Fluorescent Indocyanines for Color-Coded Surgery

I always get excited when I see an unexpected glowing spot on a TLC plate.  Fluorescence!  I’ve sent many a graduate student on a wild goose chase to try to identify the source of that glow.

My curiosity about these compounds blossomed during my time in industry, where we worked on fluorescent compounds and quenchers of fluorescence.  Adding to the fun was the fact that most small-molecule fluorophores (as opposed to materials like quantum dots) are heterocyclic compounds.

Let’s look at a current application of fluorescent heterocycles and then a bit of chemistry.

Color-Coded Surgery

Check out this recent TEDMED talk by Quyen T. Nguyen, M.D., a Professor of Surgery at UCSD, who gives an entertaining talk on the use of fluorescent dyes to aid in finding and removing every last bit of a tumor.  Her collaborator, Roger Tsien, received the Nobel Prize in Chemistry in 2008 for his work on green fluorescent protein (GFP).

In this work, cell-penetrating peptides (CPPs) composed of polycationic sequences of 6-12 consecutive arginines are covalently attached to a fluorophore (Cy5, more on that later).  These labeled CPPs can penetrate into pretty much any mammalian cell without requiring specific receptors.  To target them to the tumor, they make activatable CPPs (ACPPs).  Generalized cellular uptake of the CPPs is blocked by fusing them to a polyanionic peptide domain (featuring glutamates) via a protease-cleavable linker, neutralizing the polycation domain via the formation of an intramolecular hairpin.  For specificity, the linker is designed so that it is cleaved by certain matrix metalloproteinases that are overexpressed in tumors.  After injection, the ACPPs travel to the tumor, are cleaved by the proteinases, and accumulate in the adjacent tumor tissue.  Shine some light on the area and you can see the tumor, including the normally difficult-to-find margins.  Great stuff!

What are the fluorescent dyes being used?

Nguyen and Tsien use indocyanines (or simply “cyanines” for our purposes), which have been long-known for their fluorescent properties and are popular for photography, laser printing, non-linear optical materials, and of course labeling biomolecules.  They have the typical push-pull electronic structure of many fluorophores; the conjugated system has an electron donor at one end and an acceptor at the other.

Back to Nguyen and Tsien:  Take a look here and here for some of their work.  For many of their applications, they use Cy5 (originally Pharmacia Biotech, now GE Healthcare), a pentamethine cyanine dye shown in the scheme below, which absorbs at 650 nm and emits at 670 nm in the red region of the visible spectrum.  This is well beyond the region of background fluorescence that is found in tissue, so the signal-to-noise is good.  They’re also quite bright.  Another advantage of these long-wavelength emitters is that the light can travel through quite a bit of tissue.

Structurally, an interesting feature is the presence of the geminal dimethyl groups, which not only dictate the landscape of the conjugated pi-system, they keep the dye molecules from stacking, which would allow energy transfer and thus quenching of fluorescence.  One can vary the number of methine groups in the central conjugated region to tune the wavelengths of absorption and emission (Cy3, Cy7, etc.)

How are cyanine dyes made?

The venerable Fischer indole synthesis is used to make the 2,3,3-trimethyl-(3H)-indole starting materials.  N-alkylation with an alkyl halide produces a salt, which is used in an aldol-like double condensation reaction with 1,1,3-trimethoxy-2-propene, an equivalent of malondialdehyde.

If you would like to learn more about the chemistry of cyanine dyes, take a look at Mojzych and Henary’s review on the synthesis of cyanine dyes, or if you can’t access that, download the thesis of Jamie Gragg, who has a nice historical overview of the discovery and development of cyanines.

If fluorescent heterocycles interest you, check back here for occasional posts; there will be more!

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3 responses to “Fluorescent Indocyanines for Color-Coded Surgery”

  1. Morris Slutsky says :

    Definitely cyanines are pretty compounds. And you’re right, there’s definitely something exciting about seeing something fluorescent pop out of a reaction. That particular thrill was somewhat blunted for me when I worked on o-phenylene ethynylene oligomers – I did many, many Sonogashira couplings, and however carefully I set up the reaction, there was always some diyne byproduct. And boy that stuff was fluorescent – fractions containing diyne would actually glow from the meager bit of UV that made it through the lab windowshades. The depressing part was that this invariably meant waste of my acetylene component – which was itself often the product of many, many steps of coupling, purification, and deprotection. That pretty blue glow meant a few percent off of each coupling, and over the synthesis of a long oligomer it really cut into the overall yield quite badly. While I did save some of this byproduct here and there, I was never convinced of it’s stability, and the symmetry of the diyne linkage made NMR characterization quite difficult. A real pain overall, but an unforgettable blue glow.

  2. Will Pearson says :

    Great story. Thanks for sharing it, Morris!

  3. Peter Johnson says :

    What are the challenges ahead for this to become practical in surgery for humans and how long approximately would the process take?

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