Synthesis and NMR studies of (13)C-labeled vitamin D metabolites.
Isotope-labeled drug molecules may be useful for probing by NMR spectroscopy the conformation of ligand associated with biological hosts such as membranes and proteins. Triple-labeled [7,9,19-(13)C(3)]-vitamin D(3) (56), its 25-hydroxylated and 1 alpha,25-dihydroxylated metabolites (58 and 68, respectively), and other labeled materials have been synthesized via coupling of [9-(13)C]-Grundmann's ketone 39 or its protected 25-hydroxy derivative 43 with labeled A ring enyne fragments 25 or 26. The labeled CD-ring fragment 39 was prepared by a sequence involving Grignard addition of [(13)C]-methylmagnesium iodide to Grundmann's enone 28, oxidative cleavage, functional group modifications leading to seco-iodide 38, and finally a kinetic enolate S(N)2 cycloalkylation. The C-7,19 double labeling of the A-ring enyne was achieved by the Corey-Fuchs/Wittig processes on keto aldehyde 11. By employing these labeled fragments in the Wilson-Mazur route, the C-7,9,19 triple-(13)C-labeled metabolites 56, 58, and 68 as well as other (13)C-labeled metabolites have been prepared. In an initial NMR investigation of one of the labeled metabolites prepared in this study, namely [7,9,19-(13)C(3)]-25-hydroxyvitamin D(3) (58), the three (13)C-labeled carbons of the otherwise water insoluble steroid could be clearly detected by (13)C NMR analysis at 0.1 mM in a mixture of CD(3)OD/D(2)O (60/40) or in aqueous dimethylcyclodextrin solution and at 2 mM in 20 mM sodium dodecyl sulfate (SDS) aqueous micellar solution. In the SDS micellar solution, a double half-filter NOESY experiment revealed that the distance between the H(19Z) and H(7) protons is significantly shorter than that of the corresponding distance calculated from the solid state (X-ray) structure of the free ligand. The NMR data in micelles reveals that 58 exists essentially completely in the alpha-conformer with the 3 beta-hydroxyl equatorially oriented, just as in the solid state. The shortened distance (H(19Z))-H(7)) in micellar solutions as compared to that in the solid state is most easily rationalized on the basis that the 5(10)-torsion angle in 58 is decreased in micellar solutions as compared to that in the solid state.
Okamura
William H
WH
Department of Chemistry, University of California, Riverside, California 92521, USA. willaim.okamura@ucr.edu
Zhu
Gui-Dong
GD
Hill
David K
DK
Thomas
Richard J
RJ
Ringe
Kerstin
K
Borchardt
Daniel B
DB
Norman
Anthony W
AW
Mueller
Leonard J
LJ
D002247:Carbon Isotopes / Q000737:chemistry; D002384:Catalysis; D002776:Cholestanes / Q000138:chemical synthesis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D007553:Isotope Labeling / Q000379:methods; D008958:Models, Molecular; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D014807:Vitamin D / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000737:chemistry
D002247:Carbon Isotopes; D002776:Cholestanes; D014807:Vitamin D