October 17, 2011
Make a green fluorescent protein precursor in improved yield. Coelenteramide (1) can be used to prepare green fluorescent protein (GFP), which is partly responsible for bioluminescence in marine animals. GFP was first isolated in the early 1960s from the jellyfish Aequorea Victoria, and it has been engineered to produce color-shifted genetic derivatives that are used in live-cell imaging experiments without damaging the cell. This technique heralded a new era in cell biology: The researchers who discovered and developed GFP received the 2008 Nobel Prize in Chemistry.
Some bizarre results from introducing GFP into living organisms have been reported. Examples are green fluorescent zebrafish that were developed to detect pollution in waterways and green fluorescent mice as pets. A green cat for potential use as a model organism for diseases, particularly HIV has also been reported.
O. Shimomura and F. H. Johnson (Tetrahedron Lett. 1973, 31, 2963–2966) developed a process for preparing 1, but the yield was only 50%. Inventor I. Satoshi devised an improved synthesis shown in the figure. The condensation reaction of amine 2 with acetyl chloride 3 produces crude amide 4 in 81.5% yield; DMAP is 4-dimethylaminopyridine. After two crystallisations from EtOAc, pure 4 is isolated in 60.5% yield. Treating 4 with BBr3 and then NaHCO3 removes the ether groups to give 1 in 92.3% yield and adequate purity for GFP synthesis. Product 1 is recrystallized from EtOH to obtain the analytically pure solid.
The patent’s claims cover the preparation of many analogues of 1 and 4 that contain different substituents in place of the methoxy and phenyl groups. No examples, however, describe the synthesis of these compounds. This process is an improvement in that it gives high initial yields of the desired compound, but there are significant losses on purification. (JNC Corp. [Tokyo]. US Patent No. 8,026,363, Sept. 27, 2011; Keith Turner)