Dual isotope labeling: Conjugation of 32P-oligonucleotides with 18F-aryltrifluoroborate via copper(I) catalyzed cycloaddition

A one-pot-two-step labeling of an oligonucleotide with an ¹⁸F-ArBF₃^?(aryltrifluoroborate) radioprosthetic is reported herein. In order to characterize labeling in terms of radiochemistry, phosphorus-32 was also introduced to the 5′-terminus of the oligonucleotide via enzymatic phosphorylation. A pendant azide group was subsequently conjugated to the 5′-phosphate of the oligonucleotide. Copper(I) catalyzed [2+3] cycloaddition was undertaken to conjugate an alkyne-bearing¹⁸F-ArBF₃^? to the oligonucleotide. Following polyacrylamide gel electrophoresis, this doubly-labeled bioconjugate exhibited decay properties of both the phosphorus-32 and fluorine-18, that were confirmed by autoradiography at selected lengths of time, which in turn provided concrete evidence of successful conjugation. These results are corroborated by HPLC analysis of the labeled material. Taken together this work demonstrates viable use of ¹⁸F-ArBF₃^? prosthetics for labeling oligonucleotides for use in PET imaging.     

Simulation of an all-beta 3-icosapeptide containing the 20 proteinogenic side chains: effect of temperature, pH, counterions, solvent, and force field on helix stability

Simulations of various beta-peptides have in the last years clarified several issues concerning peptide folding equilibria and interpretation of experimental data, especially from NMR and CD spectroscopy. These simulations involved different temperatures, pH-values, ionic strengths, solvents, and force-field parameters, but a variation of these factors for one beta-peptide has not yet been done. To investigate the influence of varying these factors, we analyze the helix stability of an all-beta3-icosapeptide bearing all 20 proteinogenic amino acid side chains, which is experimentally observed to fold into a 3(14)-helix in methanol but not in water. Structural aspects, such as hydrogen-bonded rings and salt bridges, are discussed and a comparison with NMR primary (NOE distance bounds and 3J-values) and secondary (NMR derived model structures) data is made. We further investigate the reasons for the 3(14)-helix stability/instability in methanol/water. Of all factors studied, the presence of counterions seems to be the one inducing most significant effects in the simulations.     

Covalent labeling of nuclear vitamin D receptor with affinity labeling reagents containing a cross-linking probe at three different positions of the parent ligand: Structural and biochemical implications

Structure-functional characterization of vitamin D receptor (VDR) requires identification of structurally distinct areas of VDR-ligand-binding domain (VDR-LBD) important for biological properties of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). We hypothesized that covalent attachment of the ligand into VDR-LBD might alter ‘surface structure’ of that area influencing biological activity of the ligand. We compared anti-proliferative activity of three affinity alkylating derivatives of 1,25(OH)₂D₃ containing an alkylating probe at 1,3 and 11 positions. These compounds possessed high-affinity binding for VDR; and affinity labeled VDR-LBD. But, only the analog with probe at 3-position significantly altered growth in keratinocytes, compared with 1,25(OH)₂D₃. Molecular models of these analogs, docked inside VDR-LBD tentatively identified Ser237 (helix-3: 1,25(OH)₂D₃-1-BE), Cys288 (β-hairpin region: 1,25(OH)₂D₃-3-BE,) and Tyr295 (helix-6: 1,25(OH)₂D₃-11-BE,) as amino acids that are potentially modified by these reagents. Therefore, we conclude that the β-hairpin region (modified by 1,25(OH)₂D₃-3-BE) is most important for growth inhibition by 1,25(OH)₂D₃, while helices 3 and 6 are less important for such activity.