Acridinium Ester Chemiluminescence: Methyl Substitution on the Acridine Moiety

acridine moiety. Encouraged with these backgrounds, we introduced methyl groups at the 2-, 2,7- and 2,3,6,7-positions on the acridine moiety in acridinium ester derivatives with elec-Abstract: Methyl groups were introduced on the acridine moiety in chemiluminescent acridinium esters that have electron-withdrawing groups (trifluoromethyl, cyano, nitro, ethoxycarbonyl) at the 4-position on the phenyl ester. The introduction of methyl groups at the 2-, 2,7-, and 2,3,6,7-positions on the acridine moiety shifted the optimal pH that gave relatively strong chemiluminescence intensity from neutral conditions to alkaline conditions. 4-(Ethoxycarbonyl)phenyl 2,3,6,7,10-pentamethyl-10λ 4 -acridine-9-carboxylate, trifluoromethanesulfonate salt showed long-lasting chemiluminescence under alkaline conditions. Acridinium esters to determine hydrogen peroxide concentration at pH 7–10 were newly developed.

physical properties were investigated 8 14 . Acridinium ester derivatives have been particularly useful compounds in CL enzyme immunoassays 15 18 . We have recently clarified that acridinium ester derivatives having electron-withdrawing groups at the 4-position on the phenyl ester produced strong CL intensities at pH 7 and 8 19,20 . The CL intensity at higher pH alkaline conditions apparently decreased. Upon further investigations, methoxycarbonyl-phenyl 10-methyl-10λ 4 -2,7-disubstituted acridine-9-carboxylate derivatives were synthesized and their CL were measured, and the introduction of methyl groups at the 2,7-positions on the acridine moiety was effective in increasing the CL intensities at pH 9 and 10 21 .
For our present purposes, we considered that the hydrolysis of phenyl ester in acridinium ester derivatives under alkaline conditions may be related to this experimental result 11,22,23 . Before the dioxetanone is produced, the hydrolysis of phenyl ester mainly proceeds and free carboxylic acid is formed Scheme 1 . Methyl substitutions on the acridine moiety could shift the optimal pH for the CL gradually from neutral conditions to alkaline conditions. Thus, the CL intensities of acridinium ester derivatives could be changed at different pH values by introducing different numbers of methyl groups on the acridine moiety.
Encouraged with these backgrounds, we introduced methyl groups at the 2-, 2,7-and 2,3,6,7-positions on the acridine moiety in acridinium ester derivatives with elec-tron-withdrawing groups trifluoromethyl, cyano, nitro, ethoxycarbonyl at the 4-position on the phenyl ester Table 1 . CL intensities and spectra were evaluated at pH 7-10, and CL methods for measuring hydrogen peroxide at each pH value were developed.
The filtrate was evaporated in vacuo, and the residue was purified by column chromatography silica gel 60N to produce the desired acridine-9-carboxylates. These were N-methylated by methyl trifluoromethanesulfonate in dichloromethane, and the desired acridine-9-carboxylates as trifluoromethanesulfonate salts were purified by column chromatography silica gel, chloroform-methanol . Compounds 1c, 1d, and 2a-2d were obtained after washing the resulting precipitate with diethyl ether. Scheme 1 Possible chemiluminescence mechanism of acridinium ester derivatives via dioxetanone formation, and hydrolysis of phenyl ester. Table 1 Chemical structures of 1a-3d.

Apparatus
The 1 H NMR 500 MHz and 13 C NMR 125.7 MHz spectra were obtained using an Inova-500 NMR Agilent, Santa Clara, CA, USA spectrometer. High resolution HR FAB-MS positive spectra were obtained using a JMS-HX110A JEOL, Tokyo, Japan mass spectrometer. Column chromatography was performed with silica gel 60 N spherical, neutral 40-50 µm . Chemiluminescence was measured using a Lumat LB 9507 Berthold, Bad Wildbad, Germany luminometer, and CL spectra were recorded using a FP-6500 JASCO, Tokyo, Japan fluorometer.

Chemiluminescence measurement
To 50 µL of a 10 nM solution of 1a-3d in dimethyl sulfoxide was added 100 µL of a buffer solution 100 mM tris hydroxymethyl aminomethane hydrochloride Tris-HCl for pH 7 and pH 8, 100 mM glycine sodium hydroxide Gly-NaOH for pH 9 and pH 10 . The mixture was allowed to stand for 20 s, and then the CL reaction was initiated by adding 100 µL of 1 mM aqueous hydrogen peroxide solu-tion to the luminometer using an automatic injection system. The relative light unit integrated for 1 min was used to evaluate the CL intensity.

Chemiluminescence spectra measurement
To 0.5 mL of a 0.1 mM solution of 1a, 2a, and 3b in dimethyl sulfoxide in a quartz cell 10 10 50 mm was added 1 mL of a buffer solution 100 mM Tris-HCl for pH 7 and pH 8, 100 mM Gly-NaOH for pH 9 and pH 10 . The mixture was allowed to stand for 20 s, and then the CL reaction was initiated by adding 1 mL of 100 mM aqueous hydrogen peroxide solution. After 1 min, the mixture was placed in a spectrometer fluorescence bandwidth: 10 nm; response: 0.5 s; scanning speed: 500 nm/min and the CL spectra was measured.

Determination of hydrogen peroxide concentration
To 50 µL of a 10 nM solution of 1a, 3b, and 3d in dimethyl sulfoxide was added 100 µL of a buffer solution 100 mM Tris-HCl for pH 7 and pH 8; 100 mM Gly-NaOH for pH 9 and pH 10 . The mixture was allowed to stand for 20 s, and then the CL reaction was initiated by adding 100 µL of 0.01-1 mM aqueous hydrogen peroxide solution to the luminometer using an automatic injection system. The relative light unit integrated for 1 min were used to evaluate the CL intensity.

Results and Discussion
Compounds 1a-3d were new acridinium ester derivatives. Synthetic procedure of 1a-3d was shown in Scheme 2. 4-Methyl-N-phenylaniline, di-p-tolylamine or bis 3,4-dimethylphenyl amine was used as a starting material. These starting materials with oxalyl dichloride and alminium chloride give isatin derivatives. The acridine ring was formed from an isatin structure in aqueous potassium hydoxide solution. The acridine derivatives were converted to acid chloride. Subsequently, esterification of phenols in the presence of 4-dimethylaminopyridine and triethylamine, and N-methylation of acridine using methyl trifluoromethanesulfonate proceeded in dichloromethane. The yields for 1a-3d were 39 -68 .
In CL measurements, to the solution of 1a-3d in dimethyl sulfoxide was added a buffer solution, and then aqueous hydrogen peroxide solution was added to the mixture. The CL emission was measured using luminometer. The timecourses for CL development of 1d at pH 7-10 are shown in Fig. 1a. After the addition of hydrogen peroxide, the CL was flash-type and glow-type at pH 7 and pH 8, respectively. At pH 7, the intramolecular nucleophilic attack of OOH at the 9-postion on the acridine moiety to the phenyl ester may rapidly occur Scheme 1 . CL intensities at pH 7 and 8 were stronger than those at pH 9 and 10. At pH 8, the time required to reach the maximum relative light units RLU was approximately 30 s after the addition of hydrogen peroxide. The time-courses of the CL development of 1a and 1d were similar at pH 7-10. Fig. 1b shows the timecourses of the CL development of 1d, 2d, and 3d at pH 10. The descending order of CL intensities was as follows: 3d 2d 1d. Thus, an increased number of methyl groups on the acridine moiety caused an increase of CL intensities under alkaline conditions. Interestingly, an acridinium ester that showed long-lasting CL was found, i.e., 3d.
The CL intensities of 1a-3d at pH 7-10 are shown in Fig.  2. Dramatic change of CL intensities was observed at different pH values. The optimal pH value was defined as pH value that gave maximum CL intensity. The optimal pH values for the CL of 1a-1d, 2a-2d, and 3a-3d were pH 8, pH 8-9, and pH 9, respectively. The optimal pH value for CL shifted from neutral conditions to alkaline conditions. The introduction of methyl groups weak electron-donating groups at the 2-, 2,7-and 2,3,6,7-positions on the acridine moiety affected the optimal pH value for the CL of 1a-3d. As shown in Fig. 2, the CL intensities of 1a-1d decreased apparently at pH 9-10. For example, the CL intensity of 1d at pH 8 was approximately 11-fold stronger than that at pH 9. Relative to the CL intensities of 1a-1d at pH 7-10, the CL intensities of 2a-2d were higher at pH 9-10 and lower at pH 7-8. In particular, the increase of CL intensity at pH 9 was noticeable. Similar results were obtained for the CL intensities of 3a-3d, with the CL intensities of 3a-3d at pH 10 being strong. The CL intensities of 3a-3d at pH 10 were approximately 9-, 9-, 8-, and 15-fold stronger, respectively, than those of 1a-1d at pH 10. Among several CL compounds, luminol 5-amino-2,3-dihydrophthalazine-1,4-dione has high CL intensities under alkaline conditions. When the concentrations of sodium hydroxide and hydrogen peroxide were 1 mM, the maximum CL intensity of 10 nM luminol was obtained. Compared to the CL intensities of 3d 10 nM and luminol 10 nM , those of 3d at pH 9 and 10 were approximately 4-fold stronger than that of luminol.
In 1a-1d at pH 9-10, after the hydroperoxide anion reacts with the carbon at the 9-position on the acridine moiety, the hydrolysis of phenyl ester may proceed Scheme 1 11,22,23 . In contrast, in 2a-3d at pH 9-10, the dioxetanone structure could mainly be formed as a chemiluminescent pathway, and the CL intensities increase. The introduction of methyl groups on the acridine moiety may suppress the hydrolysis of the phenyl ester in acridinium Scheme 2 Synthetic procedure of 1a-3d. ester derivatives. Compounds 1a, 2a, and 3b were selected for the measurement of CL spectra, because these three had the strongest CL intensities among 1a-1d, 2a-2d, and 3a-3d, respectively. The CL spectra of 1a, 2a, and 3b were measured at pH 7-10. The maxima wavelengths of CL emission of 3b were approximately 440-450 nm Fig. 3 . The maximum wavelengths of CL emission of 1a and 2a was similar with that of 3b Fig. S1 . There was no apparent change in the maxima wavelengths with increasing number of methyl groups.
Acridinium ester derivatives synthesized in this study were applied to determine hydrogen peroxide concentra-tion at a wide range of pH conditions, including neutral. From the CL measurement of 1a-3d at pH 7-10, the CL intensities of 1a were maximal at pH 7 and 8. Compounds 3b and 3d gave maximum CL intensities at pH 9 and pH 10, respectively. Therefore, hydrogen peroxide was measured using 1a, 3b and 3d. Standard curve of hydrogen peroxide using 3d at pH 10 is shown in Fig. S2. The linear calibration ranges of hydrogen peroxide were 0.05-1 mM R 0.994 at pH 7 and 0.01-1 mM R 0.999 at pH 8 using 1a Fig. S2 . The linear calibration ranges of hydrogen peroxide were 0.01-1 mM R 0.999 at pH 9 using 3b Fig. S2 and 0.01-1 mM R 0.999 at pH 10 using 3d. A relatively long range of detectability and good linearities R 0.994 were obtained. The detection limit defined as the mean value of blank 5σ standard deviation of hydrogen peroxide at pH 10 using 3d was 4.5 mM. This CL method showed highly sensitive detection of hydrogen peroxide without an addition of catalyst. Compared to previous CL methods with an addition of catalyst for determining hydrogen peroxide using luminol hybrid Mg-Al-CO 3 layered double oxides 24

Conclusion
Acridinium ester derivatives with electron-withdrawing groups at the 4-position in the phenyl ester were synthe- Fig. 2 Chemiluminescence intensities of 1a-3d at pH 7-10. The concentration of 1a-3d and hydrogen peroxide was 10 nM and 1 mM, respectively. The relative light unit RLU integrated for 1 min was used to evaluate the chemiluminescence intensity. sized, the optimal pH values shifted from neutral conditions to alkaline conditions by introducing methyl groups at the 2-, 2,7-, and 2,3,6,7-positions on the acridine moiety. These acridinium ester derivatives can determine hydrogen peroxide concentrations under optimal conditions. Facile methyl substitutions apparently changed the CL intensities at different pH values. Moreover, acridinium ester derivatives having long-lasting CL under alkaline conditions were developed. These experimental results could be crucial information for the molecular design of useful acridinium ester derivatives at pH 7-10.