The team from University of California (UC) Berkeley, and the Lawrence Berkeley National Laboratory, led by Carolyn Bertozzi, adapted the methodology known as ‘click-chemistry‘ to the particular conditions required by ‘in vivo’ conditions. Indeed, the original ‘click’ procedures, developed by Barry Sharpless (2), involved the use of toxic copper catalysts. In their article, the authors use a copper-free click reaction to label glycans – sugars particularly abundant on the surface of cells, where they are active in cell activity signalling, as well as in response to infections – which are thought of as appealing target for molecular imaging inside living organisms.

The first step involved the injection of azide-containing sugar derivatives, which are known to metabolically label glycans with the azide function. Then, a purposedly designed molecule carrying a signalling unit as well as a function reactive towards azides, had to be injected. The click reaction proceeded and as a result, glycans could be labeled in vivo, which paves the way for future specific biomolecule labeling inside living organisms.

Click chemistry inside a mouse (reproduced from ref. 1)

References:
(1) Pamela V. Chang, Jennifer A. Prescher, Ellen M. Sletten, Jeremy M. Baskin, Isaac A. Miller, Nicholas J. Agard,
Anderson Lo, and Carolyn R. Bertozzi, “Copper-free click chemistry in living animals”, Proc. Natl. Acad. Sci. USA, published online before print January 14, 2010. doi:10.1073/pnas.0911116107

(2) H. C. Kolb, M. G. Finn and K. B. Sharpless “Click Chemistry: Diverse Chemical Function from a Few Good Reactions”, Angew. Chem., Int. Ed. 2001, 40 2004–2021. doi:10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5

It is well known that the ‘usual’ green colour is due to the presence of chlorophyll in the leaves, which harvest red and blue light to fuel photosynthetic reactions. In turn, photosynthesis allows the plant to produce carbohydrates (sugars) to sustain growth and development, together with converting carbon dioxide into oxygen. When temperatures start to decrease, and days to shorten, the amount of chlorophyll in the leaves slowly decays. Indeed, warm temperatures are required for the plant to replace the chlorophyll which is gradually decomposed over time. As the concentration of chlorophyll decreases, other dye molecules present in the leaves become more and more visible. These are essentially carotene (which gives carrots their colour) and anthocyanins (present in red grapes and wine). Depending on the tree, and on the weather conditions, the leaves become yellow or more red-brown as the green colour fades, giving rise to awesome landscapes. I spent some holiday in Japan just one year ago, and the weather forecast during this period includes very detailed maps showing the ‘red leaves forecast’!

Autumn colors in Japan, here in Shirakawa-go....

... and in Nikko.

And here are some of the molecules responsible for these various and impressive color changes:

In this context, lots of effort is dedicated to find ways to detect and destroy such compounds before they can cause harm. An appealing solution was recently proposed by a research team led by Julius Rebek, Jr. at the Scripps Institute. In an article recently published in Angewandte, they show how their novel molecules can signal the presence of organophosphorus compounds, but also render them harmless by undergoing a rapid reaction.

The sensing systems is based on an aromatic ring equipped with an oxime group (C=N-OH), which is known to react with organophosphorus compounds. The intermediate product instantaneously reacts further (which is important since at this point, the toxicity survives) to form a harmless decomposition compound and a fluorescent unit, which is used to signal the fact that the reaction has occured, and therefore the presence of toxic chemicals! Really smart approach!

References:
T. J. Dale, J. Rebek, Jr. Angew. Chem., Int. Ed. 2009, 48, 7850 –7852. DOI: 10.1002/anie.200902820

Press release: Ring Closure as Warning

Now researchers from University of Cambridge in UK and from University of Jyväskylä in Finland report in Science a tetrahedral cage-like molecule which can encapsulate tetrahedral molecules of white phosphorus. In addition of being ‘inactivated’, phosphorus was also rendered water soluble by encapsulation, and both forms, either solid or dissolved in water, were found to be literally indefinitely stable. Interestingly, the release of phosphorus from the cage can be controlled by addition of a competing guest (benzene) which expels phosphorus. Dr. Jonathan Nitschke, who led the research, underlines the potential applications of such container molecules (source: sciencedaily.com): “It is foreseeable that our technique might be used to clean up a white phosphorous spill, either as part of an industrial accident or in a war zone. In addition to its ability to inflict grievous harm while burning, white phosphorous is very toxic and poses a major environmental hazard.” In the future, this method can probably be adapted to target other harmful molecules.

For more information:
P. Mal, B. Breiner, K. Rissanen, J. R. Nitschke, Science 2009, 324, 1697. DOI: 10.1126/science.1175313

ScienceDaily, retrieved July 14, 2009.

Histamine is a very simple molecule which is present is basically every single cell of our bodies. It is produced through enzymatic decarboxylation of amino acid histidine. Histamine’s many roles include neurotransmission (particularly in the sleep regulation mechanism) and immunological response, explaining why it is involved in various immunological troubles, ranging from relatively mild allergies to severe autoimmune diseases.

Histamine

A high proportion of histamine is stored in cells called mastocytes, which are located mostly at ‘risky’ places where the outside world can come into contact with our internal tissues: skin, lungs, mouth, nose… sounds like places where we can feel allergies right? When an allergy reaction takes place, the (harmless to non-allergic people) allergen interacts at the surface of the mastocyte, inducing the release of a massive amount of histamine in the surrounding environment. This results in well known consequences, such as mucuous secretions, itchiness, conjunctivitis. To produce these effects, histamine needs to interact with particular receptors, called, not so surprisingly, histamine receptors. The easy solution to overcome these effects is to prevent the histamine+receptor interaction: this is done thanks to antihistamine molecules which are also binding to histamine receptors, but without inducing allergic symptoms (pharmacologically speaking, antihistamine is an inverse agonist of histamine).

Finally, what about the widely reported drowsiness side-effect? As stated before, histamine plays a role in the sleep regulation. Histamine metabolism is perturbated upon antihistamine injestion, and one of the side effects is a (slight) inhability to maintain vigilance. Recent drugs (including my relieving cetirizin) are supposed to possess attenuated side-effects, but in my experience it is still not perfect since I tend to feel an urge to sleep after each intake…

Cetirizine, the active compound of many antihistamine drugs.

He started his presentation by showing his first molecular square, originally prepared nearly 20 years ago [1]. This tetrameric molecule, self-assembled from Pd(II) ‘angles’ and bipyridine linkers, was the starting point of an amazing field of research. In 1995, the 2-dimensional square became a 3-d cage [2]. Again the synthesis was astonishingly simple, allowing to obtain in a high yield a nanocontainer which was found to be able to accomodate simple guests (adamantane derivatives) in its cavity. It important to realize that a ‘classical’ synthesis approach requires many more steps and complications than the simple self-assembly process devised by Fujita.

Octahedral nanocage. The vertices are palladium atoms, and four faces are occupied by tris-pyridyl ligands. Source: http://www.nature.com/materials/news/newsandviews/030522/423394a_f1.html

Interesting features of this cage are that it is water soluble (the Pd centres are charged) and its cavity can accomodate hydrophobic molecules that are not water-soluble. By varying the organic ligand, several differently shaped and sized structures were obtained by self-assembly (such as cages, bowls, boxes, tubes, catenanes, and spheres) [3].

Well it is already good to have nice-looking architectures, but then? What do you answer when your non-scientific friends asks you “So what? What is this useful for?” … Looks like people in Fujita’s group decided to tackle this question very seriously (so that they don’t have to answer ‘er well, at the moment, we don’t have practical applications but potentially it could perhaps one day blah-blah-blah…’) and thus started to investigate what their hollow macromolecules were able to do.

A striking example of application was published in 2006. The cage of the figure above was used to perform a Diels-Alder reaction between two highly hydrophobic reagents in water [4]. The first step is the inclusion of the reagents inside the cage. They are then appropriately oriented to undergo a Diels-Alder reaction, affording the final product, as indicated in the figure below. Remarkably, only one isomer is isolated (the reaction is regioselective), and it is not the isomer one obtains when the reaction is performed without the cage. Furthermore, if a bowl-shaped macromolecule is used intead of the cage to perform the same reaction, it was observed that 1) a different isomer was produced, 2) a catalytic amount of the ‘bowl’ is enough to ensure a high yield, and 3) the Diels-Alder adduct is released from the bowl after formation. This is a typically enzyme-like behaviour: once formed, the reaction product is no longer compatible with the host, and is therefore released, preventing catalyst inhibition.

Diels-Alder reactions performed inside the cage and bowl-shaped molecules.

Other impressive achievements shown during this talk included crystal engineering (a porous coordination network which is able to adapt its size and shape upon guest absorption or removal [4]) and encapsulation of biological molecules (small nucleotides duplexes formed in the hydrophobic pocket of a water-soluble cage [5]).

References:
[1] J. Am. Chem. Soc. 1990, 112, 5645. DOI: 10.1021/ja00170a042
[2] Nature 1995, 378, 469. DOI:10.1038/378469a0
[3] Acc. Chem. Rev. 2005 38 369. DOI: 10.1021/ar040153h
[4] Science 2006, 312, 251. DOI:10.1126/science.1124985
[5] Nature Chemistry 2009, 1, 53 doi:10.1038/nchem.100

Tamiflu is often refered to as a neuraminidase inhibitor. Viral neuraminidase is a protein (actually, the N in H1N1 refers to Neuraminidase) found on the surface of influenza viruses. Its activity is related to proliferation of the viruses and their reproduction inside infected cells. Oseltamivir acts by blocking this protein. The new viruses are prevented from emerging from infected cells, and if the drug is taken early enough, the spreading of the virus to the organism can be efficiently prevented. Current limitations of this type of treatment are that neuraminidase inhibitors are not renewable (they are consumed doing their job) and some resistance can occur – viral neuraminidase can be protected from the drug’s effect.

From a biomedical point of view, oseltamivir is described as a pro-drug: an ester bond has to be cleaved by enzymes in order to generate the efficient inhibitor. Several synthetic pathways are known. The commercial production starts form the biomolecule shikimic acid (see figure), which limited availability makes the large-scale production of Tamiflu complicated.

(-)-Quinic acid (1) and (-)-shikimic acid (2), two widely used starting materials for the synthesis of ostelamivir phosphate (3). They are extracted from the bark of cincona trees, and from the Chinese star anise, respectively.

Knowing that the daily dose is 150 mg per patient, one easily understands that the search for alternative synthetic routes was fueled by the need of more abundant starting materials. So far, the large scale production is still based on the original protocols developed by Gilead Sciences (I actually wonder whether this name comes from Stephen King’s novel The Dark Tower…), which starts from shikimic acid and involves several potentially hazardous steps (including azide chemistry, see here for details regarding this synthetic route). Alternative syntheses were recently developed at Roche, and by various research groups worldwide. One may cite research led by E. J. Corey, M. Shibasaki, T. Fukuyama or Barry M. Trost, which synthesis is the shortest so far (eight steps, starting from commercially available materials). Anyway, it seems industry’s requirements have not (yet?) allowed any different mode of production than the original one, but the increasing need and long production time (ca. 6 months) may well push one of the above mentioned syntheses to industrial production.

Here is a nice animation showing the mode of action of Tamiflu against influenza virus.

Mechanistic investigations have shown the importance of the Cu(I) and of the carboxylate anion for the cross-coupling to work efficiently. These two species are therefore clearly involved in the catalytic mechanism, which is shown below. The thiol ester undergoes complexation to the copper(I) carboxylate, followed by oxydative addition of the bound thiol ester to the Pd(0) catalyst (1). A copper-mediated transmetallation then allows to form the organopalladium compound (2) and eliminates the copper thiolate and boron derivative byproducts. The final ketone (3) is obtained by a reductive elimination that also regenerates the palladium catalyst.

The reaction described above uses a stoechiometric amount of the copper(I) carboxylate. To make it catalytic, it is necessary to regenerate the Cu(I) oxygenate from the eliminated Cu-SR. This was achieved by performing the reaction with an excess of boronic acid, and under air. The Cu-SR bond is broken to form a thioether with the excess boronic acid, and a copper oxygenate is made available to the system. A particular thiol ester was necessary to make the reaction work:

Ref: L. S. Liebeskind, J. Srogl, J. Am. Chem. Soc. 2000, 122, 11260;
J. M. Villalobos, J. Srogl, L. S. Liebeskind, J. Am. Chem. Soc. 2007, 129, 15734.