Parahydrogen-Induced Polarization of Amino Acids

Abstract

Nuclear magnetic resonance (NMR) has become a universal method for biochemical and biomedical studies, including metabolomics, proteomics, and magnetic resonance imaging (MRI). By increasing the signal of selected molecules, the hyperpolarization of nuclear spin has expanded the reach of NMR and MRI even further (e.g. hyperpolarized solid-state NMR and metabolic imaging in vivo). Parahydrogen (pH₂ ) offers a fast and cost-efficient way to achieve hyperpolarization, and the last decade has seen extensive advances, including the synthesis of new tracers, catalysts, and transfer methods. The portfolio of hyperpolarized molecules now includes amino acids, which are of great interest for many applications. Here, we provide an overview of the current literature and developments in the hyperpolarization of amino acids and peptides.

Keywords: amino acids; catalysis; hydrogenation; hyperpolarization; parahydrogen.

Figure 1

Parahydrogen‐based hyperpolarization: Schematic view of the spin states of dihydrogen (a,b), their populations at >300 K and ca. 25 K (c), as well as the basic reactions of hydrogenative (d) and non‐hydrogenative (e) hyperpolarization. The nuclear spins of dihydrogen form four different spin states: three ortho (triplet: T₊, T₀, T₋; a), and one para state (singlet, S; b). At room temperature, all four states are approximately equally populated (c). By cooling the H₂, the equilibrium is shifted toward the para state and almost pure pH₂ is obtained at ca. 25 K (c).[ ³³ , ⁵⁹ ] To use the NMR‐invisible para order, pH₂ is added either permanently to an unsaturated molecule (d,e), or brought into temporary contact through reversible exchange (f). Next, the spin order is converted spontaneously (free evolution) into observable polarization (pol) induced by RF pulses or variations of the magnetic field. The polarization may be observed on the added hydrogen atoms or transferred to other nuclei.

Figure 2

PHIP of AAs by direct hydrogenation. In this example, pH₂ is added to trans‐aminocrotonic acid (TACA) by homogeneous hydrogenation to yield GABA. Although the hydrogenation was successful, only a low signal enhancement was found at pH 2.54, and none was observed at neutral pH.

Figure 3

PHIP of protected AA derivatives by direct hydrogenation. In this example, methyl 2‐acetamidoacrylate is hydrogenated to N‐acetyl‐dl‐alanine methyl ester.

Figure 4

PHIP of N‐acetyldehydroamino acids by direct hydrogenation.[ ¹⁷ , ⁶¹ ] 14 μm ¹³C‐1‐N‐acetylphenylalanine precursor was hydrogenated in 350 μm CD₃OD in the presence of 0.7 μm [Rh(dppb)(COD)]BF₄. The maximum polarization for the fully deuterated precursor was P(¹H)=2.05 % and P(¹³C)=1.3 %. Other hydrogenation reactions were demonstrated for N‐acetyltyrosine, N‐acetyltryptophan, N‐acetylhistidine, and the antibiotic thiostrepton.

Figure 5

PHIP of peptides by hydrogenation of a side chain (PHIP‐label). In this example, a propargyl moiety was added to tyrosine in a peptide. The addition of pH₂ results in hydrogenation of propargyltyrosine to yield a hyperpolarized allyltyrosine peptide. In the sunflower trypsin inhibitor SFTI‐1, polarizations of P(¹H)=9.6 % were obtained in the allyl product (right).

Figure 6

PHIP of large peptides by direct hydrogenation of a side chain (PHIP‐label). SFTI‐Tyr(O‐propargyl) was hydrogenated in [D₄]MeOH with [Rh(dppb)(COD)]BF₄: A) 1D NMR (0.96 mm precursor and 4.1 mm catalyst) and B) ultrafast 2D PHIP‐TOCSY (10 s 3 bar pH₂, single scan, 5 mm precursor, and 2.5 mm catalyst). Signals marked with an asterisk (*) are impurities (adapted from Sauer et al. and Kiryutin et al. ^[67] ). For assignment of the side‐chain protons of the allyl moiety see Figure 5.

Figure 7

PHIP of AA derivatives through side‐arm hydrogenation (PHIP‐SAH). In this example, a protected alanine ethyl acrylate ester was hydrogenated to yield alanine ethyl propanoate in an aqueous solution. Polarizations of P(¹H)=0.70 % and P(¹³C,*)=4.4 % (for a deuterated vinyl group) were achieved at 80 °C and pH 6.5±0.5.

Figure 8

PHIP of AAs by heterogeneous PHIP‐SAH. Here, unprotected amino acid esters were hydrogenated over a heterogeneous catalyst (Rh nanoparticles with preadsorbed N‐acetylcysteine) by bubbling 80 % pH₂ for 30 s through the slurry at 7 bar and 80 °C (0.6–0.9 mm vinylglycine and vinylalanine in D₂O with 56 mm DCl; pH 2). P(¹H)≈1 % was observed on the resulting ethyl esters; the subsequent RF‐induced ¹H to ¹³C polarization transfer yielded P(¹³C)=0.65 % (ethylalanine) and 0.8 % (ethylglycine). The addition of a NaOD solution effected hydrolysis of the ester bond in less than 10 s and resulted in P(¹³C)=0.25 % (alanine) and 0.29 % (glycine).

Figure 9

Schematic representation of the SABRE process in which pH₂ in the presence of an Ir complex hyperpolarizes the substrate.

Figure 10

Schematic representation of the SABRE‐RELAY process. This catalytic cycle enables the hyperpolarization of a target substrate, such as the alcohol ROH, through the transfer of SABRE‐polarized protons from a transfer agent with labile protons, such as the illustrated amine R_tNH₂. Here, R is a suitable polarization receptor, e.g. CH₃ or CH₂‐phenyl.

Figure 11

Scheme of parahydrogen‐induced polarization relayed by proton exchange (PHIP‐X). First, an unsaturated transfer agent (propargyl alcohol) is hydrogenated with pH₂. Then polarization is transferred to the labile OH proton and, finally, the labile proton transfers polarization to the target molecule.