Cucurbit[7]uril–Tetramethylrhodamine Conjugate for Direct Sensing and Cellular Imaging

Abstract

This paper describes the design and synthesis of a conjugate (Q7R) comprising the synthetic host cucurbit[7]uril (Q7) linked to the fluorescent dye tetramethylrhodamine (TMR), and the characterization of its optical and guest-binding properties as well as its cellular uptake. Q7R was synthesized in two steps from monofunctionalized azidobutyl-Q7 and NHS-activated TMR. The fluorescence of Q7R is quenched upon guest binding, and this observable was used to determine equilibrium dissociation constant (K) values. Unexpectedly, the K values for guests binding to Q7R and to unmodified Q7 were essentially identical. Therefore, Q7R can directly report binding to Q7 without an energetic penalty due to the conjugated fluorophore. This result demonstrates a potentially general strategy for the design of single-component host-indicator conjugates that respond sensitively to analytes without perturbing the binding properties of the host. The unique properties of Q7R enabled measurement of K values across 3 orders of magnitude and at concentrations as low as 0.7 nM. This result is particularly relevant given the unmatched range of guests and binding affinities demonstrated for Q7. Confocal fluorescence microscopy of live and fixed HT22 neurons revealed the cellular uptake of Q7R and its punctate localization in the cytoplasm. Q7R did not alter cell growth at concentrations up to 2.2 μM over 4 days. These experiments demonstrate the feasibility of Q7R as a direct sensor for guest binding and as a cell-permeable compound for imaging applications.

Chemical Sensors Based on Cucurbit[n]uril Macrocycles

“…[22] 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 including those using strong light sources and lasers, can be established using CBn macrocycles. [34,63,64,65,66,67,68,69,70,71] Da Silva, Hubbard and co-workers demonstrated in vivo use of CB7 in tilapia fish; the encapsulation of odorants such as cadaverine by CB7 tuned down the response of the olfactory receptors. [34,63,64,65,66,67,68,69,70,71] Da Silva, Hubbard and co-workers demonstrated in vivo use of CB7 in tilapia fish; the encapsulation of odorants such as cadaverine by CB7 tuned down the response of the olfactory receptors.…”

“…[72] (However, CBn and CBn complexes can bind to certain proteins and thereby affect some enzymatic functions, e. g., that of type II endonucleases or proteases, as was shown by the groups of Nau and Pischel.) [40,71,76,77,78,79,80,81,82,83,84,85,86] Similarly, functionalisable acyclic CBn derivatives are also available, enabling the instalment of signalling units into the formally dark/transparent cucurbiturils. [40,71,76,77,78,79,80,81,82,83,84,85,86] Similarly, functionalisable acyclic CBn derivatives are also available, enabling the instalment of signalling units into the formally dark/transparent cucurbiturils.…”

Stimuli‐Responsive Cucurbit[n]uril‐Mediated Host‐Guest Complexes on Surfaces

“…[37,38] Recent studies by several groups demonstrated the potential of applying CB[n]-mediated complexes for anticancer therapies, for example as drug carrier or sensors. [39][40][41][42] In the design of dynamic systems with on-demand changes in guest binding by stimuli-responsiveness, it is attractive to exploit CB[n] since they are selective binders while being sensitive to changes in molecular structure of the guests. [43] An early reported example is the shuttling of CB [6] along a triamine chain.…”

Rational design of boron-dipyrromethene (BODIPY) reporter dyes for cucurbit[7]uril

“…Therein, the fluorescence properties of a dye are altered when encapsulated by the host, and when a competitive binder displaces the dye from the cavity, the properties of the non-encapsulated dye are regenerated. This principle has enabled, for example, real-time monitoring of enzymatic activity [18–20], the detection of membrane-transport activity [21] and membrane fusion [22], and even cellular imaging appears to be a potential future prospect [23–24]. …”

“…One possibility is the utilization of monofunctionalized CBs with outer cavity-attached fluorescent dyes [22,24]. This principally allows for the modular construction of various Förster resonance energy transfer (FRET) pairs as demonstrated with a Cy3-attached CB7, or the design of self-inclusion complexes, in which an outer cavity-attached rhodamine was intramolecularly bound in the CB7 cavity.…”