Properties of Coelenterazine and its Derivatives

The unmodified form of coelenterazine, C26H2i03N3(mass spec. M+ 423), can be crystallized from methanol as orange-yellow prisms with a melting point of 175-178°C (dec). In methanol, coelenterazine is fluorescent in yellow, and its ultraviolet absorption spectrum shows a maximum at 435 nm (s 9,800), as shown in Fig. 5.1. 

A large number of the analogues of coelenterazine have been synthesized (see Section 4.1.7). The absorption spectra of coelenterazine analogues are very similar to that of the original coelenterazine, except for e-coelenterazines. The properties of e-coelenterazines differ significantly from those of coelenterazines, particularly in their luminescence properties. The absorption spectra of e-coelenterazine (Amax 442 nm, s 9,600) and e-coelenteramide (Amax 347nm, s 19,500), both in methanol, are shown in Fig. 5.2. 

The product of the luminescence reaction of coelenterazine, coe-lenteramide, is soluble in methanol, butanol, ethyl acetate and ether, and shows a strong blue fluorescence in these solvents, although this compound is only slightly fluorescent in aqueous solutions. Coelen-teramide shows an absorption maximum at 332-333 nm (s 15,000) in methanol. 

Solubility and stability of coelenterazine. Coelenterazine is very poorly soluble in neutral aqueous buffer solutions, and the solutions are unstable in air. It can be easily dissolved in water in the presence of alkali, but the resulting solution is extremely unstable under aerobic conditions. Coelenterazine is soluble in methanol, and the solution is relatively stable. The stability is enhanced by the addition of a trace of HCl. A methanolic solution of coelenterazine can be stored for several days at — 20°C, and a methanolic solution containing 1-2 mM HCl can be stored for several months at — 70°C under aerobic conditions without significant oxidation. In many other organic solvents, coelenterazine is less stable, and spontaneously auto-oxidized at significant rates. In dimethylformamide and DMSO, it is rapidly decomposed accompanied by the emission of chemiluminescence. e-Coelenterazines are generally less stable than coelenterazines. 

The poor solubility of coelenterazine in neutral aqueous buffer solutions often hampers the use of this compound in biological applications. The simplest way to make an aqueous solution is the dilution of a methanolic 3 mM coelenterazine with a large volume of a desired aqueous buffer solution. If the use of alcoholic solvents is not permitted, dissolve coelenterazine in a small amount of water with the help of a trace amount of 1 M NaOH or NH4OH, and then immediately dilute this solution with a desired aqueous buffer solution. However, because of the rapid oxidation of coelenterazine in alkaline solutions, it is recommended that the procedure be carried out under argon gas and as quickly as possible. 

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