Figures, Schemes and SI for "Isolation and Identification of a Urinary Biomarker for Lung Cancer: 27-Nor-5β-cholestane-3α,7α,12α,24R,25S pentol glucuronide and its Deuterated Analog"
Abstract: 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 by 1H 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 24S,25R by 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 24R,25S. With the correct aglycone in hand, it was coupled with glucuronic acid, to compete the first reported synthesis of 27-nor-5β-cholestane-3α,7α,12α,24R,25S pentol glucuronide. Deuterium labeled 27-nor-5β-cholestane-3α,7α,12α,24R,25S pentol was also synthesized for use as an internal standard for MS quantitation.
Scheme 1. Retrosynthetic analysis for 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol glucuronides 1a-d.
Scheme 2. Synthesis of chiral alkene 5a.
Scheme 3. Synthesis of chiral aglycone 2a.
Scheme 4. Synthesis of 1a
Scheme 5. Synthesis of 16
Figure 1. ESI+ mass spectrum of 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol. Pseudomolecular ion m/z = 385.3+ used for detection and quantitation is marked with an asterisk (*). The peaks marked with an arrow are additional fragment ions derived from the parent molecular ion m/z = 439.3+ ([M+H]+).
Figure 2. Stereochemistry of 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol released from human urine treated with β-glucuronidase. ESI+ mass extracted chromatograms at m/z 358.5+. (A) Untreated urine. (B) Urine treated with β-glucuronidase. (C) Mixture of the four diastereoisomeric aglycones 2a-d.
Figure 3. Comparison of LCMS retention times between “561+” in urine vs standard 1a. (A) ESI- extracted mass chromatograms at m/z = 613.34+ (M-H). (B) ESI+ extracted mass chromatograms at m/z = 615.34 (M+H).
Figure 4. Identification of “561+”. (A) ESI- mass spectrum; (B) ESI+ mass spectrum. Peaks marked with an asterisk (*) are fragment ions derived from m/z 615.3+; (C) ESI+ MS/MS of m/z 615.34.
