1) Identifying molecules with long singlet lifetimes, that can be utilized as functional groups for metabolites.
The synthesis of molecules in which singlet states can be populated was approached from two different angles:
Molecules were investigated in which protons and 15N nuclei were utilized
15N nuclei:
Derivatives of triazolindione (TAD, see images for detailed structure), which contains two adjacent 15N nuclei, have been investigated.
Synthesis of three chiral TAD derivatives and one dimer was performed (Figure 1). In these molecules long T1 times were found
(up to 6 minutes for the deuterated compound) in high magnetic fields (B(0)=9.4T). However, singlet states could be populated but did not show long-lived character
(1-2 minutes at 2mT). For the protonated and deuterated compound we speculate that the scalar relaxation of the second kind is a major relaxation mechanism
that prevented the detection of the singlet state. Whereby the chlorinated compound contains two protonated methyl groups that are geometrically close to
the two 15N nuclei and serves as a dominant relaxation source. For the dimer a singlet state could not be populated because the symmetry break was too small or not existent to populate a singlet state.
1H nuclei:
Bromoacrylic acid was identified as a moiety that contains two protons in which a singlet state can be populated
even in protonated water/buffer and under atmospheric oxygen. BrAc was subsequently utilized to funcionalize glucose, alanine, serine, ethanol, propargylaclohol and the long-livety of the singelt state confirmed in aqueous buffer
under oxygen. T1 times of 1-2 seconds for the molecules have been found whereby the singlet lifetime is on the order of 12-15 seconds (Fig 3 shows a comparision of the BrAc-Alanine and BrAc). This finding represents a major result since a universal spin tag has been
identified and proven that several metabolites can be tagged with it. Although the singlet lifetimes are only on the order of 13C T1 times that are already being used in metabolic imaging experiments, we expect that the designed molecules
can now be used for in vitro cell experiments as the singlet states are long-lived in cell media. Additionally, phosphoenol pyruvate (PEP) was found to allow for populating a long-lived singlet state due to an acrylate functionality similar to the one found in BrAc.
PEP is a metabolite of the glycolysis which may be overexpressed in cancer cells and may therefore serve as a probe for cancer.
2) Hyperpolarization of molecules
In order to investigate hyperpolarization possibilities, the para-hydrogen induced polarization (PHIP) techniques was utilized. PHIP utilizes the singlet state of molecular para-hydrogen upon addition to an unsaturated bond to create hyperpolarized signals. Strategies were investigated with coworkers to hyperpolarize symmetric molecules with para-hydrogen with the future goal to generate symmetric molecules with metabolites that may be used as imaging agents. The proof-of-principle experiment showed a polarization of 9% which is well above the threshold of polarization for potential in vivo experiments (>2%). In parallel derivatives of amino acids (alanine and glycine) were synthesized that contain an unsaturated bond for hyperpolarization. The purpose is to hyperpolarize the unsaturated bond on a sidearm, transfer the polarization to the amino acid and cleave of the sidearm to yield hyperpolarized amino acids. First experiments showed that this strategy is successful in polarizing amino acids although signal enhancement factors of 3 were achieved compared to a signal at B(0)=11.7T. The future plan is to synthesize symmetric molecules that contain metabolites, can be hyperpolarized with para-hydrogen and have storage possibilities in singlet states and release hyperpolarized metabolites at a later point of time.
Overview of results:
1) Development of singlet tracers based on metabolites that contain long-lived singlet states in aqueous buffer solutions under oxygen (Publication in preparation)
2) Development of an efficent hyperpolarization strategy of symmetric tracers with parahydrogen (published work: (J. Eills et al. J Magn. Reson. 274, 163-172, 2017).)
3) Hyperpolarization of amino acids with para-hydrogen (disseminated at two conferences: ISMRM in Singapore in 2016 and the Euromar in Aarhus in 2016).
