Appl Biochem Biotechnol DOI 10.1007/s12010-015-1575-5

New Chemiluminescent Substrates of Paraoxonase 1 with Improved Specificity: Synthesis and Properties Zulipiyan Abulimite 1 & Xiaojing Mu 1 & Shangyou Xiao 1 & Min Liu 2 & Quandan Li 1 & Gang Chen 1

Received: 29 November 2014 / Accepted: 12 March 2015 # Springer Science+Business Media New York 2015

Abstract Paraoxonase 1 (PON1) is an important hydrolase, and the enzyme activity decreases in patients with liver disease, diabetes, coronary heart disease, etc. Phenyl acetate and organophosphates are usually employed as substrates for serum PON1 activity assay. However, phenyl acetate for arylesterase activity assay exhibits disadvantage of high background. According to properties of PON1, four new chemiluminescent acridinium esters were designed, prepared through three steps, and characterized with 1H NMR and mass spectrometry (MS) data, and their properties as PON1 substrates were investigated. The hydrolyses of the four compounds catalyzed by recombinant human PON1 (rhPON1) (or serum) followed firstorder kinetics within 22 min. The PON1 activator (NaCl, 0.10 mol L−1) could boost the rhPON1-mediated and serum-mediated hydrolyses of the acridinium esters to 2.01~2.26 folds, but 1.0 mol L−1 NaCl decreased the serum arylesterase activity. RhPON1 showed selectivity over other serum esterases such as lipase, acetylcholinesterase, and esterase D more than 300 folds. By using ethylene diamine tetraacetic acid (EDTA) inhibitor, the specificities of the four substrates toward serum PON1 were determined as 78.3~92.9 %, which is improved than that of the model compound 9-(4-chloro-phenoxycarbonyl)-10-methylacridinium ester triflate. Due to low toxicity, high specificity, and sensitivity of the substrates, they are useful for serum PON1 activity assay. Keywords Acridinium ester . Alkyl group . Chemiluminescence . Serum . Arylesterase activity . Stability . Specificity

* Xiaojing Mu [email protected] 1

College of Chemistry & Chemical Engineering, Chongqing University, Chongqing 400044, China

2

College of Chemistry, Huazhong University of Science and Technology, Wuhan 430074, China

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Abbreviations PON1 Paraoxonase 1 rhPON1 Recombinant human PON1 CL Chemiluminescent/chemiluminescence CPOCMA 9-(4-chlorophenoxycarbonyl)-10-methylacridinium triflate ester EDTA Ethylene diamine tetraacetic acid

Introduction Paraoxonase 1 (PON1, EC 3.1.8.1) is a Ca2+-dependent hydrolase with three carbohydrate chains [1–3]. PON1 can prevent lipid oxidation in low-density lipoprotein and thus decrease the levels of oxidized lipids that are associated with aging, anoxia-reoxygenation injury, and atherosclerosis [4–6]. Additionally, some evidences demonstrated its key role in metabolism of drugs [7–9]. Decrease of serum PON1 activities is usually related to many chronic diseases. Several papers reviewed the correlations between serum PON1 activity and diseases such as atherosclerosis, diabetes, cancers, migraine, pulmonary tuberculosis, polycystic ovary syndrome, gastroesophageal malignancies, depression, nephritic syndrome, hemodialysis, metabolic syndrome, and liver disease [10–14]. Therefore, determination of PON1 activity has a significant diagnostic value in predicting disease status. Generally, the determinations were based on the substrates mainly including phenyl acetates, organophosphates, dihydrocoumarins, and thiobutyrolactones [15–17]. There are difficulties with serum PON1 activity assay, due to interference by other serum hydrolases. For example, paraoxon for serum organophatase (OPase) activity assay and phenyl acetate for serum arylesterase activity assay showed background activity as high as 10–20 and 35–50 %, respectively [18]. Organophosphates including paraoxon, diazinon, and chlorpyrifos are well acknowledged as specific substrates due to their ability to inhibit some of other serum esterases. In our lab, a method based on the 9-(4-chlorophenoxycarbonyl)-10-methylacridinium triflate ester (CPOCMA) as a substrate was previously reported for serum PON1 arylesterase activity assay [19]. The CPOCMA showed advantages of good specificity, long wavelength emission, high sensitivity, and low toxicity. However, it was unstable in storage. Metabolism of some hydrophobic drugs was attributed to PON1 other than other blood esterases [7–9]. Additionally, different kinds of esters (such as arylesters, thiolactones, and organophosphats) can be hydrolyzed by this enzyme. It suggests that PON1 shows very good adaptability. We inferred that the substrate slightly stable against PON1-mediated hydrolysis would resist catalysis of some other serum esterases to a great extent. This would result in higher specificity. To our knowledge, acridinium esters with electron-donating substituents on the phenoxy group showed better stability against hydrolysis due to electron shielding [20]. However, the strong electron-donating groups such as−OH and−NH2 which are hydrophilic groups easily form a complex with Ca2+ in PON1 active center instead of the ester group, possibly resulting in loss of PON1 activity. Therefore, the hydrophobic alkyl groups without binding with metal ions would be introduced to modify acridinium esters. Moreover, the long aliphatic chain (alkyl group) of a PON1 substrate exhibited many degrees of freedom in docking simulations [21]. Therefore, introduction of electron-donating alkyl group to the acridinium ester would be expected to result in better stability and specificity. The new acridinium esters would be compared with the model compound (CPOCMA) in stability and specificity.

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Materials and Methods Apparatus and Materials 1

H NMR spectra were measured on a Bruker 500 spectrometer with TMS as the internal standard, and a Ruimai IFFS-A CL machine (Xi’an, China) was used for chemiluminescence (CL) tests. The solvents for flash column chromatography were redistilled before use. TLC was performed on silica gel plates and visualized by UV 254 nm. The acridine9-carboxylic acid was prepared in our lab, and its structures were confirmed by 1H NMR and MS data. Pure recombinant human PON1 (rhPON1) and human esterase D were purchased from Ximei Chemical Ltd. (Shanghai, China), which was prepared by ProSpec (Protein-Specialists), East Brunswick. Porcine pancreas lipase was purchased from Lanji Technology Co. Ltd. (Shanghai, China). Tris, CaCl2, NaCl, H2O2, CTAB, HNO3, and NaOH were of analytical grade. Distilled-deionized water was used. Human blood sera were freshly collected from healthy people in routine physical examination in the Hospital of Chongqing University. Sera were kept in a refrigerator and used up within 5 days. This study has been endorsed by Ethics Review Board of Chongqing University for human studies.

Synthesis Procedure for 9-(Phenoxycarbonyl) Acridine Ester In a flask equipped with a condenser and a CaCl2 drying tube, acridine-9-carboxylic acid hydrate was suspended in excess thionyl chloride and refluxed for 3 h. Then, the solvent was removed under vacuum to leave a yellow oil. In order to remove the thionyl chloride completely, two drops of toluene were added to the flask and then evaporated under vacuum to leave a yellow solid (acridine-9-carboxylic acid chloride). To the above residue, phenol (0.35 g) and pyridine (10 mL) were added sequentially. The mixture was stirred rigorously at room temperature for 10 h. Afterwards the volatiles were removed under vacuum to leave a brown residue. The crude product was obtained by extraction with dichloromethane (DCM), and then purified with flash column chromatography to give a pale yellow solid.

Synthesis Procedure for 9-(Phenoxycarbonyl)-10-Methylacridinium Triflate Esters To a flask flushed with nitrogen, 9-(phenoxycarbonyl) acridine ester (0.13 g) was added and followed by injection of dry DCM (3 mL) and methyl triflate (100 μL) sequentially. The mixture was stirred at room temperature for 3 h. Afterwards, the solution was condensed and subjected to silica column chromatography to give pure final product, a bright yellow solid (Rf = 0, DCM and Rf = 0.3, DCM/acetone 4:1).

Preparation of Acridinium Ester Solution A requisite amount of an acridinium ester was dissolved in acetonitrile to prepare stock solution (2.02×10−3 mol L−1). The freshly prepared stock solution was diluted gradually with acetonitrile, and the final dilution was performed with a Tris-HCl buffer (0.100 mol L−1, pH 7.5).

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Enzyme-Mediated Hydrolysis of CL Substrates Requisite amounts of enzyme (rhPON1, diluted serum, lipase, or human esterase D), Tris-HCl buffer, and acridinium ester were added sequentially to a volumetric flask and immediately diluted to 10 mL with distilled water. The test solution was incubated at 25±1 °C. Unless otherwise stated, the final concentration of all the acridinium ester was 4.02×10−11 mol L−1; the rhPON1 concentration was 1.80×10−15 mol L−1 (or the serum was 5.0×107-fold diluted). The chemiluminescence of the solution was tested at intervals of 2, 7, 12, 17, and 22 min, respectively, and the hydrolysis kinetics was determined.

CL Test A sequential injection mode was used for the tests [20]. The test solution (50 μL) and triggering reagent 1 (0.20 mL, 0.10 % H2O2 plus 0.10 mol L−1 HNO3) were added to a test tube, and then, the chemiluminescent reaction was triggered by addition of reagent 2 (1.0 mL, 1.0×10−3 mol L−1 cetyltrimethylammonium bromide plus 1.0 mol L−1 NaOH). The chemiluminescent kinetics was recorded automatically. The chemiluminescence intensity was expressed in relative light unit, and the total chemiluminescence was calculated by peak area. For each sample, three tests were paralleled, and the mean values of the data were reported.

Arylesterase Activity with Phenyl Acetate as a Substrate The rate of serum-catalyzed hydrolysis of phenyl acetate is assessed by monitoring the product phenol at 270 nm (the extinction coefficient: 1310 mol−1 cm−1 L). The reaction mixture contains 1.0 mmol L−1 phenylacetate, 1 mmol L−1 CaCl2 dissolved in 20 mmol L−1 TrisHCl buffer, pH 8.0 at 25 °C [22].

Phenotype Determination The phenotypes were determined by the method of Haagen [23]. The serum-catalyzed pnitrophenyl acetate (2.5 mmol L−1) was measured in the absence or the presence of phenyl acetate (1.0 mmol L−1) as an inhibitor. The assay solution also included 25 mmol L−1 triethanolamine hydrochloride. The activity calculation was based on the extinction coefficient of (14,000 mol−1 cm−1 L) p-nitrophenol at 405 nm, at pH 7.4. The ratio of the inhibited activity to non-inhibited activity was used to classify samples into three phenotypes as QQ, QR, and RR. One unit (1 U) of PON1 arylesterase activity is defined as 1 nmol hydrolyzed acridinium ester, 1 nmol p-nitrophenyl acetate, and 1 μmol phenyl acetate per minute, respectively.

Results and Discussion Synthetic Pathway and Structure Characterization The synthetic route for the 9-phenoxy-10-methyl acridinium triflate esters is illustrated in Scheme 1. The synthetic procedures were slightly modified according to ref [20].

Appl Biochem Biotechnol CH3CF3SO3 N

O

OH

SOCl2 Reflux,3h

N

N

N Pyridine, phenol O

Cl

r.t,10h

Methyltiflate O

R1

O

r.t,N2,3h

R2

O R1

O

R2

a Compound 1: R1=H, R2=Cl, 9-(4-chlorophenoxycarbonyl)-10-methylacridinium triflate; Compound 2: R1=H, R2=C(CH3)3, 9-(4-tert-butylphenoxycarbonyl)-10-methylacridinium triflate; Compound 3: R1=H, R2=H, 9-(4-phenyloxycarbonyl)-10-methylacridinium triflate; Compound 4: R1=H, R2=CH3, 9-(4-methylphenoxycarbonyl)-10-methylacridinium triflate; Compound 5: R1=CH3, R2=CH3, 9-(2,4-dimethylphenoxycarbonyl)-10-methylacridinium triflate.

Scheme 1 The synthetic pathway for the chemiluminescent substrates

The acridine-9-carboxylic acid chloride was not stable, and so, its structure was characterized with an IR spectrum. The absence of a band at 3500 cm−1 in IR spectra indicated the successful synthesis. The yields and 1H NMR data for the acridine esters were showed as follows: 1a (R1 =H, R2 =Cl): yield, 36 %. 1H NMR (500 M, CHCl3-d, δ, ppm): 7.42 (d, 2H, J= 8 Hz), 7.51 (d, 2H, J=8 Hz), 7.68 (apparent t, 2H, J=8 Hz), 7.86 (apparent t, 2H, J=8 Hz), 8.19 (d, 2H, J=8 Hz), 8.32 (d, 2H, J=8 Hz). 2a (R1 =H, R2 =CH3(CH3)3): yield, 63 %. 1H NMR (500 M, CHCl3-d, δ, ppm): 1.29 (s, 9H), 7.39 (d, 2H, J=8.5 Hz), 7.55 (d, 2H, J= 8.5 Hz), 7.67 (apparent t, 2H, J=8 Hz), 7.86 (apparent t, 2H, J=8 Hz), 8.24 (d, 2H, J=8 Hz), 8.32 (d, 2H, J=8 Hz). 3a (R1 =R2 =H): yield, 39 %. 1H NMR (500 M, CHCl3-d, δ, ppm): 8.26 (2H, d, J=9 Hz), 8.18 (2H, d, J=9 Hz), 7.79 (2H, dd, J=9, 7 Hz), 7.32 (1H, t, J=9 Hz), 7.41 (2H, d, J=9 Hz), 7.49 (2H, t, J=9 Hz), 7.61 (2H, dd, J=9, 7 Hz). MS (m/z), EI: 299 (M+, 78 %), 206 (97 %), 178 (100 %). 4a: (R1 =H, R2 =CH3) yield, 36 %. 1H NMR (500 M,CHCl3d, δ, ppm): 2.43 (s, 3H), 7.34 (m, 4H, J=8 Hz), 7.68 (dd, 2H, J=9, 7 Hz), 7.86 (dd, 2H, J=9, 7 Hz), 8.23 (d, 2H, J=8 Hz), 8.34 (d, 2H, J=8 Hz). 5a (R1 =CH3, R2 =CH3): yield, 70 %. 1H NMR (500 M, CHCl3-d, δ, ppm), 2.37 (s, 3H), 2.39 (s, 3H), 7.18 (m, 2H), 7.34 (d, 1H, J=8 Hz), 7.67 (dd, J=9, 7 Hz), 7.85 (dd, 2H, J=9, 7 Hz),8.28 (d, 2H, J=8 Hz), 8.33 (d, 2H, J=8 Hz). The yields and 1H NMR and MS data for the target compounds were showed as follows: 1: yield, 88 %. 1H NMR (DMSO-d6, δ, ppm): 4.96 (s, 3H), 7.73 (d, 2H, J=9 Hz), 7.85 (d, 2H, J= 9 Hz), 8.18 (dd, 2H, J=9, 7 Hz), 8.56 (dd, 2H, J=9, 7 Hz), 8.66 (d, 2H, J=9 Hz), 8.95 (d, 2H, J=9 Hz). MS (m/z), ES+: 348 ([M-CF3SO3]+, 100 %), 252 (21 %), 193 (71 %); ES−: 149 ([CF3SO3]−, 100 %). 2: yield, 93 %. 1H NMR (CH3OH-d, δ, ppm): 1.34 (s, 9H), 5.03 (s, 3H), 7.53 (d, 2H, J=8 Hz), 7.64 (d, 2H, J=8 Hz), 8.29 (apparent t, 2H, J=8 Hz), 8.55 (apparent t, 2H, J=8 Hz), 8.62 (d, 2H, J=8.5 Hz), 8.87 (d, 2H, J=8.5 Hz). MS (m/z), ES+: 424 (7 %), 370 ([M-CF3SO3]+, 100 %), 252 (21 %), 193 (68 %); ES−: 149 ([CF3SO3]−, 100 %). 3: yield, 88 %. 1H NMR (500 MHz, DMSO-d6, δ, ppm), 7.65 (1H, t, J=8 Hz), 7.81 (2H, apparent t, J= 8 Hz), 7.91 (2H, d, J=8 Hz), 8.41 (2H, dd, J=8, 7 Hz), 8.79 (2H, dd, J=9, 7 Hz), 8.89 (2H, d, J=8 Hz), 9.22 (2H, d, J=9 Hz). MS (m/z), ES+: 314 ([M-CF3SO3]+, 100 %), 193 (71 %); ES−: 149 ([CF3SO3] −, 100 %). 4: yield, 56 %. 1H NMR (CH3OH-d, δ, ppm): 2.44 (s, 3H), 5.02 (s, 3H), 7.40 (d, 2H, J=8 Hz), 7.47 (d, 2H, J=8 Hz), 8.16 (apparent t, 2H, J=8.5 Hz), 8.53

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(apparent t, 2H, J=8.5 Hz), 8.59 (d, 2H, J=8.5 Hz), 8.86 (d, 2H, J=8.5 Hz). MS (m/z), ES+: 328 ([M-CF3SO3]+, 100 %), 252 (17 %), 193 (68 %); ES−: 149 ([CF3SO3]−, 100 %). 5: yield, 83 %. 1H NMR (CH3OH-d, δ, ppm): 2.38 (s, 3H), 5.03 (s, 3H), 7.25 (m, 2H), 7.53 (d, 1H, J= 8 Hz), 8.19 (apparent t, 2H, J=8.5 Hz), 8.55 (apparent t, 2H, J=8.5 Hz), 8.63 (d, 2H, J= 8.5 Hz), 8.88 (d, 2H, J=8.5 Hz). MS (m/z), ES+: 396 (3 %), 342 ([M-CF3SO3]+, 100 %), 252 (21 %), 220 (18 %), 193 (69 %); ES−: 149 ([CF3SO3]−, 100 %).

Chemistry for the PON1 Activity Assay The acridinium esters are chemiluminescent molecules, while their hydrolysis products are non-chemiluminescence. The assay was based on the PON1-mediated hydrolysis of an acridinium ester, and the hydrolysis was monitored by CL decrease of a substrate after incubation with PON enzyme. The principle is illustrated in Scheme 2.

General Chemiluminescent Properties of the Acridinium Esters All the five acridinium esters gave off chemiluminescence upon triggering with H2O2 under basic conditions. For each acridinium ester, the intensity grew, reached a maximum, and fell gradually. The CL process finished within 4 s for the 2,4-dimethyl compound (5) and within 0.8 s for the other four compounds 1, 2, 3, and 4. All the five acridinium esters displayed a quick chemiluminescent kinetics and good CL efficiency. The within-day variation coefficients for CL intensity of the five acridinium esters at concentration of 4.02×10−11 mol L−1 were in the range 1.0~2.7 % (n=5). Accurate quantification of the substrate concentration is a basis for serum enzyme activity assay. Hence, the calibration curves for the acridinium esters are determined. It showed that for each of the acridinium esters, its chemiluminescent intensity (F/RLU) was linear over its concentration (C/1.0×10−11 mol L−1) in a range of 0.500~8.08×10−11 mol L−1. The linear equations are F=4.48×103C+17 (1, R2 =0.9994), F=1.20×104C+714 (2, R2 =0.9987), F= 1.83×104C+101 (3, R2 =0.9997), F=1.24×104C+85 (4, R2 =0.9996), F=1.36×104C+4 (5, R2 =0.9999), respectively. By using the equations, the concentration of acridinium esters can be calculated. Beyond this upper concentration limit as in the case of 2.02×10−10 mol L−1, the

CH3 CF3SO3 N

CH3 CF3SO3 N

PON1 H2O

O R1

O

O

OH

Non-Chemiluminescent R2 Chemiluminescent Scheme 2 The principle for PON1 activity assay by using the CL substrates

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CL intensity of compound 3 did not increase linearly due to that the peak was beyond the upper limit of instrumental detection range. Therefore, the acridinium ester concentration 4.02×10−11 mol L−1 within the linear range was used in the following section. Additionally, the slopes of the lines meaning the relative chemiluminescent efficiency were 0.896, 2.40, 3.66, 2.48, and 2.72×1019 RLU/mol for compounds 1, 2, 3, 4, and 5, respectively. It means the relative chemiluminescent efficiency for the five acridinium esters ranked as the following order: unsubstituted > 2,4-dimethylphenoxy > 4-methylphenoxy > 4-tertbutylphenoxy>chloro acridinium ester. This result showed that the unsubstituted acridinium ester had the highest CL efficiency and a para substituent resulted in slight decrease in CL efficiency. This conclusion is consistent with ref. [24]. In all, the new four acridinium esters (2 ~5) demonstrated good chemiluminescent quantum yields.

The Hydrolytic Stability of the Acridinium Esters The auto-degradation of the substrates should be avoided during enzyme activity assay. So, the self-hydrolysis of the acridinium esters was evaluated. The results showed that the selfhydrolysis of the five acridinium esters in Tris-HCl buffer (pH 7.5) at 25 °C was insignificant within 22 min. It means that all the five acridinium esters in the buffer were stable enough for PON1 activity assay. The stability of the acridinium esters is related to background, turnover number, and specificity in PON activity assay, and so, their difference in stability was further evaluated under different conditions. The stock solutions of the acridinium esters in acetonitrile (1.25× 10−5 mol L−1) were kept at a refrigerator (4 °C) for 12 weeks before dilution to ready-to-use solutions (4.02×10−11 mol L−1). The CL intensities of the acridinium esters after stock were compared with those freshly prepared in Fig. 1. It showed that some of the acridinium esters showed conspicuous hydrolysis after stock for 12 weeks. The stability of the acridinium esters ranked in the following order: 2,4-dimethyl>4-tert-butyl>4-methyl>unsubstituted>4-chloro. As expected the chloro (electron-withdrawing) group at the phenoxy moiety can destabilize an acridinium ester, while the electron-donating group substituted at the phenoxy moiety can

Fig. 1 The self-hydrolysis of the five acridinium esters in acetonitrile after stock in a refrigerator for 12 weeks

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stabilize an acridinium ester. By comparing the 2,4-dimethyl acrdinium ester (5) with the unsubstituted (3) or 4-methyl (4) compounds, the steric effect resulted from the substituent at the ortho position to ester bond markedly prevented an acridinium ester from hydrolysis. As expected, all the four acridinium esters (2~5) showed better stability than the model compound (1).

The Kinetics for the rhPON1-Catalyzed Hydrolysis of the Acridinium Esters PON family has three members including PON1, PON2, and PON3 [25]. Serum PON2 is not detectable, and dihydrocoumarin is the only substrate reported to date for PON2 [26, 27]. Unlike PON1, PON3 has very limited arylesterase activity and no paraoxonase activity, and human serum PON3 is about two orders of magnitude less abundant than PON1 [28–30]. Therefore, serum arylesterase activity is mainly attributed to PON1 rather than PON2 or PON3. So, in this paper, only the PON1-catalyzed and the serum-catalyzed hydrolyses of the acridinium esters were focused on. Generally, in PON1 activity assay, a weakly basic buffer ensures high turnover number. However, in basic conditions, self-hydrolysis of substrates is accelerated. In our previous investigation, self-hydrolysis of compound 1 (CPOCMA) within 20 min was not detectable in a pH 7.5 buffer, but the self-hydrolysis was observed in a pH 8.0 buffer [19]. Therefore, a pH 7.5 buffer (Tris-HCl) was employed in the enzyme-catalyzed hydrolysis of the acridinium esters. After the acridinium ester concentration was fixed as 4.02×10−11 mol L−1, the rhPON1 concentration was selected as 1.80×10−15 mol L−1 to ensure first-order hydrolysis kinetic curves for each substrate. The acridinium esters were incubated with rhPON1 in a Tris-HCl (pH 7.5) buffer at 25 °C. At intervals, the chemiluminescence was tested, and the results were illustrated in Fig. 2a. The chemiluminescence decayed after incubation of each acridinium ester with the enzyme, suggesting that the rhPON1-catalyzed hydrolysis of acridinium esters occurred. The rhPON1-mediated hydrolyses of the acridinium esters obeyed first-order reaction kinetics. V=−dC / dt=kC (1). By integration and the linear relationship between concentration and CL intensity of acridinium esters, Eq. 1 can be described as LnF=−kt+LnF0 (2), where F0 is the chemiluminescent intensity of an acridinium ester without addition of the enzyme; F is

Fig. 2 The kinetic curves for the rhPON1-catalyzed hydrolysis of the four acridinium esters. a The chemiluminescence was plotted against incubation period. b The logarithms of chemiluminescence was plotted against incubation period

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the chemiluminescent intensity of the acridinium ester after incubation with the enzyme; the t is the incubation period; and the k is a constant of hydrolysis kinetics. According to Eq. 2, LnF was plotted against t to give Fig. 2b, and the kinetic constant was calculated as the value of line slop. In Fig. 2b, the kinetic constants (k) were calculated as 0.0190, 0.0140, 0.0162, 0.0152, and 0.0129 min−1 for substrates 1, 2, 3, 4, and 5, respectively; and the values were ranked in the order 1>3>4>2>5. Based on the k values, the model substrate (1) demonstrated highest turnover number, while the 2,4-dimethyl compound (5) showed poorest turnover number. This order suggested that among the five acridinium esters, the ones susceptible to self-hydrolysis showed relatively high turnover number and the stable acridinium ester showed low turnover number.

The Kinetics for the Serum-Catalyzed Hydrolysis of the Acridinium Esters The serum-catalyzed hydrolysis of the acridinium esters was determined, and the results are presented in Fig. 3. It is clear that the serum of 5×107-fold dilution can catalyze the hydrolyses of all the five acridinium esters. The kinetic constants were calculated as 0.011, 0.0086, 0.0094, 0.0091, and 0.0078 min−1 for substrates 1, 2, 3, 4, and 5, respectively. These kinetic constant values were in the same order as that for the rhPON1-catalyzed hydrolysis: 1>3>4> 2>5. Among the five acridinium esters, the one prone to rhPON1-mediated hydrolysis was also hydrolyzed by serum easily. It should be noted that sera from different healthy subjects showed different catalytic ability toward the hydrolysis of acridinium esters. As expected, all the four acridinium esters can be hydrolyzed by rhPON1 (pure enzyme) and serum, respectively.

Hydrolysis of Acridinium Esters Catalyzed by Other Serum Esterases Besides PON1, there are other serum esterases being able to catalyze the hydrolysis of acridinium esters, causing non-specific activity (background activity). It is too arduous to evaluate hundreds of serum esterases one by one. In view of the molecular structure of

Fig. 3 The kinetic curves for the serum-catalyzed hydrolysis of the acridinium esters

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acridinium esters which are carboxylesters, it was estimated that the carboxyl esterases mainly contributed to the non-specific serum activity. Esterase D, lipase, and acetylcholinesterase are important serum carboxylesterases which have good flexibility to hydrolyze substrates of various molecular structures [31, 32]. So, their abilities for hydrolysis of the acridinium esters were investigated, respectively. The results showed that acetylcholinesterase did not hydrolyze the new acridinium esters. The kinetic curves for the esterase D-catalyzed and the lipase-catalyzed hydrolysis of the five acridinium esters were determined, respectively, and the kinetic constants for esterase D (k1) and for lipase (k2) were calculated and compared with the kinetic constants for rhPON1 (k3, from Fig. 3b) for each acridinium ester. The results were listed in Table 1. The concentrations of esterase D and of the lipase were 327 and 333 times as of rhPON1, but their catalytic abilities for the hydrolyses were in a similar level. It means that both esterase D and lipase showed far poorer activity than rhPON1 in hydrolysis of the acridinium esters. According to literatures [33, 34], it is concluded that in serum of healthy subjects, PON1 level (90.2~105.9 mg/L) is far higher than lipase level (7.7~56 μg/L). Although we did not find serum esterase D level directly from literature, its blood level of 93~155 mg/L could be calculated approximately according to literature [35], which is at a similar level with serum PON1. So, we know serum lipase and esterase D did not show detectable interference on serum PON1 activity. Smaller value of the ratios (k1/k3 and k2/k3) in Table 1 means better selectivity of rhPON1 over the estersase D or lipase. For the esterase D, the ratio (k1/k3) was in the order 1>3>4>2= 5, and for the lipase, the ratio (k2/k3) was in the order 1>3>4>5>2. The orders suggested that substrates 2 (4-tert-butyl) with a bulky group and 5 (2,4-dimethyl) with a steric group showed better selectivity toward rhPON1.

Activation Effects of NaCl on PON1 Activity and Serum Arylesterase Activity From the above discussion, we know that the esterase D and lipase contributed very low activity to the total serum arylesterase activity based on the acridinium esters. However, we were afraid that accumulative activity from hundreds of other serum hydrolases possibly resulted in high background in serum PON1 activity assay. Therefore, the specific PON1 activity was determined further with activator and inhibitor of PON1, respectively. If the serum-mediated hydrolysis of the acridinium esters was mainly aroused by serum PON1, the hydrolysis would be activated by its activator significantly; otherwise, the serum-mediated Table 1 Comparison of esterase D and lipase with the rhPON1 for hydrolysis of the five acridinium esters Parameter/comp. no.

1

2

3

4

k1a (esterase Db)/min−1

0.0188

0.0093

0.0147

0.0131

0.0085

k2a (lipaseb)/min−1

0.0230

0.0088

0.0185

0.0117

0.0086

k3a (rhPON1b)/min−1

0.0190

0.0140

0.0162

0.0152

0.0129

Ratio (k1/k3)

0.98

0.66

0.90

0.86

0.66

Ratio (k2/k3)

1.21

0.63

1.14

0.78

0.67

a

5

The k is a kinetic constant

The concentration of all the acridinium esters was fixed at 4.02×10−11 mol L−1 . The concentrations of esterase D, lipase and rhPON1 were 5.88×10−13 , 6.00×10−13 , and 1.8×10−15 mol L−1 , respectively b

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hydrolysis promoted by its activator would be trivial. It is well acknowledged that both Na+ and Ca2+ are activators of PON1. The NaCl-rhPON1-catalyzed hydrolysis of the acridinium esters was investigated. The NaCl concentration was selected as 0.100 mol L−1 according to refs. [36, 37]. The hydrolysis kinetic curves were shown in Fig. 4. By comparing the pairs of the hydrolysis kinetic constants, it can be concluded that NaCl of 0.100 mol L−1 boosted rhPON1 activity 2.10, 2.22, 2.07, 2.10, and 2.23 folds for substrates 1, 2, 3, 4, and 5, respectively. Similarly, the effects of NaCl on lipase and esterase D activity were investigated, respectively. The kinetic curves for the NaCl-lipase-mediated and NaCl-esterase Dmediated hydrolysis of the acridinium esters were obtained (unshown data). The results revealed that the NaCl (0.100 mol L −1 ) neither enhanced the lipasecontrolled hydrolysis of acridinium esters nor the esterase D-mediated hydrolysis of acridinium esters. Therefore, addition of NaCl will improve specificity in serum PON1 activity assay. The kinetic curves for the NaCl-serum-mediated hydrolysis of the substrates were determined and paralleled with those unactivated ones. The results were illustrated in Fig. 5. Clearly, addition of NaCl (0.100 mol L−1) resulted in decrease of CL and thus acceleration of the hydrolyses. The ratio of the kinetic constants (k) for each substrate was calculated. It showed that the NaCl (0.100 mol L−1) improved the k values to 2.02, 2.08, 2.07, 2.01, and 2.26 folds for the 1, 2, 3, 4, and 5, respectively. These activation effects were in a similar level to those on rhPON1. It suggested that serum arylesterase activity was mainly attributed to PON1. Otherwise, if the serum arylesterase activity was mainly attributed to the other serum hydrolases, the activation effects on serum would be far less than on rhPON1. The activation effects of NaCl at other concentrations were investigated further. The reaction kinetic constants were plotted against NaCl concentration to give Fig. 6. It demonstrated that the NaCl increased serum arylesterase activity (based on any acridinium ester) sharply until the concentration reached turning point (0.100 mol L−1).

Fig. 4 The NaCl effects on the rhPON1-mediated hydrolyses of the acridinium esters. (All the solid lines are the kinetic curves for the rhPON1-mediated hydrolyses, while the other lines are for the NaCl-rhPON1-mediated hydrolyses. The NaCl concentration was 0.100 mol L−1, and the rhPON1 concentration was 7.21× 10−16 mol L−1.)

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Fig. 5 The NaCl-serum-mediated hydrolysis of acridinium esters. (All the solid lines are the kinetic curves for the serum-mediated hydrolyses, while the other lines are for the NaCl-serum-mediated hydrolyses.)

However, Eckerson et al. had reported that NaCl (1 mol L−1) increased the activity of the Rgenotype paraoxonase activity but inhibited arylesterase activity of both genotypes based on the phenyl acetate [22]. And, this property is important for determination of the PON1 phenotype or polymorphism. Therefore, the effect of NaCl (1.0 mol L−1) on arylesterase activity based on the acridinium esters in the presence of 1.0 mmol L−1 CaCl2 was investigated. The results are shown in Fig. 7. It showed that for all the five substrates, the addition of 1.0 mol L−1 NaCl decreased the serum arylesterase activity significantly. From Fig. 7, it seems that arylesterase activities of serum 2 (QQ) were inhibited differently from those of serum 1 (QR). However, it is difficult to tell that this is due to the different phenotypes or due to other factors.

Fig. 6 The activation effects of low concentration of NaCl on serum PON1 arylesterase activity

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Fig. 7 The decrease of serum arylesterase activity by addition of NaCl (1.0 mol L−1) (The phenotypes of serum 1 (QR) and serum 2 (QQ) were identified by method in ref. [23].)

Quantification of Specific PON1 Activity by Using a PON1 Inhibitor PON1 rather than other serum esterases being mainly responsible for the serum-mediated hydrolysis of acridinium ester was validated by using a NaCl activator. To further quantify the specificity and the background, a specific inhibitor was employed to inhibit the specific PON1 activity of serum. So, the inhibited activity was attributed to PON1, and the remained activity was mainly attributed to other serum esterases. Ethylene diamine tetraacetic acid (EDTA) as a PON1 inhibitor was used to determine the background activity of serum samples [18]. The optimized concentrations of EDTA and CaCl2 were quoted according to refs. [18, 38]. The EDTA of 4.0 mmol L−1 without giving off chemiluninescence was used for the following investigation. The results were listed in Table 2. From Table 2, it is clear that addition of EDTA downregulated both the Ca2+-rhPON1mediated hydrolyses and the Ca2+-serum-mediated hydrolyses of the acridinium esters. The kinetic constants for CaCl2-EDTA-rhPON1, CaCl2-rhPON1, CaCl2-EDTA-serum, and CaCl2-

Table 2 Evaluation of background and specificity Parameter/comp. no.

1

2

3

4

5

k1a (CaCl2-EDTA-rhPON1)/min−1

0.0003

0.0002

0.0004

0.0001

k2a (CaCl2-rhPON1)/min−1

0.0223

0.0151

0.0192

0.0188

0.0139

Background for rhPON1 (k1/k2)

1.3 %

1.3 %

2.1 %

0.5 %

3.6 %

98.7 %

98.7 %

97.9 %

99.5 %

96.4 %

RhPON1 specificity (100 %-k1/k2) k3a (CaCl2-EDTA-serum)/min−1

0.0060

0.0020

0.0036

0.0036

k4a (CaCl2-serum)/min−1

0.0210

0.0139

0.0172

0.0166

0.0005

0.0008 0.0113

Background for serum (k3/k4)

28.6 %

14.4 %

20.9 %

21.7 %

7.1 %

Specificity for serum (100 %-k3/k4)

71.4 %

85.6 %

79.1 %

78.3 %

92.9 %

a

The k is kinetic constant

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serum-mediated hydrolyses were determined as k1, k2, k3, and k4 in Table 2, respectively. The background for rhPON1 and for serum was expressed as k1/k2 and k3/k4, respectively. The specificity was expressed as (100 %-k1/k2) for rhPON1 and (100 %-k3/k4) for serum, respectively. In Table 2, the substrate specificity for a serum sample ranked in the order 5>2>3≈4> 1. It seems that high stability of acridinium esters boosted substrate specificity. From Table 2, it is clear that all the five acridinium esters showed lower background than the phenyl acetate (35–50 %), and the 2,4-dimethyl ester (5) and the 4-tert-butyl ester (2) showed significantly lower backgrounds than the other acridinium esters.

Effects of Polymorphism (192) on Catalytic Efficiency and Specificity with the Acridinium Esters as Substrates The paraoxonase 1 (192) phenotypes of 10 subjects were determined by using Haagen method [23]. The ratio of the arylesterase activity based on the 4-nitrophenyl acetate inhibited by phenyl acetate to the non-inhibited activity was calculated for each serum to assign individuals to one of the three phenotypes (the individual genotypes): homozygous QQ, heterozygous QR, or homozygous RR. Our results showed that of the 10 serum samples, 5 sera are of QQ phenotype, and the other 5 sera are of QR phenotype. Unfortunately, RR phenotype was not found. We hoped that by using the two phenotypes, we could learn performance of the polymorphism (192) on the catalytic efficiency and specificity. Phenyl acetate is called a non-discriminating substrate [39], due to that the three phenotypes of paraoxonase 1 (192) do not show distinction for hydrolysis of the phenyl acetate. Therefore, the ratio of arylesterase activity based on an acridinium ester to that based on the phenyl acetate for each serum was determined. And, the mean value of the ratios for each phenotype was calculated. The results are listed in Table 3. It showed that for substrates 1, 3, and 4, the sera of QQ phenotype and those QR phenotype did not show difference in mean values of the ratio (activity with acridinium esters/activity with phenyl acetate). It means that the two phenotypes displayed similar catalytic ability, and these substrates 1, 3, and 4 are nondiscriminating substrates as phenyl acetate. However, for substrates 2 and 5, the mean values of the ratios for the QQ phenotype sera were slightly higher than those for QR phenotype sera. This means that the QQ phenotype sera hydrolyze substrates 2 and 5 in a slightly higher rate than the QR phenotype sera. However, these conclusions are based on a small number of samples, and so, they should be validated further with more samples, and RR phenotype should also be included in future work. The specificities for serum 1 (QR) and serum 2 (QQ) arylesterase activities were determined by addition of EDTA to the samples, respectively. The results showed that for each substrate, serum 1 (QR) and serum 2 (QQ) did not show significant difference in specificity.

Table 3 The mean value for ratio of the serum arylesterase activities based on the acridinium esters to those based on the phenyl acetate Ratio/Comp. no.

1

2

3

4

5

QQ (n=5) QR (n=5)

0.19±0.03 0.19±0.03

0.15±0.02 0.13±0.02

0.16±0.03 0.16±0.03

0.16±0.03 0.16±0.03

0.14±0.02 0.12±0.01

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Conclusions In PON1 activity assay, high stability of the acridinium esters is related to high selectivity but low turnover number for serum samples. The four acridinium esters with alkyl group(s) or without a substituent on the phenoxy group showed higher stability and better specificity than the model compound (CPOCMA). The 9-(4-tert-butylphenoxycarbonyl)acridinium ester triflate and the 2,4-dimethyl one showed high specificity. In the future, the MichaelisMenten constants will be determined, and new methods based on the two specific substrates for serum PON1 activity assay will be established. Acknowledgments This work was financially funded by Natural Science Foundation of Chongqing China (Nos. cstc2014jcsfglyjs0013, cstc2011BB5090) and NSFC (No.20805060). Also, thanks are given to the hospital of Chongqing University for kindly collecting human blood serum samples.

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New chemiluminescent substrates of paraoxonase 1 with improved specificity: synthesis and properties.

Paraoxonase 1 (PON1) is an important hydrolase, and the enzyme activity decreases in patients with liver disease, diabetes, coronary heart disease, et...
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