Oxyluciferin

A very active field of A-2-thiazoline-4-one chemistry concerns 2-(6 -hydroxybenzothiazol-2 -yl)-4-hydroxythiazole (171). which has appeared under the names firefly decarboxy-ketoluciferin " (401, 402) and firefly oxyluciferin (403). This later name is now commonly used (404). 

It has been suggested that the oxidation of Photinus pyralis luciferin (170) forms oxyluciferin (171) as the oxidized product (Scheme 88) (405-407), which was too unstable to be isolated and was rapidly converted in three other compounds. 

Later, fireflv oxyluciferin was successfully synthesi2ed (403. 408) and has been isolated and identified in firefly lanterns (luciola cruaciata) after the lanterns were treated with pyridine and acetic anhydride to prevent decomposition (409). In 1972, Suzuki and Goto firmly established that oxyluciferin is involved in the bioluminescence of firefly lanterns and in the chemiluminescence of firefly luciferin (403. 410).. A. mechanism involving a four-membered ring cyclic peroxide has been proposed for the reaction (406. 411). However, it was not confirmed by 0 -labelinE experiments (412). 

The following schemes represent the overall reaction of firefly bioluminescence (McElroy and DeLuca, 1978), where E is luciferase LH2 is D-luciferin PP is pyrophosphate AMP is adenosine phosphate LH2-AMP is D-luciferyl adenylate (an anhydride formed between the carboxyl group of luciferin and the phosphate group of AMP) and L is oxyluciferin. 

Fig. 1.4 Absorption spectrum of a spent luminescence solution of firefly luciferin containing luciferase-oxyluciferin after dialysis in 0.1 M potassium phosphate, pH 7.8. Replotted from the data of Gates and DeLuca, 1975, with permission from Elsevier.

Fig. 1.5 Fluorescence emission spectrum of the luciferase-oxyluciferin complex in the same solution as in Fig. 1.4 (solid line), compared with the luminescence spectrum of firefly luciferin measured in glycylglycine buffer, pH 7.6 (dotted line). The former curve from Gates and DeLuca, 1975 the latter from Selinger and McElroy, 1960, both with permission from Elsevier.

Fig. 1.12 Mechanism of the bioluminescence reaction of firefly luciferin catalyzed by firefly luciferase. Luciferin is probably in the dianion form when bound to luciferase. Luciferase-bound luciferin is converted into an adenylate in the presence of ATP and Mg2+, splitting off pyrophosphate (PP). The adenylate is oxygenated in the presence of oxygen (air) forming a peroxide intermediate A, which forms a dioxetanone intermediate B by splitting off AMP. The decomposition of intermediate B produces the excited state of oxyluciferin monoanion (Cl) or dianion (C2). When the energy levels of the excited states fall to the ground states, Cl and C2 emit red light (Amax 615 nm) and yellow-green light (Amax 560 nm), respectively.

McCapra et al. (1994) and McCapra (1997) suggested that the color variation could be caused by the conformational difference of the oxyluciferin molecule, when the plane of thiazolinone is rotated at various angles against the plane of benzothiazole on the axis of the 2-2 bond the red light would be emitted at 90° angle, reflecting its minimum structural energy. 

This finding by Branchini et al. (2002) clearly indicates that 5,5-dimethyloxyluciferin is able to emit the two different colors. This conclusion, however, does not rule out the involvement of the enolized oxyluciferin in the bioluminescence reaction of firefly. 

Orlova et al. (2003) theoretically studied the mechanism of the firefly bioluminescence reaction on the basis of the hybrid density functional theory. According to their conclusion, changes in the color of light emission by rotating the two rings on the 2-2 axis is unlikely, whereas the participation of the enol-forms of oxyluciferin in bioluminescence is plausible but not essential to explain the multicolor emission. They predicted that the color of the bioluminescence depends on the polarization of the oxyluciferin molecule (at its OH and O-termini) in the microenvironment of the luciferase active site the... 

According to Branchini et al. (2004), luciferase modulates the emission color by controlling the resonance-based charge delocalization of the anionic keto-form of oxyluciferin in the excited state. They proposed the structure C5 as the yellow-green light emitter, and the structure C6 as the red light emitter. 

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).

Fig. 3.1.4 Bioluminescence spectrum of Cypridina luciferin catalyzed by Cypridina luciferase (A), the fluorescence excitation spectrum of oxyluciferin in the presence of luciferase (B), the fluorescence emission spectrum of the same solution as B (C), and the absorption spectrum of oxyluciferin (D). The fluorescence of oxyluciferin alone and luciferase alone are negligibly weak. Measurement conditions A, luciferin (lpg/ml) plus a trace amount of luciferase in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl B and C, oxyluciferin (20 pM) plus luciferase (0.2mg/ml) in 20 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl D, oxyluciferin (41 pM) in 20 mM Tris-HCl buffer, pH 7.6, containing 0.2 M NaCl. All are at 20°C.

Luciferase turnover. The luciferase-catalyzed light-emitting reaction that forms oxyluciferin is fast, but the hydrolysis reaction of oxyluciferin into etioluciferin by luciferase is slow. The turnover rate (catalytic center activity) of luciferase was reported to be about 30/s for the luminescence reaction, and 0.03/s for the hydrolysis of oxyluciferin (Shimomura et al., 1969). 

Side reaction. The luminescence reaction of Cypridina luciferin catalyzed by luciferase involves a side reaction (Fig. 3.1.8). In the luminescence reaction, 85-90% of luciferin is converted into oxyluciferin and CO2 accompanied by light emission, whereas 10-15% of luciferin is converted directly into etioluciferin plus a keto-acid without light emission (Shimomura and Johnson, 1971). In the chemiluminescence reactions of Cypridina luciferin in organic solvents (such as diglyme, acetone, pyridine and DMSO), the proportion of the dark side reaction... 

Coelenteramide and coelenterazine. The structure of AF-350 contains the same aminopyrazine skeleton as in Cypridina etioluciferin and oxyluciferin (Fig. 3.1.8), suggesting that the bioluminescence reaction of aequorin might resemble that of Cypridina luciferin. To investigate such a possibility, we prepared the reaction product of aequorin luminescence by adding Ca2+ to a solution of aequorin. The product solution (blue fluorescent) was made acidic, and extracted with... 

Goto et al. (1974) extracted the arm photophores, and isolated a compound that is highly fluorescent in blue. They determined the structure of this compound to be coelenteramide disulfate (structure A below), and named it Watasenia oxyluciferin. Then, Inoue et al. (1975)... 

The absorption spectra of Watasenia luciferin (coelenterazine disulfate) and Watasenia oxyluciferin (coelenteramide disulfate) are shown in Fig. 6.3.2. Watasenia luciferin in neutral aqueous solutions is auto-oxidized in air more rapidly than coelenterazine, and the compound emits a strong chemiluminescence in the presence of H2O2 ( 10mM) plus Fe2+ ( 0.2mM). Watasenia oxyluciferin is strongly fluorescent in aqueous solutions (Amax 400 nm), 500 times stronger than the fluorescence of coelenteramide in aqueous media (Goto et al., 1974). 

Fig. 6.3.2 Absorption spectra of Watasenia oxyluciferin (A), coelenterazine (B), and coelenterazine disulfate (C), all in methanol. Concentrations 0.1 mM for B and C undetermined for A.

Fig. 7.2.1 Absorption spectra of Odontosyllis luciferin (solid line) and Odontosyllis oxyluciferin (dashed line), both in ethanol/water (5 6) containing 8% NaCl. To measure the latter curve, luciferin was first luminesced in the presence of luciferase, then luciferase was removed using a small column of DEAE cellulose. From Shimomura et al, 1963d, with permission from John Wiley Sons Ltd.

Fig. 7.2.2 Absorption spectra of Odontosyllis luciferin in methanol (left panel) and oxyluciferin in aqueous solution (right panel). From Trainor, 1979.

Oxyluciferin. During the luminescence reaction catalyzed by luciferase, luciferin is converted into a fluorescent compound, oxyluciferin, accompanied by the emission of greenish-blue light that spectrally matches the fluorescence of oxyluciferin (Fig. 7.2.6). The absorption spectrum of oxyluciferin is shown in Figs. 7.2.1 and 7.2.2. 

Gates, B. J., and DeLuca, M. (1975). The production of oxyluciferin during the firefly luciferase light reaction. Arch. Biochem. Biophys. 169 616-621. 

Goto, T., Iio, H., Inoue, S., and Kakoi, H. (1974). Squid luminescence I. Structure of Watasenia oxyluciferin, a possible light-emitter in the bioluminescence of Watasenia scintillans. Tetrahedron Lett., pp. 2321-2324. 

Shimomura, O., Johnson, F. H., and Masugi, T. (1969). Cypridina bioluminescence light-emitting oxyluciferin-luciferase complex. Science 164 1299-1300. 

White, E. H., and Roswell, D. F. (1991). Analogs and derivatives of firefly oxyluciferin, the light emitter in firefly bioluminescence. Photochem. Photobiol. 53 131—136. 

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