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

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 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 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 24R,25S. 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,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.


Introduction
Lung cancer, known as lung carcinoma, is the most common cause of cancer deaths in the United States [1].This is primarily because most patients are diagnosed in advanced disease states.The 5-year survival rate is between 18% and 21% [1].Early diagnosis and treatment lead to a better prognosis.An untargeted metabolomic study identified four potential urinary metabolite biomarkers for lung cancer diagnosis [2].The four biomarkers, creatine riboside (CR), N-acetylneuraminic acid (NANA), cortisol sulfate, and the indeterminate feature "561+", were found to correctly identify a patient's cancer status (+/−cancer) [2].When "561+" was isolated from human urine, m/z 561+ was discovered to be a fragment ion of m/z 615+ (see Supplementary Materials).By searching the chemical literature and MS databases for compounds with similar fragmentation patterns, "561+" was tentatively identified as 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol glucuronide of unknown C24,25 stereochemistry.
We intended to prepare an authentic standard 24S,25R pentol to confirm the identification of "561+" in our sample.During the course of our research, we discovered that the reported C24,C25 stereochemistry did not match "561+".Now, we report the synthesis of the four 24,25 diastereoisomeric alcohols of 27-nor-5β-cholestane-3α,7α,12α,24,25 pentol employing a chiral pool approach and the identity of C24,C25 stereochemistry of "561+".
The synthesis of the cross metathesis precursor alkene 5a began with the diazotization of L-allothreonine (6a) in water (Scheme 2), followed by the conversion of the carboxylic acid to a methyl ester with 7a trimethyl orthoformate [15].Diol 7a was protected as dibenzyl ether 8a before reduction of the methyl ester with DIBAL-H to afford aldehyde 9a in 60% yield.Treatment of aldehyde 9a with methyltriphenylphosphonium iodide afforded alkene 5a in 80% yield.The remaining diastereoisomeric alkenes (5b-d) were prepared in a similar manner.
Once the four diastereomeric aglycones 2a-d were prepared, we developed an LCMS method for stereochemical assignment that, unlike prior methods, does not require derivatization.Because the aglycone readily fragments in positive electrospray ionization mass spectrometry mode, the pseudomolecular ion m/z = 385.3+was used to extract chromatograms for detection and quantitation (Figure 1).

Instrumentation
NMR spectra were recorded on a Varian 400 MHz instrument (400 MHz for 1 H and 100 MHz for 13 C), and all chemical shift values referred to δ TMS = 0.00 ppm (δ (1H)) and CDCl3 (δ (13C), 77.16 ppm).LC/MS data were secured from an Agilent 1200 LC/MS spectrometer.The HRMS analysis was achieved on a Waters Xevo G2-XS using ESI ionization.

Materials
All the chemicals and solvents were procured from Sigma Aldrich unless otherwise stated.Trichloroacetimidate was purchased from Toronto Chemicals Industries.

Instrumentation
NMR spectra were recorded on a Varian 400 MHz instrument (400 MHz for 1 H and 100 MHz for 13 C), and all chemical shift values referred to δ TMS = 0.00 ppm (δ (1H)) and CDCl 3 (δ (13C), 77.16 ppm).LC/MS data were secured from an Agilent 1200 LC/MS spectrometer.The HRMS analysis was achieved on a Waters Xevo G2-XS using ESI ionization.

Materials
All the chemicals and solvents were procured from Sigma Aldrich unless otherwise stated.Trichloroacetimidate was purchased from Toronto Chemicals Industries.
(3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-17-((R)-but-3-en-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,7,12-triyl-2,2,4,4-d 4 triacetate (14).Triacetyl cholic acid-d4, 13 (1.6 g, 1 Eq, 3.0 mmol) in anhydrous benzene (63 mL) was refluxed with pyridine (0.61 g, 0.62 mL, 2.6 Eq, 7.7 mmol), copper (II) acetate (0.47 g, 0.88 Eq, 2.6 mmol) and lead tetraacetate (11 g, 8.4 Eq, 25 mmol) under argon for 2 h.The reaction mixture was cooled, and 1N HCl was added to precipitate the excess lead.The salts were filtered and washed with benzene, and the combined filtrates were washed with water and 5% NaOH (aq) to remove unreacted starting material.The organic layer was dried over MagSO4, filtered, and evaporated into a paste.The residue was chromatographed over flash silica gel.Elution with 25% ethyl acetate in hexanes yielded 14 (0.5 g, 1 mmol, 30%) as colorless foam. 1  Liquid chromatography/mass spectrometry analysis was performed on a Waters Acquity UPLC ® coupled to a Waters Xevo Q-ToF quadruple time of flight mass spectrometer operating in electrospray ionization (ESI) in positive or negative mode.The capillary and sampling cone voltages were set to 3000 and 20 V, respectively.Source and desolvation temperatures were set to 120 • C and 450 • C, respectively, and the cone and desolvation gas flows were set to 50 and 800 L/hour, respectively.To maintain mass accuracy, leucine enkephalin was injected at a concentration of 200 pg/µL in 50% acetonitrile/water containing 0.1% formic acid at a rate of 10 µL/min.Data were acquired in continuum mode from 50 to 1000 m/z.The analytes were separated by reverse phase chromatography on an Acquity UPLC ® BEH C18 (1.7 µm, 2.1 × 50 mm) column.Chromatographic separation was achieved with water (A) and acetonitrile (B) each containing 0.1% formic acid.Gradient elution, with a flow rate of 0.500 mL/min, began with an initial hold of 2% B for 0.5 min, then increased to 20% B from 0.5 to 4.0 min, 20-95% B from 4.0 to 8.0 min, 95-100% B from 8.0-8.10 min, hold for 1 min at 100%, then return to initial conditions (2% B) in 0.10 min.The column temperature was maintained at 40 • C in a column oven.Identification was performed in either positive (pseudomolecular ions) or negative mode (Table 1).Because the glucuronides readily fragment in ESI+ mode (Figure 4), it is recommended to use ESI− mode for identification and quantitation.Liquid chromatography/mass spectrometry analysis was performed on a Waters Acquity UPLC ® coupled to a Waters Xevo Q-ToF quadruple time of flight mass spectrometer operating in electrospray ionization (ESI) in positive mode.The capillary and sampling cone voltages were set to 3000 and 20 V, respectively.Source and desolvation temperatures were set to 120 • C and 450 • C, respectively, and the cone and desolvation gas flows were set to 50 and 800 L/hour, respectively.To maintain mass accuracy, leucine enkephalin was injected at a concentration of 200 pg/µL in 50% acetonitrile/water containing 0.1% formic acid at a rate of 10 µL/min.Data were acquired in profile mode from 50 to 1200 m/z.The analytes were separated by reverse phase chromatography on a Phenomenex Synergi-Hydro (2.5 µm, 2.0 × 100 mm) column.Chromatographic separation was achieved with water (A) and acetonitrile (B) each containing 0.1% formic acid.Gradient elution, with a flow rate of 0.400 mL/min, began with an initial hold of 40% B for 0.3 min, increased to 50%B over 6 min, held for 0.65 min at 95%, then returned to initial conditions (40%B) in 0.10 min.The column temperature was maintained at 40 • C in a column oven.Due to significant amounts of insource fragmentation, analytes were identified using pseudomolecular ions m/z 385.3+, 403.3+, and 461.3+ (M + Na) and retention time.