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Alzheimer's Disease

Congo Red

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.

 

35 comments on “Congo Red”

  1. Eric says:

    “suggest the binding binding mode”

    1. Derek Lowe says:

      Fixed fixed, thanks thanks!

      1. Daniel Jones says:

        Ha ha!! (Okay, that was childish of me. If it helps, I apologize twice)

  2. NHR_GUY says:

    WOW!! What a wonderful well written article Derek. Taking something as mundane as a dye like Congo Red, delving back into it’s history, explaining the history of dyes in general, then moving forward in history up to now. Finally, finishing off with Congo Red, NMR, and crystallography. Well done. The reason I still read this blog even though I’m out of the labs.

  3. b says:

    I had a project saved by Sudan Red dyes in grad school. You can use them as indicators in an ozonolysis reaction – select the right one on the reactivity spectrum and you can get selective oxidation in one part of your molecule (in this case an alkene) without oxidizing less reactive parts (in this case a benzylic methylene). You can even tune to the reactivity of different alkenes.

    I never looked into the derivation of the naming of the dyes, I always assumed they were discovered in some rock in Sudan, but I’m guessing they were named as you described above for the Congo Red dyes. Interesting.

  4. Anon says:

    Methylene blue can also bind to some of these amyloid fibrils. In fact a company named TauRx has a methylene blue based dye that inhibits Tau aggregates.

    1. Derek Lowe says:

      It needs to be noted that tau is not amyloid, and the TauRx’s drug has failed in the clinic in an Alzheimer’s trial.

      1. luysii says:

        Derek:

        Tau filaments have the classic cross beta structure of amyloid [ Cell vol. 180 pp. 633 – 644 ’20 ]. Figure 2 p. 635 is particularly revealing. Just as any brain can have a seizure, just about every protein can be made to form amyloid.

        Perhaps what you meant to say is that the tau protein and the amyloid precursor protein from whence spring the Abeta peptide and friends are different proteins.

        1. Lane Simonian says:

          Methylene blue does appear to have an effect on amyloid aggregation as well, but what effect it has is disputed–whether it increases the conversion of “toxic” amyloid oligomers into amyloid fibrils or whether it prevents the conversion of “toxic” amyloid oligomers into amyloid fibrils.

          Part of the reason for these contradictory results is that methylene blue inhibits nitration and nitration’s role in plaque formation may be variable

          https://www.jneurosci.org/content/jneuro/36/46/11693.full.pdf

          https://www.ncbi.nlm.nih.gov/pubmed/21903077

  5. gcc says:

    Great write-up, Derek! It also made me think of this very cool article from the New York Times Magazine in 2018:

    How One Man Is Recreating Lost Colors
    https://www.nytimes.com/2018/09/05/t-magazine/pedro-da-costa-felgueiras-recreating-lost-colors.html

  6. Vader says:

    So … it basically forms a congo line on the surface of the amyloid fibril?

    I’ll show myself out.

  7. Mad Chemist says:

    And we owe the existence of the dye industry to early efforts in med chem and total synthesis. William Henry Perkin discovered the first synthetic dye, mauveine when he was trying to synthesize quinine. He had no chance of actually making quinine with his approach, but organic chemistry wasn’t sufficiently advanced then to realize that.

    1. eub says:

      Yeah, it’s been deeply intertwined back and forth. The first antibiotic, Prontosil, was a red azo dye as well as being a sulfonamide, and was found by a Bayer program that tested large numbers of dyes. They got lucky, in that the color turned out to be irrelevant, leading to sulfanilamide and the sulfa drug revolution, but that’s how it goes.

  8. RTW says:

    Early in my career I uses Anthraquinone dyes as starting materials for anticancer major ot minor grove DNA binders. Started out with bright red and orange starting materials to light yellow to white products! Was really interesting chemistry. Color changes told us when most of the reactions were complete because even a small amount of starting material left would result in some pretty bright color. Could be pretty sure if it wasn’t complete and we washed the solid products we were able to remove all of the starting materials.

  9. Project Osprey says:

    Early chemistry was almost entirely a German business – everyone had chemists but they had chemical industries – but after WWI the Treaty of Versailles imposed crippling war debt on Germany which was paid in all sorts of ways, including intellectual property. Patents for asprin, dyes, surfactants, and Harber Process were licenced to the allies. In the US the Office of Alien Property Custodian seized whole factories, processes and all. You can debate the morality of if but sudden distribution of technology did give 20th century chemistry and early kick.

  10. Derek Freyberg says:

    Congo Red may not now be used as an in vivo diagnostic, but there are still dyes that are – for example Indocyanine Green (ICG), which is used as a diagnostic for liver function: give IV injection of ICG, wait a while, take blood sample, look at decrease in ICG content, and it gives you an indication of how well the liver is working.

    1. Ian Malone says:

      Additionally some of the PET tracers are also derived from these dyes, amyloid beta tracers Pittsburgh compound B and flutemetamol are both based on methylene yellow.

  11. johnnyboy says:

    Thanks for this article Derek. In my line of work (pathology) dyes such as congo red and prussian blue are used on a daily basis, but pathologists and histotechs never really learn the colourful history (and much less the chemistry) behind all the pretty hues that they impart to tissue sections.

  12. The Iron Chemist says:

    “There I was, there I was there I was, in the Congo…”

  13. AnonPatentGeek says:

    And for you patent geeks out there – the gift of Markush structures is also deeply rooted in dyes, courtesy of Eugene Markush. US Application 611,637, filed January 9, 1923. Markush was awarded a patent from the US Patent Office for “Pyrazolone Dye and Process of Making the Same” on August 26, 1924.

  14. Anonymous says:

    A couple of historical notes:
    1. http://news.bbc.co.uk/2/hi/africa/4318419.stm
    “Sudan outraged at namesake dye.
    Tests on rats have shown the dye can cause bladder and liver cancer
    The government of Sudan is angry that a cancer-causing dye at the centre of a food scare in the UK is named after the north African country.
    Sudan’s ambassador has written to the Food Standards Agency, asking it to change the name of Sudan 1 to prevent further harm to Sudan’s reputation. …”

    2. http://www.npr.org/2007/02/13/7366503/the-color-red-a-history-in-textiles
    “A Perfect Red: Empire, Espionage, and the Quest for the Color of Desire.” by Greenfield.
    For a long time, until synthetic dyes came along in the 1800s, valuable red dyes came from cochineal (carmine red).
    “According to Stevens, when the Spaniards got to Mexico in the 1500s, cochineal became the New World’s major export to Europe.” … Slave ships returned to Europe loaded with conchineal as a leg of the New World – Europe – Africa import export triangle.

    3. Didn’t Lansbury (MIT then; Harvard now) develop a Congo Red assay for amyloid fibrils? I’ve heard mixed things about its reliability.

  15. dye trying says:

    once made these photo sensitizing dyes, you’d shine the UV light on the TLC and then all these fun colors would magically appear and then fade away after the UV light was gone.

    it’s easy to track dyes all over the floor with your shoes, makes a mess.

    1. drsnowboard says:

      Best worry about the compounds you can’t see that you are seemingly tracking everywhere…

      1. Zemyla says:

        If you can avoid tracking dyes, then you can avoid tracking invisible chemicals. It’s a useful proxy.

    2. Tourettes of Chemistry says:

      Fond recollections of CR performance with plaques in the Alzheimer’s team meetings and the need to find a surrogate for this surrogate and the catalyzing of a ferret stampede down the rabbit hole (and all the jokes – ‘…constantly repeated as short term memories were not in gear during the ALZ meetings….’). The recent ‘Hits from AI’ for antibiotics post here put some of this thinking into motion as well by structural serendipity.

      As for Sudanese ire directed towards colloquial names, consider the Black Swan event of raising money for a company named ISIS.

      As for tracking invisible compounds around the lab, just make sure to keep you hands clean if you are later handling your smokes. ==> A non-obvious discovery might just be that close: CAS #: 81-07-2. Perhaps some of the bio-hackers would like to lick those floors.

      ‘The Crime and Punishment of IG Farben’ might add some trans-Atlantic perspective from the early days of dye stocks and emergent pharmaceuticals.

      1. Anonymous says:

        “Black Swan event of raising money for a company named ISIS.” – I think I’m confused. Are you talking about the antisense pharma named Isis (now Ionis) or are you talking about the terrorist group Isis? I’m trying to figure out what you mean.

        “CAS #: 81-07-2” – that is saccharin, one of many sweeteners discovered accidentally. I am quoting from wikipedia on several more:
        (1) “Saccharin was produced first in 1879, by Constantin Fahlberg, a chemist working on coal tar derivatives in Ira Remsen’s laboratory at Johns Hopkins University. Fahlberg noticed a sweet taste on his hand one evening, and connected this with the compound benzoic sulfimide on which he had been working that day.”
        (2) “Cyclamate was discovered in 1937 at the University of Illinois by graduate student Michael Sveda. Sveda was working in the lab on the synthesis of anti-fever medication. He put his cigarette down on the lab bench, and, when he put it back in his mouth, he discovered the sweet taste of cyclamate.”
        (3) “Acesulfame potassium was developed after the accidental discovery of a similar compound (5,6-dimethyl-1,2,3-oxathiazin-4(3H)-one 2,2-dioxide) in 1967 by Karl Clauss and Harald Jensen at Hoechst AG. After accidentally dipping his fingers into the chemicals with which he was working, Clauss licked them to pick up a piece of paper.”
        (4) “Sucralose was discovered in 1976 by scientists from Tate & Lyle, working with researchers Leslie Hough and Shashikant Phadnis at Queen Elizabeth College (now part of King’s College London). While researching novel uses of sucrose and its synthetic derivatives, Phadnis was told to “test” a chlorinated sugar compound. Phadnis thought Hough asked him to “taste” it, so he did. He found the compound to be exceptionally sweet.”

        I know the song “Relax” but I never even knew the group was called Frankie Goes to Hollywood. Live and learn … and, hopefully, retain.

        1. Darin Holloway says:

          One of the cell phone companies (Samsung?) Was branding their nfc payment system ISIS as the Caliphate was becoming known to Western journalists

        2. eub says:

          Great collection of cases! This one is particularly fine:
          “Phadnis was told to “test” a chlorinated sugar compound. Phadnis thought Hough asked him to “taste” it, so he did.”

          1. loupgarous says:

            Every time I read about one chemist ordering another to taste something, I remember this.

            (Note Bene: if you’re playing this through speakers and not headphones, it’s definitely NSFW. Possibly NSFF, too.)

          2. Anonymous says:

            I forgot to include another accidental “tasted it” story:
            (5) Aspartame was discovered in 1965 by James M. Schlatter, a chemist working for G.D. Searle & Company. Schlatter had synthesized aspartame as an intermediate step in generating a tetrapeptide of the hormone gastrin, for use in assessing an anti-ulcer drug candidate. He discovered its sweet taste when he licked his finger, which had become contaminated with aspartame, to lift up a piece of paper.

            Whenever I listen to the link provided by loupgarous, I think of “Don’t crush that dwarf, hand me the pliers.” (Different group, same time frame.)

          3. Tourettes of Chemistry says:

            Thanks for now putting up aspartame – still considered one of the finest moments in bio-hacking proof of concept since it first was noticed – the 20 ‘natural’ amino acids coupled in all possible combinations to give 400 dipeptides – 1 in 400 hit rate (!!!) turns aspartame into The Billion Dollar(+) Dipeptide – not bad for a weekend in the garage. In hockey, it is said, you miss all the shots you don’t take.

            Saccharin was put up as ‘first in class’ from a modern organic (coal tar) point of view as the sure to follow me too’s were presumed to be well-known as they are far more recent. For the sake of completeness the lead – lead acetate approach of Romanesque fame should be in the list as a known paleo-organic approach to the issue. Tolerance issues with lead were as anticipated if my recollection is holding up.

            For full transparency, ISIS (now dba Ionis) in Carlsbad, CA, was the proper take. Not recalling that off shore caliphate crowd being a DE company with an EIN.

            If ‘Taste’ and ‘Test’ confusion from eub is the basis of a discovery paradigm, tech transfer discussions over cell phones have got to be a similar matter based upon some of the things that are done. The saying and the hearing chasm on broad band and/or 5G rate of exchange.

          4. Tourettes of Chemistry says:

            RE: Guanine for loupgarous

            A substance first obtained from guano; it is a nucleic base and pairs with cytosine in DNA and RNA.

            Someone had to be bat-dung crazy to first find that stuff.

  16. In Vivo Veritas says:

    The book Toms River, by Dan Fagin, give a great accounting of the transformation of the dye companies into the chemical and pharma companies of today. In the process, they destroyed a quaint seaside town with billions of gallons of industrial waste they didn’t really tell anyone about. It’s a great read.

  17. David Edwards says:

    Reading this latest blog post brings back fond memories of the TV series Connections by James Burke back in the 1970s. He built a broadcasting career at the time, by tying together different historical threads in science in the same manner.

    Which allows me to introduce the phrase “serendipitous happenstance” into my post, as quite a few connections in science relied upon this.

  18. WildCation says:

    Not only was ultramarine saved for only the most precious parts of the painting, it was also one of two materials that had a separate line item on bills to patrons – the other being gold leaf.

    It’s also a collossal pain to work with, as in order to retain its pigmenting power it needs to be ground quite coarsely, giving paint a grainy texture. Synthetic ultramarine is much more tractable! Still, at least it was nontoxic, unlike vermilion (mercury (II) sulfide) or lead white.

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