APS-5: Synthesis and Application

General description

APS-5 is a white powdery substance, which is mainly used as the core reagent of chemiluminescence substrates. 

Synthetic routes

Fig. 1 The synthetic step 1 of APS-5.

9-acridinic acid (5.5 g, 0.025 mol), 4-chlorophenol (8.0 g, 0.055 mol), p-methylbenesulfonic acid (0.55 g) and methylbenzene (100 ml) were poured into 100 ml three-mouth bottle and heated for reflux reaction for 12 h. The water produced by the reaction is separated by the water separator, and the water (50 ml) and n-hexane (50 ml) are added to the pulping for 1 h. The yellow powder F (7.9 g, yield =90%) is obtained by filtering and drying [1].

Fig. 2 The synthetic step 2 of APS-5.

F (25.0g, 0.71 mol), zinc powder (9.3g, 0.14 mol) and dichloromethane (380 ml) were put into 500 ml single-mouth bottle, and then vinegar (6.6g, 0.11 mol) was added. The reaction was carried out at room temperature for 2 h, and then washed with saturated sodium bicarbonate solution (100 ml) and salt water (50 ml) successively after filtration, sodium sulfate was dried and concentrated, and methylene chloride (30 ml) was used for beating, filtration and drying to obtain light yellow powder G (20.g, yield =76%) [1].

Fig. 3 The synthetic step 3 of APS-5.

Pour G(13.0 g, 0.037 mol) and methylene chloride (250 ml) into 250 ml single-mouth bottle, Add dimethyl sulfate (11.8 g, 0. 093 mol), and react at room temperature for 12 h. After cooling, wash with saturated sodium bicarbonate solution (I00 ml), sodium sulfate dry, concentrated and condensed, and beat with ethyl acetate (30 ml) and n-hexane (60 ml). Light yellow powder H(13.0 g, yield =96%) was obtained by filtration and drying [1].

Fig.4 The synthetic step 4 of APS-5.

Pour H(10.0g, (0.027 mol) and tetrahydrofuran (100 ml) into 250 ml three-way bottle under nitrogen protection, and then drop 2 M LDA(27.3 ml, 0. 055 mol) to -80°C after the solution is dissolved. The reaction liquid becomes orange-red. After Ih reaction at _80°C, the THF (50ml) solution of phosphorus trichlorooxygen (8. 4 g, 0. 054 mol) and pyridine (25 ml) was added by drops. After 0.5 h of reaction at -80°C, the ice bath was removed. The mixture of 3-hydroxypropanitrile (9.7g, 0.14 mol) and pyridine (10 ml) was then cooled to -20°C. The ice bath was removed and the reaction was heated for 12 h. After filtration, the mixture was dried under pressure and then washed with ethyl acetate (150 ml) and water (50 mlX3). After sodium sulfate was dried and concentrated, silica gel column was passed (petroleum ether/ethyl acetate was first used to flush out impurities at 3:1, and then petroleum ether/ethyl acetate was used to flush out products at 1:2). Yellow oil I (8.0 g, yield =52%) was obtained after concentration, and purity reached 90% [1].

Application

A core reagent for chemiluminescence substrates

APS-5 is a core reagent of chemiluminescence substrate. Lectin-like oxidized low-d. lipoprotein receptor-1 (LOX-1), expressed prominently in atherosclerotic lesions, is cleaved and released as a sol. LOX-1 (sLOX-1), which is a specific biomarker to diagnose acute coronary syndrome (ACS) at an early stage.  Although sLOX-1 levels in patient's blood were successfully measured with the authors' previously established ELISA, the assay was not sensitive enough to detect normal serum levels of sLOX-1 in healthy human subjects.  The authors therefore developed sensitive and specific monoclonal antibodies (mAbs) against sLOX-1 to establish a more sensitive immunoassay.  Mice were immunized with recombinant human LOX-1 extracellular domain.  MAbs were subsequently generated by std. myeloma cell fusion techniques with a novel screening method using time-resolved fluorescence immunoassay.  Using two anti-human sLOX-1 mAbs and alk. phosphatase as a label, a sandwich chemiluminescent enzyme immunoassay (CLEIA) was developed.  In total, nine mAbs were obtained.  The dissocn. const. (Kd) values of these mAbs for sLOX-1 were 0.12-1.32 nM.  Characteristics of these mAbs were estd. and the best combination for CLEIA was selected.  The newly established CLEIA could det. sLOX-1 levels as low as 8 pg/mL, and thus, was sensitive enough to measure serum sLOX-1 levels in normal human subjects and to evaluate subtle differences.  Values for sLOX-1 measured by monoclonal CLEIA and polyclonal ELISA were highly correlated (r2 = 0.7594, p < 0.0001).  Area under the curve values of the receiver-operating characteristic curves in detecting ACS were 0.948 and 0.978 for monoclonal CLEIA and polyclonal ELISA, resp.  Thus, a more sensitive sLOX-1 CLEIA was established using newly developed mAbs against sLOX-1.  In addn. to its advantage in early diagnosis of ACS, this assay may also be useful in predicting cardiovascular disease risk in disease-free subjects [2].

E. coli O157:H7 is a pathogenic microorganism that has been implicated in numerous cases of foodborne illnesses.  A variety of rapid methods exist that show promise for the presumptive detection of this pathogen without the immediate need for incubating test samples for hours to days in microbial enrichment and culture media.  In recent years, highly sensitive chemiluminescence has become a more affordable and portable detection method.  Chemiluminescent detection has been coupled with the selectivity of antibodies, magnetic microparticle sepn./isolation, and enzymic signal amplification in order to develop a rapid method, termed enzyme-linked immunomagnetic chemiluminescence (ELIMCL)(APS-5 acts a core reagent of chemiluminescence substrate).  This work presents the application of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline with a detection limit of 7.6×103 for live cells in approx. 75 min assay time.  The blocking agent casein and the surfactant Tween 20 were used to lower background luminescence and thus maximize signal-to-noise ratios. After 5.5 h of enrichment culture, ELIMCL was demonstrated to detect E. coli O157:H7 inoculated in ground beef at 10 CFU/g in a total assay time of about 7 h [3].

References

[1] Liu Z, Xia Z. Novel method for preparation of compound and composition producing chemiluminescence by reacting with phosphatase[P]. Faming Zhuanli Shenqing, 104418887, 2015.

[2] Nakamura M, Ohta H, Kume N, et al. Generation of monoclonal antibodies against a soluble form of lectin-like oxidized low-density lipoprotein receptor-1 and development of a sensitive chemiluminescent enzyme immunoassay[J]. Journal of pharmaceutical and biomedical analysis, 2010, 51(1): 158-163.

[3] Gehring A G, Irwin P L, Reed S A, et al. Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157: H7[J]. Journal of immunological methods, 2004, 293(1-2): 97-106.