The covalent modification of therapeutic biomolecules has been broadly explored, leading to a number of clinically approved modified protein drugs. These modifications are typically intended to address challenges arising in biopharmaceutical practice by promoting improved stability and shelf life of therapeutic proteins in formulation, or modifying pharmacokinetics in the body. Toward these objectives, covalent modification with poly(ethylene glycol) (PEG) has been a common direction. Here, a platform approach to biopharmaceutical modification is described that relies on noncovalent, supramolecular host-guest interactions to endow proteins with prosthetic functionality. Specifically, a series of cucurbit [7]uril (CB [7])-PEG conjugates are shown to substantially increase the stability of three distinct protein drugs in formulation. Leveraging the known and high-affinity interaction between CB [7] and an N-terminal aromatic residue on one specific protein drug, insulin, further results in altering of its pharmacological properties in vivo by extending activity in a manner dependent on molecular weight of the attached PEG chain. Supramolecular modification of therapeutic proteins affords a noncovalent route to modify its properties, improving protein stability and activity as a formulation excipient. Furthermore, this offers a modular approach to append functionality to biopharmaceuticals by noncovalent modification with other molecules or polymers, for applications in formulation or therapy. supramolecular chemistry | protein engineering | drug delivery | protein formulation

We present a building block approach toward functionalized CB[7] derivatives by the condensation of methylene bridged glycoluril hexamer 1 and glycoluril bis(cyclic ethers) 2 and 12. The CB[7] derivatives Me2CB[7] and CyCB[7] are highly soluble in water (264 mM and 181 mM, respectively). As a result of the high intrinsic solubility of Me2CB[7], it is able to solubilize the insoluble benzimidazole drug albendazole. The reaction of hexamer 1 with glycoluril derivative 12 which bears a primary alkyl chloride group gives CB[7] derivative 18 in 16% isolated yield. Compound 18 reacts with NaN3 to yield azide-substituted CB[7] 19 in 81% yield which subsequently undergoes click reaction with propargylammonium chloride (21) to yield CB[7] derivative 20 in 95% yield which bears a covalently attached triazolyl ammonium group along its equator. The results of NMR spectroscopy (1H, variable temperature, and DOSY) and electrospray mass spectrometry establish that 20 undergoes self-assembly to form the cyclic tetrameric assembly (204) in aqueous solution. CB[7] derivatives bearing reactive functional groups (e.g. N3, Cl) are now available for incorporation into more complex functional systems.

Mixed self-assembly of ligand 1, 2, 1,6-hexanediamine (HDA) and Pd(NO3)2 afforded Fujita-type metal organic polyhedron MOP1 (diameter ≈ 8.2 nm) which is covalently functionalized with an average of 18 cucurbit[7]uril (CB[7]) units as evidenced by 1H NMR, diffusion ordered spectroscopy NMR and transmission electron microscopy measurements. By virtue of the host-guest properties of CB[7], the inner cavity of MOP can be rendered hydrophobic by using octadecyl HDA (3) as guest during the self-assembly process. The hydrophobic cavity was successfully utilized to trap the hydrophobic dye Nile Red (NR) and the anticancer drug Doxorubicin (DOX). The stimuli responsive release of encapsulated NR or DOX occurs: 1) upon addition of a competitive binder (e.g. adamantane ammonium (ADA)) for CB[7], 2) by a dual pH-chemical stimulus involving the protonation state change of adamantane carboxylate at pH 5.8, and 3) a dual pH-photochemical stimulus involving photoisomerization of trans-6 to cis-6 at pH 5.8. NR is released from NR@MOP2 within HeLa cancer cells. This body of work suggests that the covalent attachment of cucurbit[n]uril to metal organic polyhedra constitutes a promising vehicle for the development of both diagnostic and therapeutic nanoparticles.

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.

The recognition of human growth hormone (hGH) by the synthetic host molecule cucurbit[7]uril (Q7) was predicted on the basis of its N-terminal phenylalanine. An aqueous-compatible resin with covalently immobilized Q7 groups was prepared and shown to recognize native insulin and hGH in simple and complex mixtures.