Cypridina

Monooximes of a-diketones have found applicability in the synthesis of 2-aminopyrazine 1-oxides by condensation with a-aminonitriles, and this reaction was used by White and coworkers in an approach to the synthesis of Cypridina etioluciferamine (Scheme 66 R = 3-indoloyl) (73T3761). In this instance, the use of TiCU as a catalyst was essential, since the carbonyl group in 3-acylindoles is normally deactivated and the required amine/carbonyl condensation is impractically slow. Under normal circumstances the carbonyl group in simple alkyl-substituted monoximes of a-diketones is the more reactive site and the reaction is rapid, requiring no catalysis (69LA(726)loo). 

Properties of luciferin. The crystals are microscopic needles, which melt with decomposition at 205-210°C (Bitler and McElroy, 1957). It is a quite stable luciferin compared with some other luciferins, such as Cypridina luciferin and the luciferins of krill and dinoflagellates. It is not significantly affected by lOmM H2SO4 and lOmM NaOH at room temperature in air. The absorption spectral data of luciferin are shown in Fig. 1.3 (McElroy and Seliger, 1961). The molar absorption coefficient of the 328 nm peak in acidic solutions and that of the 384 nm peak in basic solutions are both 18,200 (Morton et al., 1969). Luciferin is fluorescent, showing an emission maximum at 537 nm in both acidic and basic conditions, although the intensity of the fluorescence is lower in acidic solution than in basic solution (fluorescence quantum yields 0.62 in basic condition, and 0.25 in acidic condition Morton et al., 1969). The chemical synthesis... 

In the lsO-incorporation experiment of Cypridina bioluminescence, the effects of the O atom exchange and contaminating CO2 are clearly seen in the relationship between the amount of luciferin luminesced and the amount of lsO atoms incorporated into the product CO2 (Fig. 1.14 Shimomura and Johnson, 1973a). The experiments were done in glycylglycine buffer, pH 7.8, the same buffer as chosen by DeLuca and Dempsey (1970). The total volume of the reaction mixture was 4 ml, with 40 ml of gas phase (see the reaction vessel in Fig. A.5 in the Appendix). The data of the luminescence reaction with 1802 gas in the H2160 medium indicates that at least 1 pmol of... 

Fig. 1.14 Relationship between the incorporation of 180 into product CO2 and the amount of luciferin used, in the bioluminescence reaction of Cypridina luciferin catalyzed by Cypridina luciferase. The reactions were carried out in H2160 medium with 1802 gas (solid line) in H2lsO medium with 16C>2 gas (dashed line) and the control experiment of the latter using C16C>2 gas instead of luciferin and luciferase (dotted line), all in 20 mM glycylglycine buffer, pH 7.8, containing 40 mM NaCl. From Shimomura and Johnson, 1973a. Reproduced with permission from Elsevier.

THE OSTRACOD CYPRIDINA (VARGULA) AND OTHER LUMINOUS CRUSTACEANS... 

The Ostracod Cypridina (Vargu/a) ond other Luminous Crustoceans 49... 

In this book, the classification of ostracods by Muller is used the genus names by the Poulsen classification are cited in parentheses only when appropriate. The author protests against the irresponsible shuffling of scientific names, and hopes for the revival of the Muller classification. In addition, the author believes that Cypridina luciferin is the proper name of this substance that is not necessarily bound to the genus name. 

The Ostracod Cypridina (Vorgulo) and other Luminous Crustaceans 51... 

Fig. 3.1.1 The ostracod Cypridina hiigendorfii (photo by Dr. Toshio Goto).

During the period between 1916 and the early 1950s, Harvey, his associates and students made great efforts to study various aspects of the Cypridina bioluminescence, which was described by Harvey in his book Bioluminescence (1952). It should be pointed out here... 

The Ostrocod Cypridina (Vargu/a) and other Luminous Crustoceans 55... 

Fig. 3.1.2 The apparatus used in 1956 for the methanol extraction of Cypridina luciferin. The dried Cypridina (500 g) is extracted at a temperature lower than 40°C with refluxing methanol under reduced pressure for two days. The atmosphere inside the apparatus is completely replaced with hydrogen gas that was purified by its passing through a quartz tube containing red-heated copper fragments. The temperature of the mantle heater is adjusted, the system evacuated, and then all stopcocks are closed. The extraction with refluxing methanol continues for many hours without any further adjustment. From the author s 1957 notebook.

Improved purification method of Cypridina luciferin. The purification of Cypridina luciferin became remarkably simple and easy after some of the properties of this substance were known. The following method was used to obtain the large quantity of luciferin needed for the study of its chemical structure. The method consists of three steps and takes less than eight hours to obtain crystallized luciferin. 

Extraction of luciferin with methanol from Cypridina frozen with dry ice. The chunks of frozen Cypridina are crushed into small pieces, and extracted with methanol with stirring. Small pieces of dry ice are added as needed to keep the temperature slightly below 0°C and also to prevent the oxidation of luciferin in a CO2 atmosphere. Luciferin is easily extracted into methanol. The mixture is filtered on a Buchner funnel (with suction), protecting the luciferin in a CO2 atmosphere by addition of small pieces of dry ice as needed. 

Chemical structure. The structure of the free base of Cypridina luciferin (C22H27ON7, Mr 405.50) was determined by Kishi et al. (1966a,b) as shown below (A) its sec-butyl group is in the same configuration as in L-isoleucine. The structure of oxyluciferin reported by the same authors contained an error, and the structure was corrected later as shown in Fig. 3.1.8 (McCapra and Chang, 1967 Stone, 1968). 

Thus, oxyluciferin has a molecular formula of C21H27ON72HCI. The total synthesis of Cypridina luciferin has been accomplished (Kishi et al., 1966c Inoue et al., 1969 Karpetsky and White, 1971 Nakamura et al., 2000). 

Properties. Cypridina luciferin is soluble in water, methanol and other alcoholic solvents, but not in most aprotic solvents. The ultraviolet absorption spectra of luciferin and oxyluciferin are shown in Fig. 3.1.3. Luciferin in neutral solutions is yellow (lmax 432 nm ... 

Fig. 3.1.3 Absorption spectra of Cypridina luciferin dihydrobromide (70 pM) in methanol (A), after addition of 1% volume of IN HCl (B), and oxyluciferin dihydrochloride (43 pM) in methanol (C).

Chemiluminescence. Cypridina luciferin emits light in various organic solvents in the presence or absence of a base. The most efficient... 

It should be noted that Cypridina luciferin emits a fairly strong chemiluminescence in aqueous solutions in the presence of various lipids and surfactants, even in the complete absence of luciferase. The luminescence is especially conspicuous with cationic surfactants (such as hexadecyltrimethylammonium bromide) and certain emulsion materials (such as egg yolk and mayonnaise). Certain metal ions (especially Fe2+) and peroxides can also cause luminescence of the luciferin. Therefore, great care must be taken in the detection of Cypridina luciferase in biological samples with Cypridina luciferin. 

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