Synthesis and hyperpolarisation of eNOS substrates for quantification of NO production by 1H NMR spectroscopy

Abstract

Hyperpolarization enhances the intensity of the NMR signals of a molecule, whose in vivo metabolic fate can be monitored by MRI with higher sensitivity. SABRE is a hyperpolarization technique that could potentially be used to image nitric oxide (NO) production in vivo. This would be very important, because NO dysregulation is involved in several pathologies, including cardiovascular ones. The nitric oxide synthase (NOS) pathway leads to NO production via conversion of l-arginine into l-citrulline. NO is a free radical gas with a short half-life in vivo (≈5s), therefore direct NO quantification is challenging. An indirect method - based on quantifying conversion of an l-Arg- to l-Cit-derivative by ¹H NMR spectroscopy - is herein proposed. A small library of pyridyl containing l-Arg derivatives was designed and synthesised. In vitro tests showed that compounds 4a-j and 11a-c were better or equivalent substrates for the eNOS enzyme (NO₂^- production=19-46μM) than native l-Arg (NO₂^- production=25μM). Enzymatic conversion of l-Arg to l-Cit derivatives could be monitored by ¹H NMR. The maximum hyperpolarization achieved by SABRE reached 870-fold NMR signal enhancement, which opens up exciting future perspectives of using these molecules as hyperpolarized MRI tracers in vivo.

Keywords: Hyperpolarization; MRI; Real-time imaging; SABRE; l-Arginine.

Graphical abstract

Fig. 1

In the SABRE process, the iridium catalyst mediates polarization transfer from para-hydrogen to the pyridyl substrate. This process repeats allowing the creation of a solution of hyperpolarised pyridyl substrate.

Fig. 2

Hyperpolarized ¹H spectrum for 4e (in blue) compared to its thermal ¹H spectrum (in red). Thermal spectrum is vertically enlarged by 32 times. Four hyperpolarized signals can be observed: A and C correspond to the free compound in solution, while B and D are those of the fraction bound to the catalyst.

Fig. 3

Analogue 4e was unstable during in vitro spectroscopy test, as the amide bond was cleaved producing l-arginine and 4-aminopyridine. Signals A and C belong to compound 4e, while B and D correspond to 4-aminopyridine.

Fig. 4

1D COSY corresponding to the benzylic methylene of a 1:1 mixture of 4f and the corresponding citrulline derivative 21b when overlapping aromatic signals at δ 8.70 are irradiated. Signals A, B, D and E correspond to 4f, whilst signal C belong to 21b. The broad signal at δ 4.70 is a solvent artefact.

Fig. 5

T₁ relaxation times (in seconds) for pyridyl protons in compounds 4a,e,f at pH = 7.4 and 37 °C.

Scheme 1

Reaction catalysed by NOS enzymes leading to NO production.

Scheme 2

Synthesis of first generation compounds 4a–j. Reagents and conditions: (a) HATU, TEA, CH₂Cl₂, r.t., 5 h.; (b) TFA/CH₂Cl₂ 95:5, r.t., 2 h.; (c) LiOH, THF/H₂O 4:1, r.t., overnight.

Scheme 3

Formation of 2d and 2i. Reagents and conditions: (a) SOCl₂, MeOH, r.t., overnight.

Scheme 4

Synthesis of second generation compounds 11a–c. Reagents and conditions: (a) 4-picolylamine, HATU, TEA, DCM, r.t., 5 h.; (b) CH₃SO₂Cl, Py, 40 °C, 2 h; (c) NH₂OH·HCl, K₂CO₃, EtOH, r.t., 1 h.; (d) HCl 4 M in dioxane, r.t., 1 h.; (e) SOCl₂, MeOH, r.t., overnight; (f) Boc₂O, TEA, DCM, r.t., 1.5 h.; (g) LiOH, THF/H₂O 4:1, r.t., overnight; (h) KOCN, HCl, 70 °C, 1 h.; (i) Ac₂O, NaOH, H₂O, r.t., 3 h.

Scheme 5

Synthesis of l-citrulline derivatives 21a–c. Reagents and conditions: (a) HCl 4 M in dioxane, r.t., 1 h; (b) 3-picolylamine, HATU, TEA, DCM, r.t., 5 h.; (c) 4-aminopyridine, HATU, TEA, DCM, r.t., 5 h.