Synthesis of functionalized cryptophane-A derivatives and assessment of their xenon-binding properties for the construction of hyperpolarized xenon-129 biosensors

This dissertation describes the progress in the synthesis and physical studies of cryptophane-A derivatives for use in the construction of hyperpolarized xenon-129 biosensors. Access to functionalized xenon-129 biosensors has been improved through the development of propargyl substituted cryptophanes to allow for the conjugation of bioactive moieties using the click reaction. New water-soluble cryptophane-A derivatives TAAC and TTPC have been synthesized and subjected to precise measurement of their xenon-binding affinity. These xenon binding constant determinations were made possible through the development of solution-phase xenon handling techniques which allowed access to xenon fluorescence quenching and isothermal titration calorimetry (ITC) assays. The insights gained from these studies have led the way towards an improved synthetic route to functionalizable cryptophanes and a proposed method to systematically vary the chemical shift of encapsulated xenon-129 without sacrificing xenon binding affinity. Finally, access to tri-allyl cryptophane 3.4 has allowed for crystallographic studies of cryptophane conformation as well as the interaction of xenon and other guest molecules with the cryptophane interior

Synthesis of functionalized cryptophane-A derivatives and assessment of their xenon-binding properties for the construction of hyperpolarized xenon-129 biosensors

This dissertation describes the progress in the synthesis and physical studies of cryptophane-A derivatives for use in the construction of hyperpolarized xenon-129 biosensors. Access to functionalized xenon-129 biosensors has been improved through the development of propargyl substituted cryptophanes to allow for the conjugation of bioactive moieties using the click reaction. New water-soluble cryptophane-A derivatives TAAC and TTPC have been synthesized and subjected to precise measurement of their xenon-binding affinity. These xenon binding constant determinations were made possible through the development of solution-phase xenon handling techniques which allowed access to xenon fluorescence quenching and isothermal titration calorimetry (ITC) assays. The insights gained from these studies have led the way towards an improved synthetic route to functionalizable cryptophanes and a proposed method to systematically vary the chemical shift of encapsulated xenon-129 without sacrificing xenon binding affinity. Finally, access to tri-allyl cryptophane 3.4 has allowed for crystallographic studies of cryptophane conformation as well as the interaction of xenon and other guest molecules with the cryptophane interior

Utilizing a Water-Soluble Cryptophane with Fast Xenon Exchange Rates for Picomolar Sensitivity NMR Measurements

Hyperpolarized ¹²⁹Xe chemical exchange saturation transfer (¹²⁹Xe 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 ¹²⁹Xe and rapid accumulation of depolarized ¹²⁹Xe 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 (<i>T</i>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, <i>k</i>_on ≈ 1.5 × 10⁶ M^–1s^–1 and <i>k</i>_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, <i>k</i>_off was estimated to be 1.1 × 10³ s^–1. In Hyper-CEST NMR experiments, the rate of ¹²⁹Xe 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 × 10⁴ ¹²⁹Xe atoms s^–1 (or 4.6 × 10⁴ Xe atoms s^–1, all Xe isotopes), which is more than an order of magnitude faster than <i>k</i>_off, the directly measurable Xe-TAAC exchange rate. This compels us to consider multiple Xe exchange processes for cryptophane-mediated bulk ¹²⁹Xe depolarization, which provide at least 10⁷-fold sensitivity enhancements over directly detected hyperpolarized ¹²⁹Xe NMR signals