JOURNAL OF BIOLUMINESCENCE A N D CHEMILUMINESCENCE VOL 5 1-4

(1990)

Unusual Luminescent Properties of Odd- and Even-substituted Naphthyl-derivatized Dioxetanes B. Edwards, A. Sparks, J. C. Voyta a n d 1. Bronstein Tropix Inc., 47 Wiggins Avenue, Bedford, MA 01730, USA

With the advent of enzymatically induced chemiluminescence and improved instrumentation for lurninometry, ultrasensitive detection of a wide variety of analytes is now possible using standard immunoassay and DNA probe formats. Model molecular orbital calculations and literature precedent suggest that the singlet efficiencies observed upon decomposition of dioxetanes appended with donor substituted aromatic moieties are dependent on substitution pattern. We have recently discovered, in a series of 3-(2’-spiroadamantane)-4methoxy-4-acetoxynaphth-2’-yl-l,2-dioxetanes, that enzymatic generation of a nonconjugated, charge transfer excited state results in luminescence of markedly different properties than that observed from an isomeric, conjugated excited state. An example of the former type, 3-(2’-spiroadamantane)-4-methoxy-4-(7-acetoxy)naphth-2’-yl-l,2-dioxetane (1) emitting at 550nm, not only provides an increase in @cL, but exhibits a dramatic bathochromic shift of 80-110nm from the 460nm emission of the conjugated isomer 3-(2’-spiroadamantane)-4-methoxy-4-(6”-acetoxy)naphth-2’-yl-l,2-dioxetane(2). These developments, along with the attendant glow-type luminescence kinetics displayed during the enzymatic decomposition of the new ‘odd-pattern’ dioxetane, allow the design of simple protocols capable of simultaneous or ’multichannel‘ detection of several analytes.

Keywords: Chemiluminescence; dioxetanes; multichannel immunoassay

INTRODUCTION Foreseeing the utility of chemiluminescencebased analytical systems with detection at the subattomole level, we have focused on developing enzyme cleavable dioxetane substrates displaying high signal to noise ratios, which in turn would maximize immunoassay and DNA probe assay sensitivity. Catalytic activation of chemiluminescence through enzymatic liberation of an electron-rich aromatic substituent necessitates the production of polar, charge transfer excited states. In such systems, quantum yields of chemiexcitation and fluorescence in the emitting fragment must be maximized to overcome delete08843996/90/01OOO 1-04$05.00 @ 1990 by John Wiley & Sons, Ltd

rious dipole-dipole solvent interactions and proton transfer quenching inherent in polar, protic media required for biological compatibility. Our efforts have recently been directed toward the synthesis and study of a series of dioxetane substrates derived through sensitized photooxidation of corresponding methoxy(naphth-2-y1)methylene adamantanes. The objective was to determine how changes in the position of a donor substituent on the naphthalene ring would affect chemiluminescent yield (QC-) and emission wavelength (Amax) of the excited ester fragment. (Fig. 1). The literature offers few examples from which these effects of positional isomerism can be assessed. Gagnon (1982) reported a

B. EDWARDS, A. SPARKS, J. C. VOYTA AND I. BRONSTEIN

2

Figure 1. Enzymatic production of an excited state ester via decomposition of a dioxetane anion OCH3 lower QCE, but greater stability and longer I total chemiluminescence lifetime for a meta bis( dimethylaminophenyl) tetraoxabicyclooctane when compared to the para isomer in toluene. In dimethylsulphoxide, Schaap et al. (1987a) observed a greater than 1000-fold higher QcE l a : X = 7-OAc for the 3-(2’-spiroadamantane)-4-methoxy-4(32a : X = 6-OAc oxy)phenyl-1,2-dioxetaneanion (4) than seen in 3a : X = 7-OP03Na2 the para analogue. The available scientific evidence indicates that meta substitution in phenyl n-n systems increases dioxetane stability and alters the chemiluminescence parameters. 1 As a theoretical preface to our synthetic work, X AM1 molecular orbital calculations on odd and even patterned, donor-acceptor substituted naphthalenes were performed. The calculations revealed that the largest amount of charge 1 : X = 7-OAc transfer from donor to acceptor occurs when the 2 : X = 6-OAc two groups are disposed in an odd (disjoint) 3 : X = 7-OP03Na2 relationship. On the basis of these results (Table l ) , excited states with greater charge transfer In order to evaluate the real effects of character and, therefore, chemiluminescent systems with an optimized Qc-, may be expected substitution patterns in naphthalene dioxetanes from odd-patterned naphthalene substituted and to test the predictions of the MO calculations, dioxetanes. (An odd pattern is one in which the compounds 1, 2 and 3 were synthesized for donor group’s (0-) point of attachment to the subsequent comparison of luminescence kinetics ring in relation to the ring’s point of attachment to and emission wavelengths. We also synthesized model methyl hydroxynaphthoate emitters indethe acceptor (C=O) is such that the total number of ring carbon atoms separating these points, pendently to assess the fluorescence efficiency of including the atoms at the point of attachment, is the corresponding anions in a photoexcitation mode. an odd whole number.)

-%

Table 1. Donor-acceptor substituted naphthyl model

Calculated homo-lumo gap (R=H)

Calculated net charge transfer (R=H)

Experimental aFL (MeOH) (R=CH,)

7.44 6.51 6.76

0.13 0.33 0.33

0.68

6.31 6.1 1 6.34

0.51 0.45 0.49

0.017 0.0062 0.025

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NAPHTHYL-DERIVATIZED DIOXETANES

METH0DS Dioxetane syntheses

Dioxetanes 1 , 2 (Schaap et al., 1987b), and 3 were synthesized by addition of singlet oxygen to the corresponding enol ethers ( l a , 2a, 3a) using standard photochemical techniques; details will be published in a subsequent communication. Conversion of the trisubstituted enol ethers to their corresponding dioxetanes can be monitored by loss of the ultraviolet absorption at 254 nm due to the extended vinyl-aromatic chromophore. The ‘H-NMR spectra (CDCl3) for 1 and 2 show doublets at approximately 0.97 ppm and 1.17 ppm; the ‘H-NMR spectrum ( D 2 0 ) for 3 shows doublets at 0.69 ppm and 0.98ppm. These characteristic pairs of upfield doublets correspond to the beta adamantane ring protons, shielded by the proximate aromatic ring. Kinetic luminescence measurements

A 5 x lop3mol/l solution of chromatographically pure dioxetane (1 or 2) in CH3CN ( 2 0 ~ 1 )was placed in a glass tube inside a Turner Model 20e Luminometer at room temperature. Manual injection of 4 5 0 ~ 1of 75mmolil NaOH in 20% CH3CN/H20 or 75mmol/l NaOCH3 in CH3CN induced ester cleavage and subsequent light emission. In a glass tube, 500ml of a 4 X lop4mol/l solution of chromatographically pure dioxetane 3 in HCO3-/C0?- buffer (0.05 mol/l, containing lmmol/l MgCI2; pH 9.5) was equilibrated to 30°C and treated with 2.2pg alkaline phosphatase (Biozyme ALP1 11G). The tube was placed in a Turner 20e Luminometer which was internally thermostatted at the same temperature. First order luminescent decay was recorded over a 30-minute interval. The experiment was then repeated except for the injection of 1 mol/l NaOH to pH 12, l m i n after placing the sample in the luminometer. The half lives were determined from the decay curves using graphical methods. Emission wavelength measurements

Emission wavelengths for dioxetanes 1 and 2 were recorded upon saponification of an acetonitrile or molil) aqueous acetonitrile solution (2.3 x placed in a Spex Fluorolog fluorimeter. Model

naphthyl emitters (near lo-’ mol/l; absorbance adjusted to 0.90) were deprotonated with KOH in MeOH (1 x 10p3mol/l), followed by excitation/ emission studies in a Spex Fluorolog fluorimeter. RESULTS AND DISCUSSION

Hydrolysis of acetate dioxetanes 1 and 2 resulted in two highly contrasting kinetic profiles. The naphthoxide anion of 2 gave an emission half-life (t1,2) of only 15 seconds in acetonitrile, whereas the anion of 1 showed extended chemiluminescence beyond 30 minutes in either acetonitrile or 20% acetonitrile/H20, integrating to a much larger total light output. This result is intriguing in view of the (PFL’sreported in Table 1. Because the kinetics of base-induced cleavage are similar for both esters under identical reaction conditions, the extended emission of the disjoint isomer must be due to a longer half-life of the dioxetane anion of 1. Dephosphorylation of dioxetane 3 displayed luminescence kinetics very similar to that observed for 1-emission tli2’s were 27.7min at pH 9.5 and 17.9min at pH 12. As a comparison between disjoint naphthyl and phenyl analogues, the recorded emission half-lives for dephosphorylated 6 (Bronstein et al., 1988) are 3min (pH 9.5) and 1.9min (pH 12). The surprisingly long half-lives of dioxetanes 1 and 3, yielding steady state production of light, are more conducive to reproducible light measurements in chemiluminescence-based applications than the flash kinetics associated with the conjugated oxyanion of 2 (van Dyke, 1985). 0-0

4:x=o5 : X = OAc 6 : X = OP03Na2

The emission maxima observed upon dioxetane decomposition also varied considerably with substitution pattern. Acetate hydrolysis of 1 resulted in green light emission at 555nm, red shifted by approximately 100 nm from the blue= 459nm). Enzymatic emitting isomer 2 (A,, cleavage of 1 with carboxylesterase in the presence of 0.1% BSA at pH 8 showed a blue

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B. EDWARDS, A. SPARKS, J. C. VOYTA AND I. BRONSTEIN

shift to 530 nm as expected from a chemiluminescent hydrophobic probe utilizing a charge transfer emissive state. Finally, in the course of this work, we demonstrated that the cleavage of 3-(2’spiroadamantane)-4-methoxy-4-(3-acetoxy)phenyl-1,2-dioxetane ( 5 ) [lop4mol/l, in the presence of lop3mol/l methyl 7-hydroxy-2-naphthoate (7)] by lO-*mol/l sodium methoxide in DMSO exhibited chemiluminescence at 473 nm, with no evidence of energy transfer to the green-emitting anion of 7 (A, = 551 nm). Dioxetane substrates for different enzymes, which are further differentiated by the wavelength shifting associated with substitution isomerism as reported here, would facilitate ‘multichannel’ enzyme immunoassay (EIA) of several antigens in the same sample, either simultaneously or sequentially. We are currently involved in investigations aimed at the construction of dual-channel prototypes using naphthyl and phenyl galactosides and the corresponding aromatic phosphates, with enzyme activation by galactosidase or alkaline phosphatase, followed by imaging on filtered photographic film and detection via filtered photomultiplier-tube-based luminometers.

CONCLUSIONS

Although direct measurements of QCL for dioxetanes 1-3 have yet to be performed, the phenomenon of disjoint substitution leading to steady-state ‘glow’ luminescence, in direct con-

trast to ‘flash’ luminescence observed with the conjugated isomer, suggests dioxetane decomposition can occur along several reaction coordinates, dependent on aromatic substitution patterns, with higher chemiexcitation efficiency arising from the disjoint systems. In addition, the disjoint naphthalene dioxetanes exhibit a dramatic bathochromic shift of approximately 100 nm in emission wavelength, enabling development of multichannel EIAs when used in conjunction with blue-emission dioxetanes. Acknowledgement We thank Professor Laren Tolbert at Georgia Institute of Technology for providing AM1 calculations and data.

REFERENCES Bronstein, I . , Voyta, J . C. and Edwards, B. (1988). J . Biolumin. and Chemiiumin., 2, 186. van Dyke, Knox (1985). Bioluminescence and Chemiluminescence: Instruments and Applications, CRC Press, Boca Raton, Vol. 1 , p. 5. Gagnon, S . D . (1982). Dissertation, Wayne State University. Schaap, A . P., Chen, T. S., Handlcy, R. S . , DeSilva, R. and Giri, B. P. (1987a). Chemical and enzymatic triggering of 1,2-dioxetanes. 2: Fluoride-induced chemiluminescence from tert-butyl-dimethylsilyloxy-substituted dioxetanes. Tetrahedron. Lett., 28, 1155-1 158. Schaap, A. P., Handley, R. S. and Giri, B. P. (1987b). Chemical and enzymatic triggering of 1,2-dioxetanes. 1: Aryl esterase-catalyzed chemiluminescence from a naphthy1 acetate substituted dioxetane. Tetrahedron. Lett., 28, 935-938.