Sunday, 13 October 2013

#chemclub Reviews: Cucurbiturils

This month's review is by Chad Jones, who is finishing up his PhD studying ion-neutral gas phase interactions. He blogs and podcasts at The Collapsed Wavefunction.

The most amazing type of chemistry is supramolecular chemistry. Now, you may or may not agree with that statement, but it is my goal in this review to at least convince you that supramolecular chemistry is among the most amazing. 

The Dearden group at Brigham Young University (my group) studies cucurbiturils, macrocycles so named for their distinct resemblance to a pumpkin (who are members of the plant family cucurbitaceae).

See? Pumpkins!
This review is by no means a complete look at the chemistry of cucurbiturils. There's no way I could do that - I'd like to graduate and I don't see this review making a big dent in that project. If you'd like more info I'll point you to any number of review articles4, patents5-7special journal issues, and special interest conferences

History and Overview

Robert Behrend initially synthesized cucurbiturils in 1905 by the condensation of glycoluril with formaldehyde.1 Behrend likely synthesised a range of cucurbituril derivatives including CB[5], CB[6], and CB[7]. Although he found that the newly synthesized substance had a high affinity for alkali metals and organic dyes,2 the structure of this interesting compound was not discovered for another 75 years. In 1981 Mock et al. used x-ray crystallography to show that cucurbit[n]urils, CB[n]s, are macrocyclic molecules with n repeating glycoluril monomers.3 Mock’s initial studies of cucurbiturils focused on CB[6]. Since then a number of homologues (CB[5]-CB[8], CB[10], and CB[5]@CB[10]) have been synthesized.4 A wide variety of derivatives have also been synthesized, each with a specific solubility, binding stoichiometry, and guest affinity. 

Often, very small changes in the structure leads to wildly different binding preference. As an example of this, look at cucurbit[5]uril (CB[5]) compared to decamethylcucurbit[5]uril (MC[5]). The only difference is a permethylated equator. 


Now, a passive look at these molecules says that their binding preferences should be about the same. After all most of the binding takes place in the cavity, a location that is far removed from the equatorial methyl groups. In both cases the portal diameter is about 2.4 Å. However, in the Dearden group our experience that these two cages have very different binding preferences. We have found that MC[5] is a much more rigid cage, which in turn affects the interactions inside the cavity. To me this subtle, unexpected change, is a perfect example of why cucurbiturils and their derivatives are such amazing molecules.

Cucurbituril formation and derivatives

For those interested in synthesis (which isn't really my thing) I'll quickly go over a few interesting derivatives of cucurbiturils. The original synthesis, which was later described by Mock et al., involved a two-step reaction. The condensation of glycoluril and excess formaldehyde in HCl was followed by a H2SO4 rinse. A high-yield (~82%), one-step synthesis was achieved by Buschmann by condensation in H2SO4.8 The mechanism of formation of cucurbiturils, however, was not understood until more recently.

The Isaacs group began studying the mechanism of cucurbituril formation by synthesizing a methylene bridged glycoluril dimer. To reduce reactivity, o-xylene units were attached.9,10 The result was two isomers with a pH dependent equilibrium.11 These isomers act as a type of “molecular clip” as the C-shape is able to partially encapsulate a guest, while the S-shaped isomer cannot.12 Understanding the energetics of these isomers was the first step to understanding cucurbituril formation, and led to the discovery of several cucurbituril derivatives.

Some of these derivatives include: i-CB[6] and i-CB[7], which include a single inverted glycoluril ring13; Bis-nor-seco-CB[10], a figure-eight style cucurbituril with single CH2 bridges instead of the paired bridge as seen in cucurbituril14; Nor-seco-CB[6],  a cucurbit[6]uril compound; Nor-seco-CB[6]  with a single attached phenyl group15; hemicucurbit[n]uril, compounds which can be visualized as cucurbiturils cut at the equator16; Bambus[6]uril, a combination of hemicucurbiturils and cucurbiturils17; decamethylcucurbit[5]uril, where methyl groups replace hydrogen atoms on the equator18; and CB*[5], where cyclohexane rings replace the hydrogen atoms on the equator18

Binding Properties

Cucurbiturils are highly symmetric, having two carbonyl portals with a high binding affinity for cationic guests. This binding is due to a combination of ion-dipole interaction involving the carbonyl groups and hydrophobic forces of the inner cavity. The latter is less well-understood, in part because of the lack of studies on neutral compound binding.

Image credit: see ref 24
The electrostatic potential map of CB[8] makes the cation receptor properties of cucurbiturils easy to understand. The electron dense carbonyl groups become efficient binding sites for cations. Also, the inner cavity is electron-deficient, which hints at the cavity acting as a hydrophobic region. Kaifer et al. demonstrated the importance of the inner cavity on binding preferences.19 Methylviologen binds as an inclusion complex, with a binding constant ~106 M-1 in an aqueous, salt-containing solution. This binding is due, in part, to the position of the charged nitrogens, which line up well with the carbonyl portals of cucurbiturils. Interestingly, loss of one of the two charges does not significantly affect the binding affinity. This hints that ion-dipole interactions are not the sole (and perhaps not even the primary) contributors to the binding motif. These results enforce the idea that hydrophobic interactions within the inner cavity play an important role in cucurbituril chemistry.

Understanding the unique binding of cucurbiturils requires an understanding of the microenvironment of the inner cavity.  Solvatochromic probes are useful tools to study such microenvironments. These fluorescent molecules (xanthene, coumarine, oxazine, and cyanine to name a few) have characteristic shifts in their fluorescence that are dependent on the polarity of the solvent environment.20 Nau21 and Wagner,22 using solvatochromic probes, have shown that the inner cavity of cucurbiturils is a very non-polar microenvironment. Further studies showed an environment most similar to n-octanol.23


Beyond the polarity of the inner cavity, the polarizability has also been studied using solvatochromic probes, such as 2,3-diazabicyclo[2.2.2]oct-2-ene. The inverse oscillator strength of the near-UV absorption band of DBO, a solvatochromatic dye, has a linear correlation to the polarizability of the solvent environment. Results showed that the polarizability of the inner cavity is extremely low (0.12), below that of even perfluorohexane.  As a comparison, most other macrocycles have a cavity whose polarizability is similar to that of alkanes or water. The interior of CB[7] to date has the lowest measured polarizability.

Image credit: see ref 24.

An interesting consequence of such low polarizability is a decrease in the radiative decay rate, kr, of fluorescent states.21 This decay rate is determined by the Stickler-Berg equation:

Where Ff is the fluorescence quantum yield, tf is the fluorescence lifetime, and n is the refractive index of the environment. Some fluorescent dyes, when bound to cucurbiturils, display their longest recorded fluorescent lifetimes. This effect is seen even when the fluorescent dye is too large to fit completely inside the cucurbituril, as is the case with DBO.24 

What is even more surprising, given that complexation leads to decreased radiative decay, is that the quantum yield (defined as the ratio of the radiative decay rate to the sum of all decay rates including non-radiative decay) of a dye when complexed with CB[7] increases.21 The significance of this statement is that cucurbituril complexation both decreases the radiative decay rate and increases the fluorescence quantum yield, a scenario that the Stickler-Berg equation seems to prohibit. This paradoxical result seems to suggest that cucurbituril complexation protects the dye from non-radiative relaxation pathways.21


A microenvironment with such a low polarizability that is able to affect the encapsulated molecule to this degree is interesting, albeit insufficient, evidence to support a controversial postulate by Cram that the inner cavity of molecular containers represents a new phase of matter.25

And let's face it: Nothing sounds sexier than the phrase "new phase of matter"

Further evidence for this sexy idea can be found in a more recent paper by Martin Czar. Czar used acridine orange as a probe of the inner cavity characteristics of CB[7]. He studied the fluorescence of acridine orange - both bound to CB[7] and on its own - in solution and in the gas phase. It turns out that when acridine orange is bound inside CB[7] it fluoresces as if it were in the gas phase - even if it isn't! Now that's an oversimplification, and I don't have time to describe the subtle details, but I seriously suggest you read that paper.

In case you're just skimming this GO BACK! That last paragraph (more specifically the paper I linked to in it) is one of the coolest things I've seen. It's really what I would consider one of the best papers I've read this year.

Cucurbiturils as synthetic receptors

Cucurbiturils not only have high binding affinities, but the binding can also be very specific to functional groups present in the guest. Methyl viologen (V2+) is an excellent example of this behavior. The charged nitrogens are positioned such that each is allowed to interact with the carbonyl portals. The reduction of doubly protonated methyl viologen (V2+ + e- → V+) has a smaller affect on binding affinity than would be expected if only interaction with the cucurbituril carbonyl portals were responsible for binding.


Methyl viologen (V2+)

Expecting to see this same behavior in other guests, a collaboration between Gibson, Inoue, Isaacs, Kim, and Kaifer26 instead demonstrated the unique binding of cucurbit[6]uril to ferrocenyl (Fc) guests. While FcOH and FcMe+ were found to bind strongly to CB[7],  binding to the sterically similar FcCOO- was not seen. The lack of binding can be attributed to the negative charge. Binding of FcCOO- to cucurbit[7]uril would  place the carboxylate functional group close to the electron-dense carbonyls of the cucurbituril. While this behavior of electrostatic repulsion is not at all unexpected, it does show a stark difference between cucurbiturils, which are capable of strong binding affinities and unique functional group specificity, and cyclodextrins, which in this case lack functional group specificity, binding to FcCOO- as well as FcMe+ and FcOH. It is this specificity and ultrahigh binding affinity that opens the door to possible applications in biology as synthetic receptors.

The binding of cucurbiturils to amino acids, peptides, and proteins has been studied. CB[6], as a host molecule, is too small to encapsulate amino acids.  However, Buschmann et al. have shown using isothermal titration calorimietry (ITC) that not only do amino acids have a high binding affinity (Ka ~1 × 103 M-1), but that binding affinity has little variability as the size of the amino acid is increased.27 From this data, the Buschmann group determined that the amino acids have very little interaction with the inner cavity and instead form externally bound complexes. Tao and coworkers later showed that the cavity of CB[7] was large enough to encapsulate two equivalents of phenylalanine, leucine, or tyrosine inside the cavity.28


An interesting application to cucurbiturils as synthetic receptors was demonstrated by Urbach et al. Capitalizing on the strong binding affinity of CB[7] to phenylalanine, a direct assay for insulin is reported. A fluorescent dye is first complexed with CB[7], which acts as a fluorescent quencher. When aliquots of insulin are added an increase in fluorescence is seen as the phenylalanine residue binds within CB[7], displacing the fluorescent dye. The effect is quantitative, and is seen even in a mixture of common blood proteins.29

Image credit: see ref 29.
In this review I may not have convinced you that supramolecular chemistry is the most amazing type of chemistry, but hopefully I've at least shown you something cool. 

References

1.         Behrend, R.; Meyer, E.; Rusche, F., Justus Liebigs Ann. Chem. 1905,  (339), 1-37.
2.         Meyer, E., Inaugural-Dissertation, Heidelberg, Germany. 1904.
3.         Freeman, W. A.; Mock, W. L.; Shih, N. Y., J. Am. Chem. Soc. 1981, 103, 7367.
4.         Nau, W.; Scherman, O. A., Israel Journal of Chemistry 2011, 51 (5-6), 492-494.
5.         Kim, K.; Park, K. M.; Ko, Y.; Selvapalam, N.; Nagarajan, E. R. Stationary Phase and Column Using Cucurbituril Bonded Silica Gel, and Seperation Method of Taxol Using the Column. Aug. 23, 2011.
6.         Inoue, Y.; Rekharsky, M.; Kim, K.; Ko, Y.; Selvapalam, N. Method for Determination of Presence or Absence of Peptide Compound PYY. Aug. 9, 2011.
7.         Ciaramitaro, D. A.; Zentner, B. A.; Lieux, A. J.; Merrit, A. R. Thermosetting Coatings for Particulate Materials and Methods of Application. Sep. 6, 2011.
8.         Buschmann, H.-J.; Fink, H.; Schollmeyer, E. Preparation of cucurbituril. DE19603377A1, 1997.
9.         Witt, D.; Lagona, J.; Damkaci, F.; Fettinger, J.; Isaacs, G. L., Org. Lett. 2000, 2, 755-758.
10.       Wu, A.; Chakraborty, A.; Witt, D.; Lagona, J.; Damkaci, F.; Ofori, M. A.; Chiles, J. K.; Fettinger, J.; Isaacs, G. L., J. Org. Chem. 2002, 67, 5817-5830.
11.       Chakraborty, A.; Wu, A.; Witt, D.; Lagona, J.; Fettinger, J.; Isaacs, G. L., J. Am. Chem. Soc. 2002, 124, 8297-8306.
12.       Lagona, J.; Fettinger, J.; Isaacs, G. L., J. Org. Chem. 2005, 70, 10381-10392.
13.       Isaacs, G. L.; Park, S. K.; Liu, S.; Ko, Y.; Selvapalam, N.; Kim, H.; Zavalij, P.; Kim, G. H.; Lee, H. S.; Kim, K., J. Am. Chem. Soc. 2005127, 18000-18001.
14.       Huang, W. H.; Liu, S.; Zavalij, P.; Isaacs, G. L., J. Am. Chem. Soc. 2006, 128, 14744-14745.
15.       Huang, W. H.; Zavalij, P.; Isaacs, G. L., Org. Lett. 2008, 10, 2577-2580.
16.       Miyahara, Y.; Goto, K.; Oka, M.; Inazu, T., Angew. Chem. Int. Ed. 2004, 43, 5019-5022.
17.       Svec, J.; Necas, M.; Sindelar, V., Angew. Chem. Int. Ed. 2010, 49, 2378-2381.
18.       Day, A.; Arnold, A.; Blanch, R., Molecules 2003, 8, 74-84.
19.       Kaifer, A. E.; Li, W.; Yi, S., Israel Journal of Chemistry 2011, 51 (5-6), 496-505.
20.       Reichardt, C., Solvents and Solvent Effects in Organic Chemistry. Willey-VCH: Weinheim, 1988.
21.       Nau, W. M.; Mohanty, J., Int. J. Photoenergy 2005, 7, 133-141.
22.       Rankin, M. A.; Wagner, B. D., Supramol. Chem.  2004, 16 (7), 513-519.
23.       Mohanty, J.; Nau, W. M., Angew. Chem. Int. Ed. 2005, 44 (24), 3750-3754.
24.       Nau, W. M.; Florea, M.; Assaf, K. I., Israel Journal of Chemistry 2011, 51 (5-6), 559-577.
25.       Cram, D. J., Nature 1992, 356, 29-36.
26.       Rekharsky, M. V.; Mori, T.; Yang, C.; Ko, Y. H.; Selvapalam, N.; Kim, H.; Sobransingh, D.; Kaifer, A. E.; Liu, S.; Isaacs, L.; Chen, W.; Moghaddam, S.; Gilson, M. K.; Kim, K.; Inoue, Y., PNAS 2007, 104 (52), 20737-20742.
27.       Buschmann, H. J.; Schollmeyer, E.; Mutihac, L., Thermochim. Acta 2003, 399, 203-208.
28.       Yi, J. M.; Zhang, Y.; Cong, H.; Xue, S.; Tao, Z., J. Mol. Struct. 2009, 933, 112-117.
29.       Urbach, A. R.; Ramalingam, V., Israel Journal of Chemistry 2011, 51 (5-6), 664-678.
30.       Isaacs, L. Israel Journal of Chemistry 2011, 51 (5-6), 578-591.
31.       Stancl, M.; Svec, J.; Sindelar, V., Israel Journal of Chemistry 2011, 51 (5-6), 592-599.


3 comments:

  1. Thanks a lot for the great summary! We were working with betaCD before but I never had the opportunity to look at the lifetimes of complexated molecules. If the effects are so striking perhaps the "mobility" within the cavity could be measured by monitoring the fluorescence lifetime of single molecules.

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    1. Cyclodextrins are cool molecules as well. They don't have quite the same amount of specificity that cucurbiturils do, though. Probably because CD conformations are more "flexible" than CBs.

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