Parahydrogen-induced polarization enables the single-scan NMR detection of a 236 kDa biopolymer at nanomolar concentrations

Nuclear magnetic resonance (NMR) experiments utilizing parahydrogen-induced polarization (PHIP) were performed to elucidate the PHIP activity of the synthetic 236 kDa biopolymer poly-γ-(4-propargyloxy)-benzyl-L-glutamate) (PPOBLG). The homopolypeptide was successfully hyperpolarized and the enhanced signals were detected in 11.7 T solution NMR as a function of the PPOBLG concentration. The hydrogenation with parahydrogen caused signal enhancements of 800 and more for the vinyl protons of the side chain at low substrate concentration. As a result of this high enhancement factor, even at 13 nM of PPOBLG, a single scan 1H-NMR detection of the hyperpolarized protons was possible, owing to the combination of hyperpolarization and density of PHIP active sites.

Experimental Methods

Chemicals
Chemicals used for the synthesis were purchased from the manufacturers or chemical distributors Sigma-Aldrich, Honeywell, Carl Roth, Fisher Scientific and TCI. The extra dry solvents used were taken from septa bottles, stored over molecular sieve. The n-hexane used in the NCA synthesis and purification, was dried over sodium and benzophenone under argon and then distilled. , centered in the NMR tube. The end of the capillary was close to the bottom of the tube and below the position of the detection coil so that the gas flow passes through as much of the sample as possible. For more information about the automatized PHIP-setup and the measurement conditions, see our recent publication. 2 The parahydrogen (p-H2) enrichment was performed with a parahydrogen generator from Advanced Research Systems Inc. comprising a DE204A cryostat and an ARS-4HW compressor. The cryostat is cooled to 30 K and delivers >95 % para enriched hydrogen. For the hydrogenation this parahydrogen was bubbled through the sample with a pressure of 7 bar at 25 °C. After bubbling stopped the gas flow was immediately changed to a static helium overpressure (7 bar). For time critical measurement (0,071µ and lower) the measurement was started immediately after bubbling without gas changing. Afterwards a single scan 1 H-NMR spectrum was recorded. This momentary image represents the so-called PHIP spectrum. For comparison of the enhancement, another proton spectrum is subsequently recorded, at a time point when the temporary polarization has completely decayed (thermally relaxed spectrum). All normal NMR experiments were acquired with pulse sequences from the Bruker pulse sequence library. The PHIP spectra were acquired with customized pulse sequences adjusted to the PHIP setup. 2 All chemical shifts (δ) are reported in ppm relative to TMS (δ = 0.00). In order to always achieve the maximum signal, a flip angle of 90° and 16 scans are used for the normal proton measurements. 45° flip angle and a single scan are used for the PHIP measurements. 45° is the standard excitation pulse in PASADENA PHIP for direct detection of two-spin antiphase nuclear spin order. 1

Processing of the Spectra
The spectra a processed using MestreLab Research MestReNova 14.2. If not described otherwise, the spectrum was processed as indicated in Table 1  Integration ranges 5.4-5.1 ppm 5.5-5.0 ppm*** *At concentrations below 0.66 µM a multiple point baseline correction was carried out. ** As the PHIP signals are dispersive the line broadening was reduced to minimize signal cancellation. *** At concentrations below 0.15 µM it was difficult to calculate the integral as the sum of the modulus of the respective PHIP signal due to the poor signal to noise ratio, the integrals obtained are more error loaded.

NMR of PPOBLG
No PHIP studies of the respective monomers could be performed for the following reasons: The N-carboxy anhydride (NCA), which is converted into the polymer by ring-opening polymerization, cannot be used for testing the PHIP activity due to its instability in air. The corresponding amino acid ester, which is converted into the NCA, could not be tested because it was not compatible with the selected measurement system, due to insolubility in the selected solvent. Thus only the polymer was investigated and the results will be described herein.

Precipitation
To obtain a product spectrum without catalyst residues, e.g. main paper figure 1B, the sample must be precipitated. For the precipitation experiment a mixture of 16 mg of the PPOBLG and 2.8 mg [Rh(dppb)(COD)]BF4 in 1 mL CDCl3 was used. Hydrogen was bubbled through the solution at 7 bar for 20 min. The resulting product can be precipitated out of the catalysator-containing reaction mixture by adding the reaction mixture to methanol and washing the precipitate twice with small amounts of cold water. Afterwards, the precipitated polymer can be dissolved in chloroform-d again for NMR analysis.
In addition to the two products, the spectrum obtained also contains impurities which were introduced in the course of precipitation, drying and redissolution. The signal at 2.88 ppm may be assigned to water in acetone which is typical for a freshly rinsed but not yet completely dried NMR tube. However, the impurity has no influence on further measurements and comparability.

Information about the catalyst
The catalyst is commercially available with the CAS number: 79255-71-3. 1  The catalyst complex is activated by hydrogenation. The COD ligand is detached which slightly changes the structure of the complex and its resulting NMR spectrum.
Assignment after reaction:

Extrapolation of the effective enhancement related to the thermal signal
To estimate an enhancement factor Ɛ for those concentrations where no thermal signal is available for calculation, we plotted the results of the intensity of the thermal signals of the series of concentrations and fitted the data with a second order polynomial. The obtained equation ( = 0.4316 2 + 0.4469 + 0.0004) is used to estimate the intensity of thermal signals at the low concentrations of 0.071 and 0.053 µM. Below those concentrations no thermal signals could be determined. Figure 6: Control of the reaction progress during the hydrogenation, in order to observe the change of the signals over time. This also allowed to determine the end point of the reaction with 16 mg PPOBLG, which is then precipitated. Due to the presence of the catalyst, the signals of the newly formed allyl and alkyl moiety may be shifted compared to the assignment of the precipitated products. Between single 1 H experiments in the pseudo-2D experiments a hydrogenation time of 10 s was used. There is no additional delay between the acquisition of the spectra. (500MHz, 298K, in CDCl3).