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.
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!
Having worked in the oligonucleotide area where one often wants to snap on a label such as a fluorophore in a site-specific way, I came to appreciate the need for the “ideal reaction;” things are hard enough in this area without messy labeling methods.
Here’s what an ideal conjugation reaction might look like:
- One step; no protecting group manipulations.
- Fast at room temperature.
- Specific. No cross-reactivity at other sites, double labeling, etc.
- No catalyst, no additive, no external stimulus.
- No by-products hanging around that have to be removed.
In the context of bigger molecules such as polymers and biopolymers, one also has to consider compatibility with their chemistry and synthesis. Let’s stick with oligonucleotides for the moment, though this discussion applies pretty readily to peptides.
In this and the next post, we’ll consider some methods that approach this ideal.
The traditional methods for labeling and bioconjugation are no fun.
As it stands, the classic approach to oligonucleotide modification is a challenge. First, you synthesize an oligonucleotide that has a protected amine or thiol hanging off somewhere, namely from a nucleobase, the 3’-phosphate terminus, or the 5’-alcohol terminus. You then deprotect it and treat the resultant amine or thiol with the desired labeling agent, e.g., an NHS ester (for amine labeling) or a maleimide or iodoacetamide (for thiol labeling). Such reactions are quite imperfect, proceeding in modest yield and selectivity. A tedious purification is then required to remove the desired labeled material from unreacted starting material, labeling by-products, and other oligonucleotides that somehow aren’t the right ones. What a blast!
There has to be a better way…
The ideal reaction: What fits the bill?
An attractive approach is a spontaneous reaction between the label and a handle that you’ve installed synthetically (and site-specifically) on the oligonucleotide. Let’s stick with the simplicity of addition reactions, recognizing that there are some other great approaches such as Bertozzi’s Staudinger ligation.
To start with, you would synthesize an oligonucleotide with a handle on it so you have a chance to zero in on that site and that site only. Check.
Now we need a label that bears a moiety that will recognize the handle (and only the handle) and snap onto it quickly and without any help. The term “spring-loaded” often gets used.
Sharpless’s concept of click chemistry readily comes to mind. The de facto standard click reaction, virtually synonymous with the term “click,” is the copper-catalyzed Huisgen 1,3-dipolar cycloaddition between azides and alkynes, an addition reaction that leaves behind a robust triazole linkage. This reaction has enabled some wonderful things in the world of biomolecule labeling and conjugation. But still, there’s that copper. Not quite ideal. We’ll hear more about this from Jack Hodges in the next post.
Okay, what about metal-free click reactions? There are a growing number of copper-free azide-alkyne cycloadditions, e.g., with strained cycloalkynes (Bertozzi and others); a great idea. Again, more on that in the next post.
Inverse-Demand Diels-Alder reactions of tetrazines
Here’s another addition reaction that I like, which draws on the 50+ year-old inverse-demand Diels-Alder reaction between tetrazines and strained alkenes to give dihydrodiazines. Okay, technically, this is an addition-elimination reaction that produces dinitrogen, but I think we can live with that as a by-product. On the oligonucleotide front, Jäschke and co-workers showed that norbornenes could be incorporated into the nucleic acid using normal phosphoramidite chemistry. Subsequent reaction with various 1,2,4,5-tetrazines, e.g., the dansyl-bearing compound shown, occurred simply by mixing at room temperature in neutral aqueous solution; no catalyst, no other reagents, no help required. (See also O’Reilly’s very recent example involving polymer functionalization.)
Things are starting to look rather ideal!
For those of you that need to modify biomolecules and polymers, what’s your favorite approach to the ideal reaction?