Many roots of organic chemistry, and of medicinal chemistry in particular, often originate in what might seem like an unlikely place: the dyestuff industry of the late 19th century. I had already known this to some degree, but writing the historical vignettes in The Chemistry Book really brought it home to me. And if you go further back, dyes and pigments in general have been a huge driving force in the development of what would eventually become the science of chemistry. You can start with ochre cave paintings and work your way up: interesting, vivid, and unusual colors have had value throughout human history, and people have devoted a lot of time and effort into seeking them out and learning how to exploit them. Tyrian purple was so distinctive and so extravagantly costly that its use was reserved for the Roman elite, and in European art you can spot the most important part of a scene in a religious painting from the Middle Ages by where the artist chose to break out the expensive blue “ultramarine” paint, which contained lapis lazuli sourced from what’s now Afghanistan. Why not use the technique for those little vivid blue Egyptian statues? Well, we forgot how to make those.
The discovery and production of things like Prussian Blue and Perkin’s mauve were industrial breakthroughs that made fortunes, and they demonstrated that it was possible to make new dyes and pigments that had never been extracted from plants (or mollusks!) nor ground out of colorful rocks. The Ruhr area in Germany became the center of this business, using the byproducts of the coal-mining industry as chemical feedstocks. The real mining and extraction started to take place in the labs and in the deposits and seams of chemical knowledge, as witness the land rush that occurred as chemists learned how to turn anilines into azo dyes. Those growing collections of synthetic compounds and intermediates became the first screening libraries in the early 20th century – in a weird and unprecedented situation, there were now enough new chemicals that had actually been produced by human hands to make it worth looking through them to see what else they might do other than stain fabric.
There are a lot of stories that pick up at that point, but let’s look at an incident in 1922, when Herman Bennhold was investigating stains for microscopy. The prehistoric search for dyestuffs that would be particularly bright, selective, or colorfast had moved into culture dishes and tissue slices. Iodine’s blue color reaction with starch (well, triiodide) had been discovered in 1814 and had been used since the 1830s to show starchy deposits in plant samples, and microscopy had been tangled up with the dyestuff industry ever since. In the early 1880s, for example, Hans Christian Gram discovered the bacterial staining method that everyone still uses to divide those organisms by the structures of their outer membranes. But the relationships of dyes and what they would stain was still rather fuzzy: the principles for textiles were (a bit) more worked out, but why different cellular structures picked up different colors was impossible to understand in detail. So there was a tremendous amount of sheer experimentation, and in 1922 Bennhold found that the dye Congo Red (CR) was particularly good for amyloid deposits in pathology samples.
Congo Red had been around since the 1880s, and is a story by itself. Bayer wasn’t interested when their chemist Paul Böttiger discovered the compound, so he patented it himself and sold it to rival AGFA. Those were the days! The dye was actually a big hit and nearly knocked Bayer out of the dye business before they ripped off the technique and made their own version. The Congo name appears to have been nothing more than a marketing ploy, taking advantage of popular interest in West Africa at the time, but a whole list of other dyes had “Congo” attached to them once it took off. Bayer and AGFA ended up forming a cartel, but a massive patent lawsuit then tied everything up for a while and enriched the lawyers; it was a spectacular mess.
Now, amyloid had been noticed for centuries as an odd substance that was found in the organs (liver and spleen especially) of elderly patients and those who had died of other causes. Gradually people realized that it was a protein deposit, but no one was sure why or how it formed. Bennhold himself introduced CR as an alarming in vivo diagnostic tool for suspected amyloidosis. This straightforward procedure involved administering an i.v. dose, waiting an interval, then withdrawing plasma to see if the dye had been particularly quickly cleared by its binding to amyloid, and such was the state of medicine that variations on this were used for decades. Meanwhile, back in 1906, Alois Alzheimer had described the odd cerebral histopathological profile of the disease that now bears his name, and application of the CR technique to such samples in the 1920s showed that amyloid protein was involved. It not only stains such proteins red, but undergoes a striking color change to green (birefringence) as the angle of polarized light is changed, a phenomenon also realized around this time.
Congo Red is still used for that purpose today, actually, even though we have numerous other techniques to distinguish proteins in tissue samples. But what’s been unclear is how it actually binds to the amyloid fibrils. Amyloids have been studied since the 1930s by intrepid X-ray crystallographers, whose early work showed that the proteins were orderly enough to provide some data even with the equipment available at the time – much too orderly in a biological context, of course. Over the years there have been numerous proposals for the binding mode of CR to amyloid, but there is no X-ray structure that settles the matter.
This new paper, though, has what may be the best spectral evidence to date. A combination of NMR experiments, visible absorbance spectra, and DFT calculations suggest the binding mode shown at right, which has a different stoichiometry than the previous one from 2011. This proposal also features overlap between the different CR molecules, pi-stacking between the naphthalenes and biphenyls, which seems to do a better job of explaining the optical effects noted in the complex. I doubt that the question will be completely settled by this new publication, but we seem to be getting closer to figuring out how a dye from the 1880s, which invented to improve on plant pigments from antiquity, and was found to stain this protein in the 1920s, might actually be doing it. Science is long and science is hard and complicated and detailed beyond anyone’s ability to really describe, but it does lead somewhere. All kinds of places, in fact.