1 SCIENTIFIC REPORTS | (2019) 9:19683 | https://doi.org/10.1038/s41598-019-56063-0 www.nature.com/scientificreports [ 13 C 6 ,D 8 ]2-deoxyglucose phosphorylation by hexokinase shows selectivity for the β-anomer Gal Sapir, Talia Harris, Sivaranjan Uppala, Atara Nardi-Schreiber, Jacob Sosna, J. Moshe Gomori & Rachel Katz-Brull * A non-radioactive 2-deoxyglucose (2DG) analog has been developed here for hyperpolarized magnetic resonance investigations. The analog, [ 13 C 6 ,D 8 ]2DG, showed 13% polarization in solution (27,000- fold signal enhancement at the C 1 site), following a dissolution-DNP hyperpolarization process. The phosphorylation of this analog by yeast hexokinase (yHK) was monitored in real-time with a temporal resolution of 1 s. We show that yHK selectively utilizes the β anomer of the 2DG analog, thus revealing a surprising anomeric specifcity of this reaction. Such anomeric selectivity was not observed for the reaction of yHK or bacterial glucokinase with a hyperpolarized glucose analog. yHK is highly similar to the human HK-2, which is overexpressed in malignancy. Thus, the current fnding may shed a new light on a fundamental enzyme activity which is utilized in the most widespread molecular imaging technology for cancer detection – positron-emission tomography with 18 F-2DG. Te hexokinase (HK) family of enzymes carry out the frst step in glycolysis and therefore are of key importance in cellular metabolism 1 . HKs play an important role in malignancy: one of the earliest observations in malignant transformation is an increased glycolytic fux 2 , which is largely attributed to changes in the activity of HKs. For example, in rapidly growing tumor cells, HK expression is markedly elevated and most of the enzyme is localized to the mitochondrial membrane 3,4 , which provides HK with increased ATP availability resulting in increased activity 5 . HK isoenzymes vary in function and in organ expression. In health, HK-4 (glucokinase, GK) is important in glucose sensing in the pancreas and in the liver 6 while HK-1 and HK-2 mainly function in other tissues such as muscle and brain. Increased or decreased HK expression has been found to correlate with the clinical outcome in a number of disease states 7–20 . Elevated HK-2 expression is correlated with higher histological grade in hepato- cellular carcinoma (HCC) 7 as during the process of HCC tumorigenesis, normal GK expression is silenced, and HK-1 and HK-2 are overexpressed 8,9 . Since the afnity of HK-1 and HK-2 for glucose is higher than that of GK, their expression results in increased glucose utilization which supports HCC tumorigenesis. HK-2 expression was shown to be correlated with the survival of patients with HCC, gastric and colorectal cancer, and WHO grade IV glioblastoma 10–13,20 . Aggressive types of cancer including pancreatic ductal adenocarcinoma 14 , renal cell carcinoma 15 , medulloblastoma 16 , and mouse models of lung and breast cancer 17 are characterized by high levels of HK-2. Other non-malignant diseases with increased HK-2 expression in the liver include fatty liver disease 18 , and hepatitis C virus infection 19 . [ 18 F]Fluoro-2-deoxy-D-glucose (FDG) is the most widely used tracer in positron emission tomography (PET) for detecting, staging, and monitoring of various malignancies 21 . It was previously suggested that the increased HK-2 activity forms the basis for the utility of FDG-PET imaging of malignant tumors 5 . Nuclear magnetic reso- nance (NMR) has been used for the study of glucose and 2-deoxyglucose (2DG) metabolism 22–25 . In 13 C-NMR, as opposed to PET, the chemical evolution of 2DG to 2DG-6-phosphate (2DG6P) can be discerned. Tis property could be useful for diferentiating the efects of glucose transporters expression from those of HK expression in vivo. For the above reasons, glucose and 2DG imaging has been a desired target in magnetic resonance imaging, which does not involve ionizing radiation. MR investigations of glucose and 2DG have been carried out using the gluco-CEST approach 22,26,27 , thermal equilibrium 13 C-NMR with carbon-13 labeled substrates 28 , and deuterium metabolic imaging (DMI) 29 . Department of Radiology, Hadassah Medical Center, Hebrew University of Jerusalem, The Faculty of Medicine, Jerusalem, Israel. *email: rkb@hadassah.org.il OPEN