Hoover HS, Blankman JL, Niessen S, Cravatt BF. (2008)Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based proteinprofiling.
Bioorg Med Chem Lett. 18(22):5838-41.
Patricelli MP, Cravatt BF. (2001) Proteins regulating the biosynthesis and inactivation ofneuromodulatory fatty acid amides.
Vitam Horm. 62:95-131. Review
Further optimization using phase transfer catalyst, 18-crown-6, to improve the yield did not prove successful. Given the concise synthetic route (only two steps with an overall yield of 23 %) and commercially available starting materials, our synthesis represents a very streamlined approach to obtaining C3 labeled morphine.
The fact that the chemical modification did not significantly alter the binding mode of (−)-morphine (1) to its receptor TLR4 is supported by the in silico simulation, NMR spectroscopy, and whole cell assays. Using a molecular mechanics (MM) simulation model with a Merck Molecular Force Field (MMFF), a total of 2704 conformers of biotinylated (−)-morphine (2) were scanned to search for the most favorable structure (). Energy minimization of the (−)-morphine (1) and biotinylated (−)-morphine (2) suggests that the morphine substructure in compound 2 retains much of its rigidity after the addition of biotin (), with a root-mean-square-deviation (RMSD) of less than 0.7 Å.
Our straightforward synthesis of biotinylated (−)-morphine (2) is outlined in . The intermediate 7 can be readily obtained in good yield (65 %) by the classical substitution reaction between deprotonated biotin (5) and commercially available 1,2-bis-(2-iodoethoxy)-ethane (6). It is observed that the choice of base is pivotal for this reaction since compound 6 is unstable under strong basic conditions. Compound 7 was then subject to react with (−)-morphine (1). We found that the conditions had to be carefully tuned to selectively alkylate the C3 phenolic hydroxyl group. It is also important to prevent elimination of HI from compound 7. Several bases (NEt3, KOH, or NaH) and solvents (Et2O, DMF, CHCl3, or DMSO) were tested (), each of which had different effects in promoting the reaction. Using weaker bases, such as K2CO3 and NEt3, and less polar solvents, no reaction occurred and the reactants could be recovered completely. When the reaction was subjected to the strong inorganic base KOH, compound 7 partly degraded due to the fragile nature of compound 7 under strong basic conditions. Finally, optimal conditions were obtained by using NaH to deprotonate the phenol of (−)-morphine (1) in DMSO which subsequently reacted with compound 7 to provide biotinylated (−)-morphine (2) in 35 % yield.
There are commercially available suppositories of morphine,hydromorphone, and oxymorphone. Medications can also be placed in acolostomy or similar stoma, provided that the flow of effluent is slowenough to allow the drug to be absorbed via the mucosa Whenconverting from the oral to the rectal route, start with the same amountas the oral dose and titrate as needed.
In summary, a practical and concise approach to the synthesis of biotinylated (−)-morphine (2) was developed. The sequence involves two synthetic steps with an overall yield of 23 %. As the C3 hydroxy group is a conserved functional group, this method could potentially be generalized to other opioid derivatives. Also, in silico simulation, NMR, and cellular assays were performed, showing that biotinylated (−)-morphine (2) binds to the same region of TLR4 as (−)-morphine (1) and that biotinylated morphine (2) acts on TLR4 with similar biological activities.
Using AutoDock 4.0 (), in silico docking experiments were carried out to search for potential binding sites on TLR4 (). Lamarckian Generic Algorithm (LGA) and the torsion angles of the ligand were varied using AUTOTORS. First, the high-resolution structure of TLR4 was used as the target receptor after modification of Gasteiger charges and surface solvation. The full-length of TLR4 is split into two parts to which the opioid molecules were docked in order to carry out more accurate calculation. Both receptor and ligand were allowed flexibility when running morphine against TLR4. Energy minimized biotinylated (−)-morphine (2) was used in the docking and allowed to have seventeen of its bonds able to rotate within its structure to simulate realistic binding of this ligand to TLR4. A total of 100 runs were carried out and the results were grouped into clusters using an rmsd-tolerance of 2.0 Å. Finally, the resulted clusters were ranked using a force field scoring function (). The credibility of the docking results was evaluated by estimating binding energy and the distribution of the independent runs in clusters (fewer binding modes predicted is preferred). Among those, the highest ranked docked structure was used for molecular visualization.
Meier J, Niessen S, Hoover H, Foley TL, Cravatt BF, Burkart MD. (2009) An orthogonal active siteidentification system (OASIS) for proteomic profiling of natural product biosynthesis.
ACS Chem Biol. 4(11):948-57.
A generally applicable strategy of chemically labeling (−)-morphine (1) is described. The synthesis starts from commercially available starting materials and can be completed in two steps with an overall yield of 23 %. In silico simulation and NMR results show that the binding of (−)-morphine to one of its molecular targets, toll-like receptor 4 (TLR4), was not affected by the modification. Secreted Embryonic Alkaline Phosphatase (SEAP) reporter assay results demonstrate that C3 biotinylated and unmodified (−)-morphine show similar biological activities in live cells. To our knowledge, these studies provide the first practical and concise method to label various opioid derivatives, a group of important therapeutics in pain management, for biochemical/pharmacological studies.
Adachi S, Cognetta AB 3rd, Niphakis MJ, He Z, Zajdlik A, St Denis JD, Scully CC, Cravatt BF, Yudin AK. Facile synthesis of borofragments and their evaluation in activity-based protein profiling. Chem Commun (Camb) 2015 Feb 12;51:3608-11.