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Heterocycles via alkyl-Heck-type reactions

How do you make saturated heterocycles via construction of the 3,4-C-C bond?

Erik Alexanian and co-workers at UNC have just published a nice report on the use of alkyl iodides in intramolecular Heck-type reactions, producing oxygen and nitrogen heterocycles by such a bond construction.  This is a significant result for several reasons, so let’s take a look.

A bit of background on this bond construction

You have a few basic choices as indicated in the electronic disconnection shown below, where ionic and radical chemistry are considered.

While there are examples of each, this general disconnection is usually not a first choice.  β-hetero anions are prone to β-elimination; β-hetero cations may cyclize onto the heteroatom, and β-hetero radicals… well, they involve radical chemistry.  Though having said that, β-hetero radicals are some of the best radical cyclizations out there, adding to tethered alkenes at rates higher than the benchmark 5-hexenyl radical.

The most powerful way to make this bond involves organometallic chemistry, generally involving two unsaturated sites (Heck chemistry, ring-closing metathesis, eneyne chemistry).  But what if we want one partner to be an sp3 carbon?  Specifically, what if we want to do a transition-metal-catalyzed reaction where a metal resides on the sp3 carbon?

Enter the Heck reaction

The classic Heck reaction comes to mind (hey, Nobel Prize and all), but primary organopalladium species tend to β-eliminate before they can cyclize (if there is a suitably-disposed β-hydrogen).

In 2007, Firmansjah and Fu reported intramolecular alkyl-Heck-type reactions in carbocyclic systems by tinkering with the palladium catalyst, finding that the rate of cyclization could beat out β-hydride elimination.  However, the reaction appears to be limited to primary halides and terminal alkenes.

The current work

Now, Alexanian and co-workers have shown that the alkyl-Heck reaction can be accomplished with good old tetrakis(triphenylphosphine)palladium.  Below you can see the use of a primary iodide with a tethered alkene, producing hexahydroindoles in good yield.  A variety of alkene types are tolerated. Note the use of 10 atm carbon monoxide; more on that later.

In contrast to Fu’s work, which was believed to involve a two-electron oxidative addition of the palladium(0) into the C-halogen bond, Alexanian has evidence that the UNC chemistry proceeds via one-electron chemistry.  Hence, single-electron transfer (SET) from the Pd(0) to the alkyl halide results in C-I bond cleavage to produce a β-hetero radical, which cyclizes and captures Pd(I)-iodide to give the organopalladium intermediate shown.  Normal β-hydride elimination, the last part of a classic Heck cyclization, then ensues.

It’s interesting to note that carbon monoxide is required for primary halides to work.  But secondary halides don’t need it, as shown below. [Edit – Second structure revised to correct missing oxygen.]

This is some promising work for sure, providing a nice alternative to known methods involving more classical radical conditions.  Let’s hope the Alexanian group will be able to tune the reaction a bit to make it as practical as possible; it would really fill a niche.

Happy holidays, everyone!

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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!

Toward the ideal reaction – Part 2

Toward the Ideal Reaction – Part 2

In Part 1, the concept of an ideal reaction for biomolecule labeling was outlined:  One step, no protecting groups, fast, efficient, specific, no catalyst or reagents, no external stimulus, and easy cleanup.  Jäschke’s work on oligonucleotide labeling via the inverse-demand Diels-Alder cycloaddition of tetrazines with norbornenes was featured.

Let’s look at some other chemistry and invite an expert guest, Jack Hodges, to chime in.  He also has a few things to say about big pharma versus small companies.

What about click chemistry?  Well, about that copper…

When one thinks of simple addition reactions for biomolecule labeling, click chemistry comes to mind.  Sharpless’s copper-catalyzed dipolar cycloaddition of azides with alkynes to produce triazoles is the de facto standard click reaction, but, well, it uses copper.  Not ideal.  Plus, the copper may cause problems in the applications we’re focusing on.

Naturally, ingenious chemists have jumped on that problem.

Copper-free click reactions – dial in some strain, maybe change the dipole

How do you speed up dipolar cycloadditions to alkynes without using copper?

You can fool around with the electronics of the alkyne, but then you run the risk of making it susceptible to unwanted reactions.

Alternatively, it’s well-known that dipolar cycloadditions are faster with strained dipolarophiles.  Bertozzi harnessed this effect for click chemistry via cyclooctynes (so-called SPAAC reactions, for strain-promoted alkyne-azide cycloadditions).  Others have joined the fray.

Another approach is to swap out the azide for a speedier nitrone dipole (SPANC reactions), as reported by the groups of van Delft and McKay.

Here’s an example of what we’re talking about, using van Delft’s bicyclononynes (BCNs):

Insights from a guest expert:  Dr. Jack Hodges

To give us an inside look at the state of the art in this area, we have with us my colleague Jack Hodges, ex-WLPD/Pfizer, who now leads the chemistry effort at Berry & Associates, Inc. (B&A), a firm that specializes in nucleic acids chemistry.  Some of you may recall Jack’s prominence in the early days of combinatorial chemistry, where he published seminal articles on the use of polymer-supported scavengers.  He has recently negotiated a scientific and business relationship between B&A and SynAffix, B.V. to provide a line of SPAAC reagents for the oligonucleotide field that are derived from van Delft’s BCN.

Will Pearson:  Jack, what are some of the challenges of using click chemistry in the oligonucleotide field?  

Jack Hodges:  There are plenty of examples of people who have used Cu-catalyzed Click reactions on oligonucleotides but there are also other examples where the Cu(I) catalyst has been reported to cause partial degradation of the oligo.  The partial degradation paper we always note in our product literature is Kanan, M.W., et al., Nature, 2004, 431, 545-9.  There are probably other similar reports.  This problem doesn’t seem too surprising to me since both Cu(I) and Cu(II) can coordinate to heteroatoms, and nucleic acids are full of heteroatoms.  Whether or not you believe Cu(I) is a serious problem around oligos, my feeling is that if you don’t need it, why bother?  There are numerous recipes for making Cu(I) in-situ for traditional Click reactions and often it takes a fair amount of investigation to find the one that will work for your application.  The Cu-free approach does away with all that.  Just put the strained alkyne and the azide together and the triazole forms.  The old fashioned expression is “dump and stir”.  Somehow “dump and stir” doesn’t have as nice a connotation as “Cu-free Click” or the sound of the quirky acronym “SPAAC” (short for strain-promoted azide/alkyne cycloaddition), but in practice it is just that simple.

WP:  For copper-free click reactions of azides with alkynes, there are quite a few competing techniques.  Would you please tell us about your path to selecting van Delft’s BCNs, giving us a little perspective on the various technologies that are available?  What made BCNs your top choice for commercialization?  (B&A also sells compounds based on Schultz & Pigge’s MFCOs.)

JH:  There are two things that attracted us to BCN.  First the BCN synthesis looks attractive from a commercial standpoint.  It starts with 1,5-cyclooctadiene (which is about as cheap an 8-membered ring compound as one can buy) and requires relatively few synthetic steps, each of which look to be scalable.  Second, BCN has the lowest calculated LogP value among the other popular cyclooctynes in the literature.  This makes BCN desirable in terms of maintaining the viable biological properties for oligos and other biological macromolecules to which it is attached.  DIFO is pretty close to BCN in terms of LogP but it is much harder to synthesize.  To be fair, BCN-OH is still hugely more lipophilic than propargyl alcohol.  So if you absolutely need to avoid lipophilicity, maybe the traditional Cu-catalyzed Click is your best bet.

WP:  What should we know about IP and licensing in this field?

JH:  So far as I am aware, the only issued US Patent for Cu-free Click reagents and methods belongs to UC Berkeley (US 7,807,619).  There are other published patent applications that may well lead to additional patent coverage in this field.  It seems pretty clear that SPAAC reagents and methods fall outside the broad coverage of the Scripps patents that cover Sharpless’s Cu-Catalyzed Click methodology.

WP:  The classic ways to attach things to oligonucleotides involve amine acylation or thiol alkylation.  Besides click chemistry, what other bioorthogonal conjugation methods do you believe are useful for the modification of oligonucleotides?

JH:  Another Click reaction, oxime formation, is gaining steam with oligos.  B&A now sells some oxyamine and aldehyde reagents that can be incorporated during oligo synthesis or via post-synthetic modification.  Based upon their current popularity, we will be expanding our oxyamine product offerings.

WP:  While I’ve got you here, is there anything you’d like to say about your experience of working in a small chemistry company versus your time in big pharma?

JH:  On the whole both experiences have been very positive.  I had a great run in the pharma industry when it was still a vibrant place for doing top notch research.  For most of my 22-year career in pharma, drug companies embraced the financial risk of drug discovery.  That made it fun to be a chemist working in big pharma.  I don’t think the same fun is there today because drug companies got too big via mergers.  As they grew, the lower productivity of research staff that occurred while they were distracted immediately following a merger (when there is an inevitable re-evaluation, repositioning, and reorganization of research endeavors) somehow became part of the justification for the industry to reduce the overall amount of research investment.  To me, pharma companies today have all caught the same disease.  They all seem downright fearful of research investment.  To the scientists in the trenches, it can appear as if their management puts as high a priority on trying to minimize risk as it does trying to discover drugs.  Perhaps Bruce Roth’s recent article in C&E News says it better than I do here. (WP:  Links here and at Chemjobber here; see also Firestone’s perspective here and at In the Pipeline here.)

Today, I am very happy working in a privately-held specialty chemical company.  The ‘you eat if you can sell it’ attitude of small company life certainly keeps a scientist’s daily activities interesting, especially in a world where foreign competitors seem to always have a lower labor cost.  This environment drives Berry & Associates to work on the most difficult problems of our customers.  There we can provide products of tremendous value to cutting edge biological technologies.  To highly educated and experienced chemists, such work is as fun as it is profitable.

WP:  You’ve been working with heterocycles for most of your career.  Looking back so far, what accomplishment in that field do you feel is most significant?

JH:  Ten years later, I still get inquiries about my lithiooxazole paper that was published in JOC in 1991.  It didn’t feel like super-remarkable chemistry at the time but people still read that paper.  Editorially speaking, I guess that is a darn good reason to publish results that initially surprise you.  You can save others from the same surprises!

Toward the ideal reaction – Part 1

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.
  • Quantitative.
  • 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?