Parahydrogen hyperpolarized NMR detection of underivatized short oligopeptides.

Parahydrogen hyperpolarization has evolved into a versatile tool in NMR, allowing substantial sensitivity enhancements in analysis of biological samples. Herein we show how its application scope can be extended from small metabolites to underivatized oligopeptides in solution. Based on a homologous series of alanine oligomers, we report on an experimental and DFT study on the structure of the oligopeptide and hyperpolarization catalyst complexes formed in the process. We demonstrate that alanine oligomers coordinate to the iridium carbene-based catalyst in three different ways, each giving rise to distinctive hydride signals. Moreover, the exact structures of the transient oligopeptide-catalyst complexes are oligomer-specific. This work gives a first insight into how the organometallic iridium-N-heterocyclic carbene-based parahydrogen hyperpolarization catalyst interacts with biopolymers that have multiple catalyst binding sites. A preliminary application example is demonstrated for oligopeptide detection in urine, a complex biological mixture.

Understanding Parahydrogen Hyperpolarized Urine Spectra: The Case of Adenosine Derivatives.

Parahydrogen hyperpolarization has emerged as a promising tool for sensitivity-enhanced NMR metabolomics. It allows resolution and quantification of NMR signals of certain classes of low-abundance metabolites that would otherwise be undetectable. Applications have been implemented in pharmacokinetics and doping drug detection, demonstrating the versatility of the technique. Yet, in order for the method to be adopted by the analytical community, certain limitations have to be understood and overcome. One such question is NMR signal assignment. At present, the only reliable way to establish the identity of an analyte that gives rise to certain parahydrogen hyperpolarized NMR signals is internal standard addition, which can be laborious. Herein we show that analogously to regular NMR metabolomics, generating libraries of hyperpolarized analyte signals is a viable way to address this limitation. We present hyperpolarized spectral data of adenosines and give an early example of identifying them from a urine sample with the small library. Doing so, we verify the detectability of a class of diagnostically valuable metabolites: adenosine and its derivatives, some of which are cancer biomarkers, and some are central to cellular energy management (e.g., ATP).

Parahydrogen hyperpolarization of minimally altered urine samples for sensitivity enhanced NMR metabolomics.

Parahydrogen hyperpolarization has been shown to enhance NMR sensitivity in urine analysis by several orders of magnitude if urine samples are prepared by solid phase extraction (SPE). We present a different approach, developed for minimal sample alteration before analysis. Removing SPE from the workflow allows to retain a wider range of metabolites and paves the way towards more universal hyperpolarized NMR metabolomics of low abundance metabolites.

Developing Analytical Applications for Parahydrogen Hyperpolarization: Urinary Elimination Pharmacokinetics of Nicotine.

Nuclear magnetic resonance spectroscopy (NMR) is a valuable analytical tool with applications in a vast array of research fields from chemistry and biology to medicine and beyond. NMR is renowned for its straightforward data interpretation and quantitative properties, making it attractive for pharmacokinetic applications, where drug metabolism pathways, concentrations, and kinetics need to be evaluated. However, pharmacologically active compounds and their metabolites in biofluids often appear in minute concentrations, well below the detection limit of NMR. Herein, we demonstrate how parahydrogen hyperpolarization overcomes this sensitivity barrier, allowing us to detect mid-nanomolar concentrations of a drug and a drug metabolite in a biofluid matrix. The performance of the method is demonstrated by monitoring nicotine and cotinine urinary elimination, reflected by their concentrations in urine during the onset and withdrawal from nicotine consumption. An NMR limit of detection of 0.1 µM and a limit of quantitation of 0.7 µM is achieved in a practical pharmacokinetics scenario where precise quantitative and qualitative analysis is desired.

Diastereoselective multicomponent cascade reaction leading to [3.2.0]-heterobicyclic compounds.

A general three-component triple cascade reaction through an iminium-enamine-iminium sequential activation initiated by a hetero-Michael addition to α,ß-unsaturated aldehydes affords [3.2.0]heterobicycles in high diastereoselectivity. The rate and diastereoselectivity of the reaction depended on the (E)-4-heterocrotonate and size of the secondary amine. The enantiomers of the major diastereoisomer of oxa- and azabicyclo[3.2.0]heptane derivatives were separated by enzymatic kinetic resolution with immobilized Candida antarctica Lipase B (CALB), with E values up to 153. The absolute configuration of the nonacylated enantiomer of oxabicyclo[3.2.0]heptane was determined by single crystal X-ray analysis.

Chemoenzymatic synthesis and evaluation of 3-azabicyclo[3.2.0]heptane derivatives as dopaminergic ligands.

New 3-azabicyclo[3.2.0]heptane derivatives were synthesized using a multicomponent reaction. Racemic compounds were efficiently resolved by kinetic resolution with immobilized lipase B of Candida antarctica (Novozym 435). The obtained compounds demonstrated greater binding affinity at D(2L) and D(3) dopamine receptors compared to D(1) binding sites, and individual enantiomers of the same compound possessed distinct affinities.

Synthesis and biological activity of bimorpholine and its carbanucleoside.

A new enantiomerically pure carbacyclic nucleoside analogue with bimorpholine as a nonaromatic nucleobase was synthesized. The nucleoside analogue and bimorpholine were tested for cytotoxicity using an MTT assay and the xCELLigence System. Both assays revealed that compound 3 was highly cytotoxic at a 50 µM concentration while the cytotoxic effect of compound 1 was much less prominent. No antiretroviral activity was detected for this compound. In contrast, it acted as a potent inhibitor of hepatitis C virus (HCV) replication. Most likely this effect originates largely from the cytotoxicity of the compound; however, it is possible that a specific mechanism of HCV inhibition also exists.

rac-2,2'-Bipiperidine-1,1'-diium dibromide.

In the title compound, C(10)H(22)N(2) (2+)·2Br(-), a precursor in the synthesis of organocatalysts, the bipiperidinium ion is located on a twofold rotation axis which passes through the mid-point of the central C-C bond. The piperidinium ring adopts a chair conformation. In the crystal, the cations are linked together by Br(-) ions through N-H⋯Br hydrogen bonds, forming layers parallel to the ab plane.

A novel diastereoselective multicomponent cascade reaction.

A novel multicomponent cascade reaction led to the formation of a strained 3-azabicyclo[3.2.0]heptane derivative 4. The unstable ester 4 was reduced in a one-pot procedure to a stable alcohol 6. The formation of the bicyclic product is highly diastereoselective, predominantly affording one diastereoisomer. The obtained azabicycloheptanes are important pharmacophores.

Enantioselective organocatalytic Michael addition of aldehydes to beta-nitrostyrenes.

The utility of C(2)-symmetric bipiperidine and bimorpholine derivatives as organocatalysts in the Michael addition of enamine intermediates formed from aldehydes to nitroolefins has been demonstrated. The best results were obtained when the reaction was run in the presence of (2R,2'R)-N-iPr-bipiperidine. The products were formed via an enamine intermediate with high diastereo- and enantioselectivity with relatively short reaction times.