Utilizing a water-soluble cryptophane with fast xenon exchange rates for picomolar sensitivity NMR measurements.

Hyperpolarized (129)Xe chemical exchange saturation transfer ((129)Xe Hyper-CEST) NMR is a powerful technique for the ultrasensitive, indirect detection of Xe host molecules (e.g., cryptophane-A). Irradiation at the appropriate Xe-cryptophane resonant radio frequency results in relaxation of the bound hyperpolarized (129)Xe and rapid accumulation of depolarized (129)Xe in bulk solution. The cryptophane effectively "catalyzes" this process by providing a unique molecular environment for spin depolarization to occur, while allowing xenon exchange with the bulk solution during the hyperpolarized lifetime (T(1) ≈ 1 min). Following this scheme, a triacetic acid cryptophane-A derivative (TAAC) was indirectly detected at 1.4 picomolar concentration at 320 K in aqueous solution, which is the record for a single-unit xenon host. To investigate this sensitivity enhancement, the xenon binding kinetics of TAAC in water was studied by NMR exchange lifetime measurement. At 297 K, k(on) ≈ 1.5 × 10(6) M(-1) s(-1) and k(off) = 45 s(-1), which represent the fastest Xe association and dissociation rates measured for a high-affinity, water-soluble xenon host molecule near rt. NMR line width measurements provided similar exchange rates at rt, which we assign to solvent-Xe exchange in TAAC. At 320 K, k(off) was estimated to be 1.1 × 10(3) s(-1). In Hyper-CEST NMR experiments, the rate of (129)Xe depolarization achieved by 14 pM TAAC in the presence of radio frequency (RF) pulses was calculated to be 0.17 µM·s(-1). On a per cryptophane basis, this equates to 1.2 × 10(4)(129)Xe atoms s(-1) (or 4.6 × 10(4) Xe atoms s(-1), all Xe isotopes), which is more than an order of magnitude faster than k(off), the directly measurable Xe-TAAC exchange rate. This compels us to consider multiple Xe exchange processes for cryptophane-mediated bulk (129)Xe depolarization, which provide at least 10(7)-fold sensitivity enhancements over directly detected hyperpolarized (129)Xe NMR signals.

Shorter synthesis of trifunctionalized cryptophane-A derivatives.

Efficient syntheses of trisubstituted cryptophane-A derivatives that are versatile host molecules for many applications are reported. Trihydroxy cryptophane was synthesized in six or seven steps with yields as high as 9.5%. By a different route, trihydroxy cryptophane modified with three propargyl, allyl, or benzyl protecting groups was synthesized with yields of 4.1-5.8% in just six steps. Hyperpolarized (129)Xe NMR chemical shifts of 57-65 ppm were measured for these trisubstituted cryptophanes.

Multiple hindered rotators in a gyroscope-inspired tribenzylamine hemicryptophane.

A gyroscope-inspired tribenzylamine hemicryptophane provides a vehicle for exploring the structure and properties of multiple p-phenylene rotators within one molecule. The hemicryptophane was synthesized in three steps in good overall yield using mild conditions. Three rotator-forming linkers were cyclized to form a rigid cyclotriveratrylene (CTV) stator framework, which was then closed with an amine. The gyroscope-like molecule was characterized by (1)H NMR and (13)C NMR spectroscopy, and the structure was solved by X-ray crystallography. The rigidity of the two-component CTV-trismethylamine stator was investigated by (1)H variable-temperature (VT) NMR experiments and molecular dynamics simulations. These techniques identified gyration of the three p-phenylene rotators on the millisecond time scale at -93 °C, with more dynamic but still hindered motion at room temperature (27 °C). The activation energy for the p-phenylene rotation was determined to be ~10 kcal mol(-1). Due to the propeller arrangement of the p-phenylenes, their rotation is hindered but not strongly correlated. The compact size, simple synthetic route, and molecular motions of this gyroscope-inspired tribenzylamine hemicryptophane make it an attractive starting point for controlling the direction and coupling of rotators within molecular systems.

Crystallographic observation of 'induced fit' in a cryptophane host-guest model system.

Cryptophane-A, comprised of two cyclotriguaiacylenes joined by three ethylene linkers, is a prototypal organic host molecule that binds reversibly to neutral small molecules via London forces. Of note are trifunctionalized, water-soluble cryptophane-A derivatives, which exhibit exceptional affinity for xenon in aqueous solution. In this paper, we report high-resolution X-ray structures of cryptophane-A and trifunctionalized derivatives in crown-crown and crown-saddle conformations, as well as in complexes with water, methanol, xenon or chloroform. Cryptophane internal volume varied by more than 20% across this series, which exemplifies 'induced fit' in a model host-guest system.

Substituent effects on xenon binding affinity and solution behavior of water-soluble cryptophanes.

A water-soluble triacetic acid cryptophane-A derivative (TAAC) was synthesized and determined by isothermal titration calorimetry and fluorescence quenching assay to have a xenon association constant of 33,000 M(-1) at 293 K, which is the largest value measured for any host molecule to date. Fluorescence lifetime measurements of TAAC in the presence of varying amounts of xenon indicated static quenching by the encapsulated xenon and the presence of a second non-xenon-binding conformer in solution. Acid-base titrations and aqueous NMR spectroscopy of TAAC and a previously synthesized tris(triazole propionic acid) cryptophane-A derivative (TTPC) showed how solvation of the carboxylate anions can affect the aqueous behavior of the large, nonpolar cryptophane. Specifically, whereas only the crown-crown conformer of TTPC was observed, a crown-saddle conformer of TAAC was also assigned in aqueous solution.

Cryptophane xenon-129 nuclear magnetic resonance biosensors targeting human carbonic anhydrase.

(129)Xe NMR biosensors are promising agents for early disease detection, especially when their interactions with target biomolecules can perturb (129)Xe chemical shifts well beyond the typical field inhomogeneity of clinical MRI. We introduce human carbonic anhydrase (CA) as a single-binding-site enzyme for studying xenon biosensor-protein interactions. A xenon-binding cryptophane was substituted with linkers of varying lengths to p-benzenesulfonamide to yield nondiastereomeric biosensors with a single (129)Xe NMR resonance. X-ray crystallography confirmed binding of the eight-bond-linked biosensor containing a single xenon atom in the CAII active site. Biosensor dissociation constants (K(d) = 20-110 nM) were determined by isothermal titration calorimetry (ITC) for isozymes CA I and II. The biosensor-CA complexes yielded "bound" hyperpolarized (129)Xe NMR resonances of narrow line width that were shifted by 3.0-7.5 ppm downfield, signifying much larger shifts than seen previously. Moreover, isozyme-specific chemical shifts clearly differentiated CA I and II, despite their similar structures. Thus, xenon biosensors may provide a powerful strategy for diagnosing human diseases characterized by the upregulation of specific CA isozymes and other protein biomarkers.

Designing 129Xe NMR biosensors for matrix metalloproteinase detection.

Xenon-129 biosensors offer an attractive alternative to conventional MRI contrast agents due to the chemical shift sensitivity and large nuclear magnetic signal of hyperpolarized (129)Xe. Here, we report the first enzyme-responsive (129)Xe NMR biosensor. This compound was synthesized in 13 steps by attaching the consensus peptide substrate for matrix metalloproteinase-7 (MMP-7), an enzyme that is upregulated in many cancers, to the xenon-binding organic cage, cryptophane-A. The final coupling step was achieved on solid support in 80-92% yield via a copper (I)-catalyzed [3+2] cycloaddition. In vitro enzymatic cleavage assays were monitored by HPLC and fluorescence spectroscopy. The biosensor was determined to be an excellent substrate for MMP-7 (K(M) = 43 microM, V(max) = 1.3 x 10(-)(8) M s(-1), k(cat)/K(M) = 7,200 M(-1) s(-1)). Enzymatic cleavage of the tryptophan-containing peptide led to a dramatic decrease in Trp fluorescence, lambda(max) = 358 nm. Stern-Volmer analysis gave an association constant of 9000 +/- 1,000 M(-1) at 298 K between the cage and Trp-containing hexapeptide under enzymatic assay conditions. Most promisingly, (129)Xe NMR spectroscopy distinguished between the intact and cleaved biosensors with a 0.5 ppm difference in chemical shift. This difference most likely reflected a change in the electrostatic environment of (129)Xe, caused by the cleavage of three positively charged residues from the C-terminus. This work provides guidelines for the design and application of new enzyme-responsive (129)Xe NMR biosensors.