Category Archives: Chemistry (relatively pure)

Remember entropy? — Take II

Organic chemists have a far better intuitive feel for entropy than most chemists. Condensations such as the Diels Alder reaction decrease it, as does ring closure. However, when you get to small ligands binding proteins, everything seems to be about enthalpy. Although binding energy is always talked about, mentally it appears to be enthalpy (H) rather than Gibbs free energy (F).

A recent fascinating editorial and paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 4278 – 4280, 4424 – 4429 ’17 ]shows how the evolution has used entropy to determine when a protein (CzrA) binds to DNA and when it doesn’t. As usual, advances in technology permit us to see this (e.g. multidimensional heteronuclear nuclear magnetic resonance). This allows us to determine the motion of side chains (methyl groups), backbones etc. etc. When CzrA binds to DNA methyl side chains on the protein move more, increasing entropy (deltaS) and as well all know the Gibbs free energy of reaction (deltaF) isn’t just enthalpy (deltaH) but deltaH – TdeltaS, so an increase in deltaS pushes deltaF lower meaning the reaction proceeds in that direction.

Binding of Zinc redistributes these side chain motion so that entropy decreases, and the protein moves off DNA. The authors call this dynamics driven allostery. The fascinating thing, is that this may happen without any conformational change of CzrA.

I’m not sure that molecular dynamics simulations are good enough to pick this up. Fortunately newer NMR techniques can measure it. Just another complication for the hapless drug chemist thinking about protein ligand interactions.

A recent paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 6563  – 6568 ’17 ] went into more detail about measuring side chain motions  as a surrogate for conformational entropy.  It can now be measured by NMR.  They define complete restriction of  the methyl group symmetry axis as 1, and complete disorder, and state that ‘a variety of models’ imply that the value is LINEARLY related to conformational entropy making it an ‘entropy meter’.  They state that measurement of fast internal side chain motion is largely restricted to the methyl group — this makes me worry that other side chains (which they can’t measure) are moving as well and contributing to entropy.

The authors studied some 28 protein/ligand systems, and found that the contribution of conformational entropy to ligand binding can be favorable, negligible or unfavorable.

What is bothersome to the authors (and to me) is that there were no obvious structural correlates between the degree of conformation entropy and protein structure.  So it’s something you measure not something you predict, making life even more difficult for the computational chemist studying protein ligand interactions.

Entangled points

The terms Limit point, Cluster point, Accumulation point don’t really match the concept point set topology is trying to capture.

As usual, the motivation for any topological concept (including this one) lies in the real numbers.

1 is a limit point of the open interval (0, 1) of real numbers. Any open interval containing 1 also contains elements of (0, 1). 1 is entangled with the set (0, 1) given the usual topology of the real line.

What is the usual topology of the real line? (E.g. how are its open sets defined) It’s the set of open intervals) and their infinite unions and their finite intersection.

In this topology no open set can separate 1 from the set ( 0, 1) — e.g. they are entangled.

So call 1 an entangled point.This way of thinking allows you to think of open sets as separators of points from sets.

Hausdorff thought this way, when he described the separation axioms (TrennungsAxioms) describing points and sets that open sets could and could not separate.

The most useful collection of open sets satisfy Trennungsaxiom #2 — giving a Hausdorff topological space. There are enough of them so that every two distinct points are contained in two distinct disjoint open sets.

Thinking of limit points as entangled points gives you a more coherent way to think of continuous functions between topological spaces. They never separate a set and any of its entangled points in the domain when they map them to the target space. At least to me, this is far more satisfactory (and actually equivalent) to continuity than the usual definition; the inverse of an open set in the target space is an open set in the domain.

Clarity of thought and ease of implementation are two very different things. It is much easier to prove/disprove that a function is continuous using the usual definition than using the preservation of entangled points.

Organic chemistry could certainly use some better nomenclature. Why not call an SN1 reaction (Substitution Nucleophilic 1) SN-pancake — as the 4 carbons left after the bond is broken form a plane. Even better SN2 should be called SN-umbrella, as it is exactly like an umbrella turning inside out in the wind.

What is docosahexenoic acid and why should you care?

Why should drug chemists care about docosahexenoic acid — it’s a fairly trivial organic structure as these things go – a 22 carbon straight chain carboxylic acid with 6 double bonds — https://en.wikipedia.org/wiki/Docosahexaenoic_acid. However the structure is decidedly non-random (see later)

Docosahexenoic acid turns out to be crucial for the function of the blood brain barrier (BBB), something that makes it very difficult to get drugs into the brain. Years of work have shown that the only drugs able to get through the BBB are small lipid soluble molecules of mass under 400 kiloDaltons with fewer than 9 hydrogen bonds. Certainly not a large group of drugs. The more we know about the BBB, the more likely we’ll be able to figure out something to circumvent it.

The BBB was known to exist more than 100 years ago. Ehrlich found that dyes injected into the circulation were rapidly taken up by all organs except the brain. His student E. Goldmann found that dye injected into the CSF stained the brain but not other organs.

The barrier has at least two components — (1) a very tight seal between the cells lining brain blood vessels (e.g. the endothelium) — see the end of the post — (2)very low transfer across the endothelial cell from the vessel lumen. The latter is called transcytosis and involves formation of small vesicles at the lumenal surface of the endothelium, migration across the endothelial cell with release of vesicle content on the other side.

In general there are two mechanisms of transcytosis — clathrin coated pits, and caveolae. Brain endothelium shows very low rates of transcytosis. There aren’t any coated pits (no explanation I can find) and the rate of caveolar transcytosis is very low.

Dococsahexaenoic acid is the reason for the low rate of caveolar transcytosis. Here is why.

[ Nature vol. 509 pp. 432 – 433, 503 – 506, 507 – 511 ’14 Neuron vol. 82 pp. 728 – 730 ’14 ] An orphan transporter, MFSD2a (Major Facilitator Superfamily Domain containing 2a) is selectively expressed in the BBB endothelium. It is REQUIRED for formation and maintenance of BBB integrity. Animals lacking MFSD2a show uninhibited bulk transcytosis across the endothelium. The animals show no obvious defects in the junctions between the endothelial cells. Pericytes (cells in the brain layer after the endothelium) are important in keeping the levels of MFSD2a at normal levels as animals lacking them show the same defects in the BBB as those lacking MFSD2a. Even though knockouts don’t have much of a BBB, they have normal patterning of vascular networks.

MFSD2a is the major transporter of docohexaenoic acid (DHA), an omega3 fatty acid (more later). DHA isn’t made in the brain and must be transported into it. Knockouts have reduced levels of DHA in the brain accompanied by neuronal loss in the hippocampus and cerebellum and microcephaly. Human cases due to mutation are now known (11/15). Transport of DHA and fatty acids into the brain across the BBB occurs only in the form of esters with lysophosphatidylcholines (LPCs) but not as free fatty acids in a sodium dependent manner. The phospho-zwitterionic headgroup of of LPC is essential for transport. MFSD2a ‘prefers’ long chain fatty acids (oleic, palmitic), failing to transport fatty acids with chain lengths under 14.

So MFSD2a inhibits transcytosis at the same time it promotes fatty acid transport into the brain. Major Facilitator Superfamily (MFS) proteins use the electrochemical potential of the cell to transport substrates. The best known MFSs are the glucose transporters (GLUT1 – 4).

So the blood brain barrier is due in part to the lipid transport activity of MFSD2a which gives BBB endothelium a different lipid composition (with lots of docosahexenoic acid) ) than others, inhibiting caveolar transport. Increased DHA levels are associated with membrane cholesterol depletion, as well as displacement of caveolin1 (the major protein involved in this form of transcytosis) from caveolae.

It is likely that MFSD2A acts as a lipid flippase, transporting phospholipids, including DHA containing species from the outer to the inner plasma membrane leaflet (where caveolin1 binds).

What is so hot about docosahexenoic acid — 22 carbons all in a row, a carboxyl group and 6 double bonds. We’re not talking fused ring systems, alkaloids, bizarre functional groups etc. etc.

Half the answer is that the double bonds are NOT randomly arranged. The 6 occur all in a row (but with methylene groups between them). This tells the chemist that they are not conjugated, hence the chain is probably not straight. Think how unlikely the arrangement is considering the way 6 double bonds and 9 methylenes COULD be arranged in a chain (2^15). Answer 5 ways depending on where the arrangement starts relative to the end of the chain.

The other half is that all the double bonds are cis, making it very unlikely that the 21 carbon chain can straighten out and cross the membrane. Lots of DHA means a very disordered membrane, which may be impossible to caveolin1 (and clathrin) to bind to.

So even though it’s years and years since I left organic chemistry, it permits the enjoying of the biochemical esthetics of the blood brain barrier.

The tight junctions between endothelial cells are primarily responsible for barrier function. These tight junctions are found only in the capillaries and postcapillary venules of the brain. Endothelial cells of the brain have few pinocytotic vesicles and fenestriae. [ Neuron vol. 71 p. 408 ’11 ] The brain vasculature has the thinnest endothelial cells, with the tightest junction and a higher degree of pericyte coverage coverage (‘up to’ 30%). [ Neuron vol. 78 pp. 214 – 232 ’13 ] The tight junctions are made from occludin, claudins and junctional adhesion molecules, and are closer to the lumen than the adherens junctions (which also link endothelial cells to each other) made by the cadherins (E, P and N). (ibid p. 219) TLR2/6 specific stimuli.

Remember entropy?

Organic chemists have a far better intuitive feel for entropy than most chemists. Condensations such as the Diels Alder reaction decrease it, as does ring closure. However, when you get to small ligands binding proteins, everything seems to be about enthalpy. Although binding energy is always talked about, mentally it appears to be enthalpy (H) rather than Gibbs free energy (F).

A recent fascinating editorial and paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 4278 – 4280, 4424 – 4429 ’17 ]shows how the evolution has used entropy to determine when a protein (CzrA) binds to DNA and when it doesn’t. As usual, advances in technology permit us to see this (e.g. multidimensional heteronuclear nuclear magnetic resonance). This allows us to determine the motion of side chains (methyl groups), backbones etc. etc. When CzrA binds to DNA methyl side chains on the protein move more, increasing entropy (deltaS) and as well all know the Gibbs free energy of reaction (deltaF) isn’t just enthalpy (deltaH) but deltaH – TdeltaS, so an increase in deltaS pushes deltaF lower meaning the reaction proceeds in that direction.

Binding of Zinc redistributes these side chain motion so that entropy decreases, and the protein moves off DNA. The authors call this dynamics driven allostery. The fascinating thing, is that this may happen without any conformational change of CzrA.

I’m not sure that molecular dynamics simulations are good enough to pick this up. Fortunately newer NMR techniques can measure it. Just another complication for the hapless drug chemist thinking about protein ligand interactions.

The incredible combinatorial complexity of cellular biochemistry

K8, K14, K20, T92, P125, S129, S137, Y176, T195, K276, T305, T308, T312, P313, T315, T326, S378, T450, S473, S477, S479. No, this is not some game of cosmic bingo. They represent amino acid positions in Protein Kinase B (AKT).

In the 1 letter amino acid code K is lysine T, threonine, S serine, P proline, Y tyrosine.

All 21 amino acids are modified (or not) one of them in 3 ways. This gives 4 * 2^20 = 4,194,304 possible post-translational modifications. Will we study all of them? It’s pretty easy to substitute alanine for serine or threonine making an unmodifiable position, or to substitute aspartic acid for threonine or serine making a phosphorylation mimic which is pretty close to phosphoserine or phosphothreonine, creating even more possibilities for study.

Most of the serines, threonines, tyrosines listed are phosphorylated, but two of the threonines are Nacetyl glucosylated. The two prolines are hydroxylated in the ring. The lysines can be methylated, acetylated, ubiquitinated, sumoylated. I did take the trouble to count the number of serines in the complete amino acid sequence and there are 24, of which only 6 are phosphorylated — so the phosphorylation pattern is likely to be specific and selected for. Too lazy do the same for lysine, threonine, tyrosine and proline. Here’s a link to the full sequence if you want to do it — http://www.uniprot.org/uniprot/P31749

The phosphorylations at each serine/threonine/tyrosine are carried out by not more than one of the following 8 kinases (CK2, IKKepsilon, ACK1,TBK1, PDK1, GSK3alpha, mTORC2 and CDK2)

AKT contains some 481 amino acids, divided (by humans for the purposes of comprehension) into 4 regions Pleckstrin Homology (#1 – #108), linker (#108 – #152) catalytic –e.g. kinase (#152 – #409),regulatory (#409 – #481).

This is from an excellent review of the functions of AKT in Cell vol. 169 pp. 381 – 3405 ’17. It only takes up the first two pages of the review before the functionality of AKT is even discussed.

This raises the larger issue of the possibility of human minds comprehending cellular biochemistry.

This is just one protein, although a very important one. Do you think we’ll ever be able to conduct enough experiments, to figure out what each modification (along or in combination) does to the many functions of AKT (and there are many)?

Now design a drug to affect one of the actions of AKT (particularly since AKT is the cellular homolog of a viral oncogene). Quite a homework assignment.

Is Martin Burke the anti-Christ for synthetic organic chemistry?

Will a machine put synthetic organic chemists out of business. Is its proponent and inventor Martin Burke the anti-Christ? 2 years ago he thought that he’d need 5,000 building blocks to make 282,487 natural products. Now he’s down to 1,400, 20 years and 1 Billion dollars [ Science vol. 356 pp. 231 – 232 ’17 ].

Back in the day we studied the zillions of terpene natural products built from various machinations of just the isopentyl group. Does he really need another 1,399?

The synthesis is a modification of the Suzuki synthesis in which R-B(OH)2 and R’ – X are coupled by palladium to form R -R’. It uses MIDA (HOOC CH2 NCH3 CH2 COOH — N-MethylIminodiAcetic acid) which wraps itself around the boron and shuts down further synthesis.

In 2008 Burke found that MIDA boronates stick to silica when methanol and ether are both present, and then drop off when tetrahydrofuran (THF) is present. This allows catch and release. For purification they can run the compounds through a silica containing vial.

In 2015 some 200 building blocks with the halogen and MIDA capped boronic acid were availablle commercially.

Burke hooked up with a computer scientist to look at the structures of the 282,487 and break them down into fragments needing only carbon carbon bond formation — a fascinating problem in graph theory.

Derek did a post on this a few years ago. Hopefully he’ll do another.

Because they aren’t there

George Mallory tried 3 times to be the first to climb Everest dying on his last attempt. When asked why he was so obsessed, he achieved immortality by saying “because it’s there”. Chemists have spent 60 years trying to synthesize carbon nanobelts “because they aren’t there”.

Well a group of Japanese chemists finally did it [ Science vol. 356 pp. 172 -175 ’17 ] It’s not quite the ultimate belt because the 6 membered rings are staggered as they are in phenacenes –https://en.wikipedia.org/wiki/Phenacene. There are 6 three ringed phenacenes in the structure, and the diameter of the ring is 8.324 Angstroms. There is no question that they got the compound as they crystallized it and have bond lengths for all.

If you look at the paper, this is a zig zag structure rather than a linear poly anthracene. The bond lengths show that every other ring has symmetric bond lengths midway between sp2 and sp3 (e.g. it’s aromatic), while the other ring clearly is not.

It be interesting to measure the chemical shifts of the C-H bonds over the center of the ring — if they could make a paraCyclophane-type molecule bridging the diameter by a (CH2)n moiety.

As long as we’re on the subject what about putting a twist in the ring and making a mobius belt. Mobius molecules are known — http://www.scs.illinois.edu/denmark/wp-content/uploads/gp/2008/Collins-1.pdf — is a very nice review — with a lot of pictures.

The authors think that their work has potential applications — “our synthesis of carbon nanobelt 1 could ultimately lead to the programmable synthesis of single- chirality, uniform-diameter CNTs (30–32) and open a field of nanobelt science and technology”. I think they were just having fun as chemists are wont to do.

Did these guys just repeal the second law of thermodynamics and solve the global warming problem?

Did these guys just repeal the second law of thermodynamics and solve the global warming problem to boot? [ Science vol. 355 pp. 1023 – 1024, 1062 -1066 ’17 ] Heady stuff. But they can put a sheet of metamaterial over water during the day in Arizona and cool it by 8 degrees Centigrade in two hours!

How did they do it? Time for a little atmospheric physics. There is nothing in the Earth’s atmosphere which absorbs light of wavelength between 8 and 13 microns (this is called the atmospheric window). So anything radiating energy in this range sends it out into space. This is called radiative cooling. It doesn’t work during the day because most materials absorb sunlight in the visible and near infrared range (.7 -2.5 microns) heating them up. Solar power density overwhelms the room temperature radiation spectrum shorter than 4 microns. So for daytime cooling you need a material reflecting all the light shorter than 4 microns, while being fully emissive for longer wavelengths.

This work describes a metamaterial– https://en.wikipedia.org/wiki/Metamaterial — in which small (average diameter 4 microns) spheres ofSiO2 (glass) are randomly dispersed in a polymer matrix transparent to visible and infrared light. The matrix is 50 microns thick. The whole shebang is backed by a very thin (.2 micron) silver mirror. So light easily passes through the film and is then bounced back by the mirror without being absorbed.

Chemists have already studied the Carnot cycle, which gives the maximum efficiency of a heat engine. This is always proportional to the temperature difference between phases of the cycle. That’s why the biggest thing about a nuclear power plant is the cooling tower (and almost as important). Well few things are colder than the cosmic microwave background (2.7 degrees Centigrade above absolute zero).

So while the entropy of the universe increases as the heat goes somewhere, locally it looks like the second law of thermodynamics is being violated. No work is done (as far as i can tell) yet the objects spontaneously cool.

Perhaps the physics mavens out there can help. I seem to remember Feynman and Wheeler once saying something to the effect that radiation is impossible without something around to absorb it. If I haven’t totally garbled the physics, it almost sounds like emitter and absorber are entangled.

Anyway beaming heat out into space through the atmospheric window sounds like a good way to combat global warming.

No wonder DARPA supported this research.

The humble snow flea teaches us some protein chemistry

Who would have thought that the humble snow flea (that we used to cross country ski over in Montana) would teach us a great deal about protein chemistry turning over some beloved shibboleths in the process.

The flea contains an antifreeze protein, which stops ice crystals from forming inside the cells of the flea in the cold environment in which it lives. The protein contains 81 amino acids, is 45% glycine and contains six  type II polyProline helices each 8 amino acids long (https://en.wikipedia.org/wiki/Polyproline_helix). None of the 6 polyProline helices contain proline despite the name, but all contain from 2 to 6 glycines. Also to be noted is (1) the absence of a hydrophobic core (2) the absence of alpha helices (3) the absence of beta turns (4) the protein has low sequence complexity.

Nonethless it quickly folds into a stable structure — meaning that (1), (2), and (3) are not necessary for a stable protein structure. (4) means that low sequence complexity in a protein sequence does not invariably imply an intrinsically disordered protein.

You can read all about it in Proc. Natl. Acad. Sci. vol. 114 pp. 2241 – 2446 ’17.

Time for some humility in what we thought we knew about proteins, protein folding, protein structural stability.

Ring currents ride again

One of the most impressive pieces of evidence (to me at least) that we really understand what electrons are doing in organic molecules are the ring currents. Recall that the pi electrons in benzene are delocalized above and below the planar ring determined by the 6 carbon atoms.

How do we know this? When a magnetic field is applied the electrons in the ring cloud circulate to oppose the field. So what? Well if you can place a C – H bond above the ring, the induced current will shield it. Such molecules are known, and the new edition of Clayden (p. 278) shows the NMR spectra showing [ 7 ] paracyclophane which is benzene with 7 CH2’s linked to the 1 and 4 positions of benzene, so that the hydrogens of the 4th CH2 is directly over the ring (7 CH2’s aren’t long enough for it to be anywhere else). Similarly, [ 18 ] Annulene has 6 hydrogens inside the armoatic ring — and these hydrogens are even more deshielded. Interestingly building larger and larger annulenes, as shown that aromaticity decreases with increasing size, vanishing for systems with more than 30 pi electrons (diameter 13 Angstroms), probably because planarity of the carbons becomes less and less possible, breaking up the cloud.

This brings us to Nature vol. 541 pp. 200 – 203 ’17 which describes a remarkable molecule with 6 porphyins in a ring hooked together by diyne linkers. The diameter of the circle is 24 Angstroms. Benzene and [ 18 ] Annulene have all the carbons in a plane, but the picture of the molecule given in the paper does not. Each of the porphyrins is planar of course, but each plane is tangent to the circle of porphyrins.

Also discussed is the fact that ‘anti-aromatic’ ring currents exist, in which they circulate to enhance rather than diminish the imposed magnetic field. The molecule can be switched between the aromatic and anti-aromatic states by its oxidation level. When it has 78 electrons ( 18 * 4 ) + 2 in the ring (with a charge of + 6) it is aromatic. When it has 80 elections with a + 4 charge it is anti-aromatic — further confirmation of the Huckel rule (as if it was needed).

On a historical note reference #27 is to a paper of Marty Gouterman in 1961, who was teaching grad students in chemistry in the spring of 1961. He was an excellent teacher. Here he is at the University of Washington — http://faculty.washington.edu/goutermn/