key: cord-0949438-1f396qxs
authors: Jeong, Hye Jin; Min, Sein; Kim, Sarah; Namgoong, Sung Keon; Jeong, Keunhong
title: Hyperpolarization study on remdesivir with its biological reaction monitoring via signal amplification by reversible exchange
date: 2022-02-02
journal: RSC advances
DOI: 10.1039/d2ra00062h
sha: a58fdf9999f3f5738c68d962e066666f51986020
doc_id: 949438
cord_uid: 1f396qxs

Our experiments indicate hyperpolarized proton signals in the entire structure of remdesivir are obtained due to a long-distance polarization transfer by para-hydrogen. SABRE-based biological real-time reaction monitoring, by using a protein enzyme under mild conditions is carried out. It represents the first successful para-hydrogen based hyperpolarization application in biological reaction monitoring.

and used without further purification (Its purity was confirmed via 1 H-NMR). Methanol-d 4 (CD 3 OD, 99.8 atom %D, Eurisotop) and dimethyl sulfoxide-d 6 (CD 3 SOCD 3 , 99.8 atom %D, Eurisotop) were also used in the form obtained. In the experiment using Pre-catalyst1 (IMes-Ir, [Ir(IMes)(COD)Cl], 2 mg, 3.1x10 -3 mmol), remdesivir (18 mg, 3.1x10 -2 mmol) was dissolved in CD 3 OD (900 μL) and CD 3 SOCD 3 (900 μL), individually 1 . Pre-catalyst2 (Crabtree's-Ir, [Ir(COD)(PCy 3 )(py)]PF 6 , 2 mg, 2.5x10 -3 mmol) was resolved to solution of remdesivir(15 mg, 2.5x10 -2 mmol) in CD 3 OD (900 μL).

Signal. 1 H NMR spectra used for the characterization of remdesivir was acquired on a Bruker Avance ̊̊̊ NMR spectrometer operating at a 1 H resonance frequency of 300MHz and referenced to the residual CH 3 peak of methanol-d 4 (δ=3.31) or CH 3 peak of dimethyl sulfoxide-d 6 (δ =2.50). The para-hydrogen generator was composed as a home-built instrument, in which hydrogen gas (Hanmi gas, >99.9%, a mixture the spin isomers ortho-hydrogen and para-hydrogen) was allowed to pass through a heat exchanger filled with a FeO(OH) catalyst (Sigma Aldrich) 2-4 . This instrument was filled with liquid nitrogen in a Dewar flask generating ca. 50% para-hydrogen. In each experiment, parahydrogen continuously flowed into the drug candidate sample at a rate of 6mL/min at 23 ̊C and 1 atm. The following system was established and developed to gain various magnetic field data: the power supply was GPS-1850D (Bench Power Supply, Linear DC). A shielded coil wound with copper-coated wire and a shielded coil on top was 200 mm in diameter and 190 mm in height. The magnetic field through the shielded coil was controlled by setting the current, which was in the range of 0-5 A. The magnetic field produced by the modulated current was measured using a Lakeshore Gauss meter.

For the calculation of the 1 H signal enhancement factor (fold), the signals of samples amplified through hyperpolarization and those of non-amplified samples were compared by using the below equation 1 . The enhancement factor was estimated using the raw integral of the hyperpolarized and non-polarized spectra. 

The esterase from the porcine liver (PLE, lyophilized powder, 15 units/mg) was purchased from Sigma-Aldrich.

The PLE (20mg, 300units) was suspended in deuterium oxide (1mL). The sample for reaction monitoring was prepared with remdesivir (18mg, 3.1x10 -2 mmol) and pre-catalyst1 (IMes-Ir, [Ir(IMes)(COD)Cl], 2mg, 3.1x10 -3 mmol) in CD 3 SOCD 3 (800μL) by adding enzymic stock solution (100μL). The sample solution was kept constant at pH 8 by the addition of 0.1N sodium hydroxide solution [5] [6] [7] . The reaction mixture was moved to a 5mm NMR tube and activated by para-hydrogen for 20 min at 110G. The bubbling process by para-hydrogen substituted the stirring of solution for homogeneity. After activation, individual hyperpolarized spectrum was obtained by using the same method of hyperpolarized signal measurement procedure described above, at 10min intervals over 2 hours. 

Organic Process Research and Development

 Figure S3 1 H spectra of remdesivir: enzymatic hydrolysis after 120 min (black spectrum) and amplified through hyperpolarization (red spectrum) at the same time (left); Amplification number of protons from hyperpolarized enzymatic hydrolysis of remdesivir after 120min (right).