One-pot non-enzymatic formation of firefly luciferin in a neutral buffer from p-benzoquinone and cysteine

Firefly luciferin, the substrate for the bioluminescence reaction of luminous beetles, possesses a benzothiazole ring, which is rare in nature. Here, we demonstrate a novel one-pot reaction to give firefly luciferin in a neutral buffer from p-benzoquinone and cysteine without any synthetic reagents or enzymes. The formation of firefly luciferin was low in yield in various neutral buffers, whereas it was inhibited or completely prevented in acidic or basic buffers, in organic solvents, or under a nitrogen atmosphere. Labelling analysis of the firefly luciferin using stable isotopic cysteines showed that the benzothiazole ring was formed via the decarboxylation and carbon-sulfur bond rearrangement of cysteine. These findings imply that the biosynthesis of firefly luciferin can be developed/evolved from the non-enzymatic production of firefly luciferin using common primary biosynthetic units, p-benzoquinone and cysteine.


S4
was subjected to HPLC separation with a Develosil ODS-UG-5 column (× 250 mm; Nomura Chemical, Aichi, Japan), a multiwavelength detector (MD-2010 Plus, Jasco), and a fluorescence detector (FP-1520, Jasco). The HPLC conditions were as follows: mobile phase, linear gradient of methanol in water containing 0.1% (v/v) formic acid from 10 to 100% for 45 min; flow rate 0.6 mL/min; UV detection, 327 nm; fluorescence detection, excitation/emission, 330/530 nm. The fraction eluted at a retention time of 35.0 to 37.0 min was concentrated to dryness under a nitrogen stream at room temperature and further dried in vacuo. The residue was dissolved in CD3OD and the solution was analyzed by using an NMR spectrometer (600 MHz) equipped with a cryogenically cooled probe.

Calibration curve for quantification of firefly luciferin
Calibration curve was determined by chiral HPLC analysis of authentic D-firefly luciferin dissolved in methanol. The amount of authentic firefly luciferin was determined spectrophotometrically, using an absorption coefficient of 18,620 M -1 cm -1 at 327 nm in methanol 1 .

Quantification of firefly luciferin produced by the one-pot reaction in various buffer concentrations
To 90 L of 50, 100, 250, 500 mM ammonium acetate (pH 7.0) or Tris-HCl (pH 7.5) in a 1.5 mL micro tube was added 5 L of an 80 mM aqueous solution of L-cysteine and 5 L of an 80 mM aqueous solution of p-benzoquinone. The L-cysteine and p-benzoquinone solutions were both prepared immediately prior to being used. After being stirred by a micro mixer (model E-36; Taitec) in high speed mode for 3 h at 30 C, the resultant solution was diluted with 400 L of water and acidified with 10 or 20 L of 3 M HCl to below pH 3. The pH of the aqueous solution was confirmed by using pH test paper (Toyo Roshi). The acidified solution was extracted with ethyl acetate (500 L × 2). The combined organic layer was concentrated to dryness under a nitrogen stream at room temperature and dissolved in 100 L of water by a sonicator (model UT-206; Sharp, Osaka, Japan). The aqueous solution was centrifuged at 17,400 ×g for 3 min at 4 C and 10 L of the supernatant was subjected to chiral HPLC analysis.

Quantification of firefly luciferin produced by the one-pot reaction at various temperature
To 500 L of a stirred 8 mM solution of L-cysteine in 180 mM ammonium acetate (pH 7.0) in a glass test tube (model ST 13-100; Nichiden-Rika, Kobe, Japan) was added a single portion 500 L of an 8 mM aqueous solution p-benzoquinone. The L-cysteine and p-benzoquinone solutions were both prepared immediately prior to being used. After being stirred for 3 h at various temperatures (4, 30, 60, 90 C), 100 L of the resultant solution was pretreated as described in the previous section, and 10 L of the resultant solution was subjected to chiral HPLC analysis. Reaction temperature was controlled using an ultra-cooling reactor (UCR-150N; Techno Sigma, Okayama, Japan) or oil bath (model W-1; Nippon Rikagaku Kikai, Tokyo, Japan). The reaction without stirring was also performed at 30 C by the same method.

Quantification of firefly luciferin produced by the one-pot reaction under various atmospheres
An 8 mM solution of L-cysteine in 180 mM ammonium acetate (pH 7.0) and an 8 mM aqueous solution of p-benzoquinone were both degassed by successive freeze-pump-thaw using a nitrogen gas.
These solution were both prepared immediately prior to being used. To 2 mL of the L-cysteine solution was added a single portion mL of the p-benzoquinone solution using a common syringe technique.
After being stirred for 3 h at 30 C under balloon pressure of a nitrogen gas or 95% oxygen gas, 100 L of the resultant solution was pretreated as described in the previous section, and 10 L of the resultant solution was subjected to chiral HPLC analysis.

Reaction of compound 1 or compound 2 with D-cysteine
To 50 L of a 10 mM solution of D-cysteine in 180 mM ammonium acetate (pH 7.0) in a 1.5 mL micro tube was added a single portion 50 L of a 10 mM solution of compound 1 or compound 2 suspended aqueous solution, respectively. All solutions were prepared immediately prior to being used.
After being stirred by a micro mixer (model E-36; Taitec) in high speed mode for 3 h at 30 C, 100 L of the resultant solution was pretreated as described in the previous section, and 10 L of the resultant solution was subjected to chiral HPLC analysis.

LC/ESI-TOF-MS analysis of the one-pot reaction products from p-benzoquinone and L-cysteine
A 40 mM solution of L-cysteine in 100 mM ammonium acetate (pH 7.0) and a 40 mM aqueous solution of p-benzoquinone were both degassed by successive freeze-pump-thaw using a nitrogen gas.
These solution were both prepared immediately prior to being used. To 2.2 mL of the L-cysteine

Synthesis of compound 2
The synthetic route is summarized in the following scheme. solids, the reaction mixture was filtered through a filter paper on a Büchner funnel with aspirator suction. The filtrate diluted with tap water (100 mL) and the mixture was extracted with ethyl acetate (100 mL× 1, 50 mL × 2). The combined organic layer was washed with brine (× 2) and filtered through a Celite pad on a Büchner funnel with aspirator suction, and dried over anhydrous Na2SO4.

Preparation of ester 3 from p-benzoquinone:
The solution was concentrated to dryness using a rotary evaporator and further dried in vacuo. The residue was purified by column chromatography (silica gel 96 g; hexane-ethyl acetate, 2:1v/v). The Sigma) cooled at -88 C under a nitrogen atmosphere. The mixture was stirred at same temperature for S11 The reaction mixture was quenched with ice-cooled methanol (0.65 mL) and stirred for 3 min. To the quenched solution was added a saturated aqueous potassium sodium tartrate solution (15 mL). The mixture was warmed to room temperature and stirred for 140 min. The resultant suspension was extracted with CH2Cl2 (15 mL × 3). The combined organic layer was washed with brine (70 mL × 1) and dried over anhydrous Na2SO4. The solution was concentrated to dryness using a rotary evaporator and further dried in vacuo. The residue was purified by column chromatography (silica gel 13 g; hexane-ethyl acetate, 20:1v/v) to give aldehyde 5 (122 mg, 46%) as a light yellow oil. 1