Isolation and Identification of a Urinary Biomarker for Lung Cancer: 27-Nor-5β-Cholestane-3α,7α,12α,24 R ,25 S Pentol Glucuronide and Its Deuterated Analog.

An untargeted metabolomic study identified four potential lung cancer diagnostic biomarkers in human urine. One of the potential biomarkers was an unidentified feature possessing a m / z value of 561+. "561+" was isolated from human urine and tentatively identified as 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol glucuronide with unknown C24,25 stereochemistry using 1 H NMR and mass spectrometry. In a prior report, the C24,25 stereochemistry of the aglycone, 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol, was found to be 24 S ,25 R through GC analysis of the acetonide-TMS derivative. An authentic sample was prepared and found not to have the same stereochemistry as "561+". To identify the C24,25 stereochemistry, four C24,C25 diastereoisomeric alcohols of 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol were prepared from chiral amino acids. Using an LCMS method, the C24,C25 stereochemistry of the "561+" aglycone was determined to be 24 R ,25 S . With the correct aglycone in hand, it was coupled with glucuronic acid to complete the first reported synthesis of 27-nor-5β-cholestane-3α,7α,12α,24 R ,25S pentol glucuronide. Deuterium labeled 27-nor-5β-cholestane-3α,7α,12α,24 R ,25 S pentol was also synthesized for use as an internal standard for MS quantitation.