Feasibility of sensing small molecule thiols using hyperpolarized [13C]cyanate
Cornelius von Morze1, Chloe Najac1, Robert R Flavell1, David E Korenchan1, Pavithra Viswanath1, Lucas Carvajal1, John Kurhanewicz1, Sabrina M Ronen1, Daniel B Vigneron1, and David M Wilson1

1Department of Radiology and Biomedical Imaging, University of California, San Francisco, San Francisco, CA, United States


The purpose of this study was to show basic feasibility of non-invasively detecting small molecule thiols using hyperpolarized [13C]cyanate. We detected rapid formation of the expected hyperpolarized S-[13C]carbamyl thiol adduct after adding cysteine to liquid hyperpolarized [13C]cyanate samples. This work demonstrates a new non-enzymatic approach for detecting small molecule thiols such as reduced glutathione, which could be very useful for research on oxidative stress.


Cyanate, which exists in chemical equilibrium with urea in aqueous urea solutions, interacts with certain nucleophilic functional groups, in particular rapidly attaching to (“carbamylating”) thiol groups in a reversible manner1 (Fig. 1A). The purpose of this study was to show the feasibility of non-invasively monitoring small molecule thiol levels by temporary attachment of the hyperpolarized (HP) 13C label in situ via this reaction. Small molecule thiols can be selectively detected using this method, while adducts formed with larger molecular weight thiols such as cysteine residues in albumin are not detected due to short T1 times. Reduced glutathione (GSH) is the most prominent small molecule thiol in vivo (cellular concentration ~0.5-10mM)2. Since GSH is a key cellular antioxidant, non-invasive methods for GSH measurement could be very useful for biological research on oxidative stress. We have hyperpolarized [13C]cyanate and detected a well-resolved small molecule HP S-carbamyl thiol adduct in vitro, showing basic feasibility of this HP MR approach for small molecule thiol detection.


Potassium [13C]cyanate (Icon Isotopes, Summit, NJ) was dissolved to ~4M in H2O/glycerol, and hyperpolarized via dissolution DNP to yield 40mM aqueous HP [13C]cyanate. For proof of principle, 20mM cysteine was added to the sample after dissolution. Non-localized HP spectra were acquired in a clinical 3T MRI scanner. Liquid HP samples were transported to the scanner in the field of a handheld magnet, to minimize loss of polarization due to scalar coupling with fast-relaxing 14N 3. Thermal spectra of authentic unlabeled product S-carbamyl cysteine (MP Biomedicals, Burlingame, CA) as well as the 13C-labeled raw material were also acquired at 11.7T to confirm the identity of the observed HP product peak.


The solution T1 of [13C]cyanate was 58s at 3T and polarization level was 3% (back-calculated to time of dissolution). In addition to the primary cyanate peak and some impurities, a small HP peak was observed at ~8ppm downfield from the urea reference (Fig. 1B), corresponding to the expected thiol adduct S-[13C]carbamyl cysteine. This peak was absent from samples lacking thiol and the chemical shift of this peak matched the 11.7T thermal spectrum of authentic product S-carbamyl cysteine (Fig. 1C). The peak was also clearly observed in thermal spectra of the thiol-containing samples, obtained after the HP experiment.


Formation of a temporary HP thiol adduct is a new, non-enzymatic approach for detection of small molecule thiols. We are working to determine whether sufficient cellular uptake of cyanate would occur to facilitate this reaction in vivo or in cells on the timescale of a HP experiment. Cyanate has relatively low toxicity in small doses (much lower than cyanide). In a prior study, no adverse effects were observed in 60 mice given daily i.p. injections of 32mg/kg potassium cyanate over a five-month period.4


We gratefully acknowledge support from NIH K01DK099451 and P41EB013598.


1. Stark GR. J Biol Chem. 1964. 2. Meister & Anderson. Ann Rev Biochem. 1983. 3. Shang H et al. MRM. 2015. 4. Cerami A et al. J Pharmacology & Experimental Therapeutics. 1973.


Fig. 1. Reversible formation of S-carbamyl thiol adduct (A) and detection of this adduct using HP 13C NMR (B). Resonance corresponding to S-carbamyl cysteine adduct (173ppm) was observed only after addition of cysteine to HP cyanate dissolution (B, left spectrum). Identity of this product was confirmed by high resolution NMR of authentic product (C).

Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)