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. 2019 Sep 24;10(45):10607-10619.
doi: 10.1039/c9sc03450a. eCollection 2019 Dec 7.

Rapid 13 C NMR Hyperpolarization Delivered From para-hydrogen Enables the Low Concentration Detection and Quantification of Sugars

Free PMC article

Rapid 13 C NMR Hyperpolarization Delivered From para-hydrogen Enables the Low Concentration Detection and Quantification of Sugars

Peter M Richardson et al. Chem Sci. .
Free PMC article


Monosaccharides, such as glucose and fructose, are important to life. In this work we highlight how the rapid delivery of improved 13C detectability for sugars by nuclear magnetic resonance (NMR) can be achieved using the para-hydrogen based NMR hyperpolarization method SABRE-Relay (Signal Amplification by Reversible Exchange-Relay). The significant 13C signal enhancements of 250 at a high field of 9.4 T, and 3100 at a low field of 1 T, enable the detection of trace amounts of these materials as well as the quantification of their tautomeric makeup. Using studies on 13C and 2H isotopically labelled agents we demonstrate how hyperpolarization lifetime (T 1) values can be extended, and how singlet states with long lifetimes can be created. The precise quantification of d-glucose-13C6-d 7 at the millimolar concentration level is shown to be possible within minutes in conjunction with a linear hyperpolarized response as a function of concentration. In addition to the measurements using labelled materials, low concentration detection is also illustrated for millimolar samples with natural abundance 13C where isomeric form quantification can be achieved with a single transient.


Fig. 1
Fig. 1. Schematic for the SABRE-Relay process, with a carrier molecule first undergoing SABRE and subsequently passing polarization into glucose via proton exchange.
Fig. 2
Fig. 2. (a) Single scan 13C{1H} hyperpolarized NMR spectrum (bottom spectrum, ×16) and a thermal reference NMR spectrum (top spectrum) of d-glucose 13C6 acquired with 512 transients, measured at 9.4 T (400 MHz). (b) Analogous 1 T (43 MHz) measurement; the thermal reference spectrum now needed 10 000 transients (bottom spectrum, which has also been vertically scaled ×500 relative to the upper trace). The structures of the two isomeric forms of glucose (α and β) that are detected are shown inset.
Fig. 3
Fig. 3. (a) Single-shot pulse sequence used to measure the hyperpolarized lifetime (T1) values, in this study; 15 points were measured per experiment with a starting angle of 15° which gives 25.8% of the polarization for each point. (b) Comparison of the repolarization and single-shot methods of measuring the hyperpolarized T1 which give the same decay, although the single shot measurement involves significantly less signal intensity. A comparison of hyperpolarization lifetimes between d-glucose-13C6-d7 and d-glucose-13C6 is displayed at (c) 9.4 T and (d) 1 T, using the manual repolarization method in each case. All samples contained 2.5 mg of agent, 4.8 mM of catalyst 3 with 23.8 mM benzyl-d7-amine in 0.65 mL DCM-d2 : DMF (1.6 : 1).
Fig. 4
Fig. 4. SABRE-Relay hyperpolarized 13C NMR spectra. (a) 13C{2H} NMR spectrum (top) and a 13C NMR spectrum (bottom) of 2.5 mg (21.1 mM) of mM d-glucose-1-13C1-d1 (structure inset) with 4.8 mM of 3 and 23.8 mM benzyl-d7-amine in 0.65 mL DCM-d2 : DMF (1.6 : 1) measured at 9.4 T. (b) Corresponding results showing a 13C spectrum of d-glucose-1-13C1-d1 (top) and a 13C{1H} spectrum of d-glucose-1-13C1 (bottom) measured at 1 T. The inset spectrum reflects the boxed region of interest and highlights the presence of two isomers whose signals overlap. (c) Hyperpolarized (top) and 900 scan thermal reference scan (bottom, ×100 scaling) for a 21.1 mM d-glucose-1,2-13C2 sample with 4.8 mM of 3 and 23.8 mM benzyl-d7-amine in DCM-d2 : DMF (1.6 : 1) (0.6 mL) acquired at 1 T. The inset of (c) shows the region of interest and demonstrates that both isomers can be distinguished at 1 T. (d) Structures of the glucose variants used in this study along with their corresponding hyperpolarized T1 values at 1 T. In the case of the 13C6 variants all peaks were analyzed together due to spectral overlap.
Fig. 5
Fig. 5. (a) Pulse sequences used to create and then probe singlet spin order. I produces an anti-phase magnetization, while II refocuses this response to create the more usual in-phase signal; delays are as defined in text. (b) Normalized signal intensities plotted against sample storage time to determine the lifetime of singlet state (9.9 ± 2.3 s) using method II. The sample was repolarized between each data point (blue curve); longitudinal magnetization decays with a lifetime of 1.6 ± 0.2 s. (c) Region of these 13C{1H} NMR spectra showing the hyperpolarized signal derived from d-glucose-1,2-13C2 – (i) method I, (ii) method II and (iii) signal acquired from longitudinal thermal magnetization.
Fig. 6
Fig. 6. (a) Structures of the ten different catalysts used in collecting the SABRE-Relay 13C signal enhancements for d-glucose-13C6-d7 shown in (b); the largest enhancement was observed for d22-5. (c) Corresponding 13C signal enhancement factors achieved using pre-catalyst d22-5 and the indicated NH containing materials ammonia (NH3), benzylamine (BnNH2), benzyl-d7-amine (d7-BnNH2), phenethylamine (PEA) and phenethyl-1,1,2,2,d4-amine (d4-PEA); all samples contained 10.3 mM of the NH source and 4.8 mM of d22-5 in a DCM-d2 : DMF (1.6 : 1) solution that was shaken at 60.6 G for ten seconds. (d) Polarization transfer field dependence on the signal gain for a d-glucose-13C6-d7 sample with 4.8 mM of 3 in a DCM-d2 : DMF (1.6 : 1) solution measured at 1 T; PTF obtained using a set of bespoke Halbach arrays. (e) Signal gain as a function of shake time for a d-glucose-13C6-d7 sample using 4.8 mM of d22-5 in a DCM-d2 : DMF (1.6 : 1) solution; a bi-exponential equation was used to fit the data which highlights the process of polarization build up versus p-H2 consumption in the NMR tube.
Fig. 7
Fig. 7. Intensity of SABRE-Relay hyperpolarized 13C{2H} signal response for d-glucose-13C6-d7 from samples containing 23.8 mM benzylamine-d7 and 4.8 mM of d22-5 in 0.65 ml of DCM : DMF (1.6 : 1 mixture) at 9.4 T as a function of glucose concentration. In each case the sample was shaken for 10 s in a 60.6 G magnetic field. These data have been fitted to a single exponential build-up function (red line) and a linear change (black line) over the range 0–20 mM. The fitted lines have been extrapolated to show the potential theoretical signal increase with concentration. Inset shows an expansion to highlight the linear range.
Fig. 8
Fig. 8. (a) SABRE-Relay hyperpolarized 13C{2H} NMR spectrum of 19.9 mM of 13C6-d7-fructose with 23.8 mM benzyl-d7-amine and 4.8 mM [IrCl(COD)(IMes)] of d22-5 in a 0.65 ml DCM-d2 : DMF (1.6 : 1) mixture measured at 9.4 T (top spectrum) with the corresponding thermal reference, averaged over 512 scans (bottom spectrum, 18 h). (b) Expansion of (a) to highlight the different isomeric forms indicated by their C-2 resonances.
Fig. 9
Fig. 9. (a) 13C{1H} NMR spectra acquired for 40 mM of d-fructose (natural 13C abundance) with 23.8 mM benzyl-d7-amine and 4.8 mM of d22-5 in a 0.65 ml DCM-d2 : DMF (1.6 : 1) mixture measured at 9.4 T. The bottom spectrum shows the result of signal averaging over 1024 scans (approx. 17 h), the middle spectrum represents the single scan SABRE-Relay hyperpolarization measurement and the bottom spectrum shows the 16 SABRE-Relay accumulation. (b) shows an analogous measurement for 40 mM d-glucose (natural 13C abundance) with 23.8 mM benzyl-d7-amine and 4.8 mM of d22-5 in a 0.65 ml DCM-d2 : DMF (1.6 : 1) mixture measured at 9.4 T. The bottom spectrum in (b) shows a single scan hyperpolarized SABRE-Relay experiment while the top spectrum shows the result from the accumulation of 16 hyperpolarized FIDs. The vertical scaling of the three spectra was selected to establish comparable levels of noise.

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