinking has also raised questions about kinase inhibitors in general; are they efficacious because of or inspite of their promiscuity?

2. In one study, it was found that the promiscuity of ligands inversely correlates with their molecular weight. This is not too surprising, considering that more complex ligands will have to fulfil multiple site interactions with the receptor, the probability of which will be lower than that for fewer points of interaction. To me, this also translates into the entropic difficulties of binding to receptors, with more complex ligands having to pay greater entropic penalties. Of course, 'more complex' in terms of higher molecular weight does not necessarily mean ligands with more rotatable bonds; it can just mean more flat rings. But the problem of multiple site interaction is still valid even for these. For nature, the best ligands would be those which have a highly favourable enthalpy of interaction with the receptor at a few well-defined points, without having to pay a high entropic cost. That is one of the reasons why so many of the potent molecules that nature has designed are small, planar, heterocyclic ligands. This concept also spills over into fields like kinase inhibitor design. For selective design, we usually try to strike some golden mean in terms of ligand complexity.

3. There are also promiscuous proteins, with the most well-known example being cytochrome P450. Tubulin is also a pretty promiscuous protein. Sadly, I don't think we really understand what features of a protein or ligand make it promiscuous. Hydrophobicity seems to be a common criterion, but the very word "common" indicates that it is spread across a wide range of protein and ligand classes. One interesting feature of another promiscuous protein, PXR, is that it seems to bind multiple configurations of the same ligand. I have always wanted to find an example of such a protein. Whether this is a strong criterion for promiscuity would be a very interesting proposition.

To me, it appears that promiscuity first of all is not as widely observed as we would think, especially in terms of ligand lipophilicity. Consider Taxol. To my knowledge, there is not a single protein other than Tubulin to which it binds so strongly, inspite of it being quite lipophilic. But clearly, lipophilicity itself cannot be a sufficient criterion for ligand promiscuity. Also, the high selectivity of taxol may be related to its relatively high molecular weight, with reference to the MW-promiscuity study. The point seems to be that although we know some examples of promiscuous proteins and promiscuous drugs, their general features seem not much different from other non-promiscuous molecules.

It seems then, that to have effective promiscuous drugs, what we need is not just rampant promiscuity but selective promiscuity (I know, sounds like an oxymoron). The way I think about it, it won't work if the drug is flagrantly promiscuous across diverse chemical classes. Simply throwing in a greasy compound into the cocktail of body chemistry may get you a wildly promiscuous inhibitor, but that would not be what you want for a drug.
Consider kinase inhibitors. We can almost take it for granted that an ideal, conpletely promiscuous kinase inhibitor that targets dozens of kinases across different classes cannot be made into a working drug. But one can think of a particular subset of kinases, intricately linked to each other through signal transduction pathways, very sensitive to modulation of each other and to modulation by small molecules, implicated in a disease such as cancer, and expressed in cancer cells constitutively. If, and this is a big if, one can design an inhibitor that will inhibit this unique chosen subset of kinases, then one could potentially have a very potent drug.

In light of the very selective and stringent promiscuity criteria above, I would think that if anything, it may be more difficult to design a truly potent and effective promiscuous inhibitor, compared to a highly selective inhibitor, except by serendepity. In the latter case, you have to selectively target one protein. In the former case, you may have to selectively target three of four proteins. That just sounds so difficult through rational drug design. Naturally, designing such inhibitors would entail an intimate understanding of signal transduction pathways.

"I run from reality"

Yes, that's why I prefer to do computational chemistry and virtual screening too. Presenting UCSF's Brian Shoichet

Slippery questions

Here are a few questions about water that I have either read, or which recurringly occured to me through reading about the stuff that life's made from. Some of them are easy to answer, some are difficult. Some are general questions, others are subsets of these general questions. Most of them are connected. Many are of the "simple-yet-extremely-tricky-to-answer" kind. I know the answer to some of the questions, don't know those to others, and am not sure about yet other ones.

1. What is the strength of an average hydrogen bond in bulk water?
2. What is the strength of an average hydrogen bond in "biological water" (solvating proteins for example) and the strength of a hydrogen bond in the first hydration shell?
3. What is the lifetime of an average hydrogen bond in bulk water? In biological water?
4. Is hydrogen bond exchange between water a discrete phenomenon or a concerted one?
5. How many hydrogen bonds on an average does a water molecule form in bulk? In confinement? In biological water?
6. Is there a long-range "hydrophobic bond"?
7. Can one always predict de novo whether displacement of a water molecule from a hydrophobic pocket of a protein by a ligand will lead to greater binding affinity? How?
8. How does enthalpy-entropy compensation work in case of water?
9. What is the difference between hydration dynamics of bulk water and biological water?
10. What is the exact difference between hydrogen bonding structure and dynamics in ice and liquid water?
11. How does the molecular structure and hydrogen bond network of liquid water change according to the temperature? What is the difference between these properties for bulk water at room temperature and at 4 degrees celsius (at maximum density)? For biological water? Corollary of this question: what changes does the molecule structure of water undergo at low temperatures in biological systems and around biomolecules?
12. How do we explain the Hofmeister effect- the differential ability of various ions to precipitate proteins?
13. How does the hydrophobic effect depend on size of solute? On conformation?
14. Can we develop a comprehensive first principles water model?

More to come as I think about this. Some of these questions have been addressed before at the Water in Biology blog.