Chemiluminescent reagent for the detection of alkaline phosphatase

Abstract

Described herein are 1,2-dioxetanes useful as chemiluminescent probes, diagnostic agents, and imaging agents. Compositions containing such compounds and methods of using them are also described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 369,879, filed July 29, 2022, which is incorporated by reference as if fully set forth herein. [Background technology]

[0002] Chemiluminescent dioxetanes are strained cyclic peroxides that can undergo rapid decomposition to generate excited transient species that subsequently decay to the ground state via the emission of light.

[0003] Such compounds are useful as luminescent probes in a variety of assays, including enzyme activity assays, immunoassays, and DNA detection assays. Chemiluminescence-based assays can offer superior sensitivity because, unlike fluorescence absorption-based assays, no light excitation is required.

[0004] Dioxetanes can be generated in situ at the time of their use, or they can be prepared in advance into a stable form and then activated. When generated in situ via the oxidation of a precursor alkene, chemiluminescent dioxetanes can also function as a detection or imaging method for reactive oxygen species (ROS). One example of a stable chemiluminescent dioxetane is 4-methoxy-4-(3-phosphatephenyl)spiro[1,2-dioxetane-3,2'-adamantane]. This compound, also known as LUMIGEN® PPD, can be activated upon treatment with alkaline phosphatase (ALP). ALP is an enzyme that catalyzes the hydrolysis of phosphate groups. Upon activation, the resulting compound subsequently undergoes cleavage of the 1,2-dioxetane ring, emitting light and thus functioning as a luminescent probe in ALP-labeled assays.

[0005] Dioxetane compounds have been developed that are sensitive and strongly radioactive under non-aqueous conditions, but such compounds suffer from the drawback that they are weakly emissive in aqueous media and can take a long time to reach maximum emission after contact with the desired analyte. Summary of the Invention

[0006] There is a need for dioxetanes that provide rapid response in the presence of analytes and analyte concentrations, or both. There is also a need for dioxetanes that are strongly radioactive and suitable for use in aqueous environments. This disclosure describes compositions comprising a combination of a dioxetane and one or more selected surfactants that unexpectedly provide improved radiation results in aqueous environments. Certain combinations can result in large relative differences (greater than 20-fold) in brightness in relative light units (RLU), depending on the combination of dioxetane and surfactant. One important benefit of the compositions described herein is that they can improve the detection limit for alkaline phosphatase. For example, using the compositions described herein, it is possible to detect 1×10 -12 mol / μL to 1×10 -23 For example, in some embodiments, the compositions described herein can detect alkaline phosphatase concentrations of at least 1×10 mol / μL. -23 mol / µL, at least 1 x 10 -22 mol / µL, at least 1 x 10 -21 mol / µL, at least 1 x 10 -20 mol / µL or at least 1 x 10 -17 Additionally, or alternatively, the compositions described herein can be used to detect ALP in compositions having an ALP concentration of up to (i.e., at most) 1×10 -21 mol / μL, up to 1×10 -20 mol / μL, up to 1×10 -19 mol / μL, up to 1×10 -18 mol / μL, up to 1×10 -15mol / μL, up to 1×10 -10 mol / μL or up to 1×10 -5 In some exemplary embodiments, the compositions described herein can be used to detect ALP in a composition having an ALP concentration of 1.2×10 mol / μL. -15 AP4 with an ALP concentration of 1.2 x 10 mol / µL -17 AP6 with an ALP concentration of 1.2 × 10 -19 AP8 with an ALP concentration of 1.2 × 10 mol / µL; and 1.2 × 10 -20 For example, using the compositions described herein, a composition containing 5 μL to 50 μL of alkaline phosphatase composition (e.g., 10 μL of AP9) diluted in 50 μL to 250 μL (e.g., 100 μL) of buffer solution, such as 1×10 -11 mol~1×10 -22 1 x 10 mol alkaline phosphatase in a composition -11 mol ~ 1 × 10 -22 mol of alkaline phosphatase (e.g., 1 × 10 -14 mol, 1 × 10 -15 mol, 1 × 10 -16 mol, 1 × 10 -17 mol, 1 × 10 -18 mol, 1 × 10 -19 mol, 1 × 10 -20 mol, 1 × 10 -21 mol, 1 × 10 -22 mol, 1 × 10 -23 mol; 1×10 -13 mol~1×10 -22 mol, 1 × 10 -11 mol ~ 1 × 10 -20 mol, 1 × 10 -13 mol~1×10 -18 mol, 1 × 10 -15 mol ~ 1 × 10 20 or 1×10 -14 mol~1×10 -17 mol of alkaline phosphatase can be detected.

[0007] The present disclosure provides compounds of formula I and salts thereof

[0008] [ka] (In the formula, R 1 and R 2 Each of these is independently C3 to C 10 alkyl or R 1 and R 2 together with the carbon to which they are attached, C5 to C 10 providing a cycloalkyl ring, R 3 is C1~C 10 Alkyl, C6-C 10 is aryl or heteroaryl, R 4 is C2~C 10 is alkenyl, R 5 is H or C1~C 10 is alkyl, X is phosphate), and at least one surfactant.

[0009] The terms "surfactant" and "enhancement agent" are used interchangeably herein. Examples of such surfactants include, but are not limited to, phosphonium surfactants, which are also interchangeably referred to herein as "phosphonium enhancers." Phosphonium surfactants include polymeric phosphonium surfactants and small molecule phosphonium surfactants. Examples of polymeric phosphonium surfactants include polyvinyl-type polymers with pendant quaternary phosphonium groups, as disclosed in U.S. Pat. No. 5,393,469.

[0010] Exemplary polymeric phosphonium enhancers include polyvinylbenzyltributylphosphonium chloride copolymers with polyvinylbenzyltrioctylphosphonium chloride and polyvinylbenzyltributylphosphonium chloride, as well as dicationic compounds with two quaternary ammonium or phosphonium groups, such as those disclosed in U.S. Pat. No. 5,451,347.

[0011] Examples of polymeric phosphonium enhancers include repeating units (A), repeating units (B), or both.

[0012] [ka] (In the formula, "Bu3" refers to "tributyl" and "Oct3" refers to "trioctyl") The molar ratio of repeating unit (A) to repeating unit (B) can be in the range of 1:100 to 100:1 (e.g., 100:1 to 1:1, including, for example, 80:1, 70:1; 60:1, 50:1; 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 70:30, 75:25, 60:40, or 50:50). The weight average molecular weight (Mw) can be, for example, in the range of 100,000 g / mol to 400,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary embodiments, Mw can be 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, 360,000, 380,000, or 400,000 g / mol. In some embodiments, Mw is 220,000 g / mol. In some embodiments, Mw is 240,000 g / mol. In some embodiments, Mw is 260,000 g / mol. In some embodiments, Mw is 280,000 g / mol. In some embodiments, Mw is 300,000 g / mol. The copolymers can be random copolymers, block copolymers or random-block copolymers.

[0013] Examples of polymeric phosphonium enhancers include those of the general formula

[0014] [ka] (In the formula, m and n each independently represent an integer of 0 to 1000 (for example, 1 to 500, 250 to 1000, 300 to 900, 50 to 500, or 250 to 750). The m:n ratio can be, for example, 100:1 to 1:1, including m:n ratios of, for example, 80:1, 70:1; 60:1, 50:1; 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 70:30, 75:25, 60:40, or 50:50, where m+n is in the range of 100 to 1000 (e.g., m+n can be 100 to 600, 200 to 500, or 300 to 600), and the weight average molecular weight (Mw) can be, for example, in the range of 100,000 g / mol to 400,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary embodiments, Mw can be 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, 360,000, 380,000, or 400,000 g / mol. In some embodiments, Mw is 220,000 g / mol. In some embodiments, Mw is 240,000 g / mol. In some embodiments, Mw is 260,000 g / mol. In some embodiments, Mw is 280,000 g / mol. In some embodiments, Mw is 300,000 g / mol. The copolymers can be random copolymers, block copolymers or random-block copolymers.

[0015] Exemplary polymeric phosphonium enhancers / surfactants include those having the structure

[0016] [ka] (In the formula, z is an integer of 2 to 1000 (e.g., 2 to 500, 100 to 600, 450 to 800, 300 to 600, 2 to 250, 50 to 250, 100 to 300, 150 to 400, 175 to 400, 50 to 300, 100 to 500, 150 to 400, or 10 to 200) The weight average molecular weight (Mw) can be in the range of, for example, 500 g / mol to 500,000 g / mol, 500 g / mol to 100,000 g / mol, 50,000 g / mol to 100,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary embodiments, Mw can be 50,000, 60,000, 70,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, or 350,000 g / mol. In some embodiments, Mw is 70,000, 80,000, 100,000, or 220,000 g / mol. In some embodiments, Mw is 240,000 g / mol. In some embodiments, Mw is 260,000 g / mol. In some embodiments, Mw is 280,000 g / mol. In some embodiments, the Mw is 300,000 g / mol. Examples of such polymeric phosphonium enhancers / surfactants include those having the structure

[0017] [ka] (In the formula, z is an integer of 2 to 1000 (e.g., 2 to 500, 100 to 600, 450 to 800, 300 to 600, 2 to 250, 50 to 250, 100 to 300, 150 to 400, 175 to 400, 50 to 300, 100 to 500, 150 to 400, or 10 to 200) The weight average molecular weight (Mw) can be in the range of, for example, 500 g / mol to 500,000 g / mol, 500 g / mol to 100,000 g / mol, 50,000 g / mol to 100,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary embodiments, Mw can be 50,000, 60,000, 70,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, or 350,000 g / mol. In some embodiments, Mw is 70,000, 80,000, 100,000, or 220,000 g / mol. In some embodiments, Mw is 240,000 g / mol. In some embodiments, Mw is 260,000 g / mol. In some embodiments, Mw is 280,000 g / mol. In some embodiments, the Mw is 300,000 g / mol.

[0018] Exemplary small molecule phosphonium enhancers / surfactants include those of the formula

[0019] [ka] (In the formula, R 12 ~R 14 are each independently C1 to C 10 is alkyl, R 15 is arylalkyl, X - is the counterion (e.g., chloride) Examples of small molecule surfactants include R 15 is a C2-C8 alkenyl group or a group of the formula

[0020] [ka] (wherein k is 0, 1, or 2; R 16~R 18 are each independently C1 to C 16 alkyl) R may be an arylalkyl substituted with a group 16 ~R 18 are each independently C4 to C 10 The C2-C8 alkenyl group can be represented by the formula

[0021] [ka] (In the formula, R 19 is C1-C8 alkyl, C2-C8 alkenyl, or C2-C8 alkynyl) It can be a group of the formula:

[0022] Examples of surfactants contemplated herein for use with the dioxetane compounds described herein include fluorescein surfactants (also referred to interchangeably herein as "fluorescein enhancers"), such as those of Formula V / VI

[0023] [ka] which typically includes

[0024] [ka] (In the formula, R 6 is C6~C 20 Alkyl groups, such as C6-C 15 , C8~C 16 , C 10 ~C 14 and C 12 ~C 20 alkyl group) Examples of compounds of Formula V / VI include 5-dodecanoylaminofluorescein, 5-hexadecanoylaminofluorescein, 5-stearylaminofluorescein, etc. The present disclosure also provides compounds having a peak RLU greater than 8,000 (e.g., at 37°C); and / or an emission half-life (T) of 3 minutes or less.1 / 2 ) (e.g., at 37°C).

[0025] Combinations of any of the foregoing surfactant / enhancers (e.g., combinations of two or more small molecule phosphonium, polymeric phosphonium, and fluorescein surfactant / enhancers) are also contemplated herein, however, the use of only one surfactant is also contemplated herein.

[0026] The present disclosure also provides a method for determining at least one of the presence and concentration of an analyte (e.g., ALP, or ALP conjugated to an analyte of interest) in a sample, comprising contacting the sample with a compound of formula I and monitoring the sample for luminescence.

[0027] Advantages, some of which are unexpected, are achieved by various embodiments of the present disclosure. For example, when compounds of Formula I were first developed, electron-withdrawing groups were selected for bright chemiluminescent probes. 4 It has been common knowledge that R is required in the compound of formula I. See, for example, ACS Cent. Sci. 2017, 3, 349-358. Based on this common knowledge, those skilled in the art would understand that R is required in the compound of formula I. 4 Because of the lack of electron-withdrawing groups at the R 4 It has an electron-donating vinyl group at the position.

[0028] The various compounds and compositions described herein can advantageously provide rapid and intense luminescence signals in non-aqueous media, aqueous media, or both. Because the maximum luminescence signal is achieved more rapidly, assays involving such compounds or compositions can be performed faster than those with compounds lacking the properties of the currently described compounds and compositions, which is an important advantage. Moreover, the compounds and compositions of the present disclosure provide increased luminescence intensity, including in aqueous media. The compounds and compositions of the present disclosure also provide improved signal-to-noise ratios and have reduced background signals. Due to these advantageous properties, various embodiments of the present disclosure can provide methods or kits that can detect analytes in aqueous or non-aqueous samples in less than 3 minutes, less than 1 minute, less than 30 seconds, or less than 15 seconds. [Brief explanation of the drawings]

[0029] [Figure 1] 1 is a plot of relative luminescence units as a function of time (minutes) for the compounds "VPPD," "VMPD," and "PPD" using the fluorescein surfactants used in LP530. The fluorescein surfactants used in LP530 include the fluorescein surfactants of formula V / VI and cetyltrimethylammonium bromide (CTAB). "VPPD," "VMPD," and "PPD" have structures as further described herein. [Figure 2-1] FIG. 2A is a plot of relative light emission as a function of time (minutes) for the compounds “VPPD,” “VMPD,” and “PPD” using “EXL surfactants” having structures further described herein. [Figure 2-2] Figure 2B is a plot of RLU as a function of time at various concentrations of "VPPD" (in the range of 50 mg / L to 500 mg / L) while keeping the EXL concentration constant (in the range of 100 mg / L to 250 mg / L). [Figure 2-3]Figure 2C is a plot of RLU as a function of time at various "EXL surfactant" concentrations (ranging from 100 mg / L to 500 mg / L) while keeping the VPPD concentration constant (ranging from 50 mg / L to 500 mg / L). [Figure 3] 1 is a plot of the relative light emission as a function of time (minutes) for the compounds "VPPD," "VMPD," and "PPD" in EXL surfactant, or "VPPD" in the fluorescein surfactant used in LP530, "VMPD" in the fluorescein surfactant used in LP530, or LP530 (i.e., PPD in the fluorescein surfactant), normalized to the intensity of VPPD in EXL surfactant. [Figure 4] 1 is a plot of relative light output as a function of time (min) for VPPD in various phosphonium surfactants; relative light output as a function of time (min) for LP530 is also shown. [Figure 5] 1 is a plot of RLU as a function of moles of ALP for "VPPD" in a polymeric phosphonium surfactant. [Figure 6] Figure 1 shows plots of RLU as a function of time (minutes) for VPPD in EXL surfactant and for LP530. In this experiment, 100 μL of VPPD and polymeric phosphonium enhancer (EXL) solution was mixed with 10 μL of AP10 (1.2 × 10 mol / μL ALP) and incubated at 37°C, and 100 μL of LP530 solution was mixed with 10 μL of AP8 (1.2 × 10 mol / μL ALP) and incubated at 37°C. Notably, AP8 has a 100-fold greater concentration of ALP than AP10. That is, with LP530, a 100-fold higher ALP concentration was required to obtain approximately the same light signal as achieved with VPPD and EXL. [Figure 7]1 is a plot of RLU as a function of time (min) for VPPD EXL and LP530 background, where "background" refers to the signal obtained for a VPPD composition containing EXL without any ALP or LP530, respectively. The signal is generated from thermal decomposition of the dioxetane when incubated at 37° C. DETAILED DESCRIPTION OF THE INVENTION

[0030] Reference will now be made in detail to certain embodiments of the presently disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the presently disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0031] The compounds of the present disclosure are useful in chemiluminescence applications, such as alkaline phosphatase (ALP) assays and chemical probes.One important benefit of the compositions described herein is that they can improve the ALP detection limit.For example, the compositions described herein can detect 1×10 -12 mol / μL to 1×10 -23 For example, in some embodiments, the compositions described herein can detect alkaline phosphatase concentrations of at least 1×10 mol / μL. -23 mol / µL, at least 1 x 10 -22 mol / µL, at least 1 x 10 -21 mol / µL, at least 1 x 10 -20 mol / µL or at least 1 x 10 -17 Additionally, or alternatively, the compositions described herein can be used to detect ALP in compositions having an ALP concentration of up to 1×10 mol / μL. -21 mol / μL, up to 1×10 -20 mol / μL, up to 1×10 -19 mol / μL, up to 1×10 -18 mol / μL, up to 1×10-15 mol / μL, up to 1×10 -10 mol / μL or up to 1×10 -5 In some exemplary embodiments, the compositions described herein can be used to detect ALP in a composition having an ALP concentration of 1.2×10 mol / μL. -15 AP4 with an ALP concentration of 1.2 x 10 mol / µL -17 AP6 with an ALP concentration of 1.2 × 10 -19 AP8 with an ALP concentration of 1.2 × 10 mol / µL; and 1.2 × 10 -20 For example, using the compositions described herein, a composition containing 5 μL to 50 μL (e.g., 10 μL of AP9) of alkaline phosphatase composition diluted in 50 μL to 250 μL (e.g., 100 μL) of buffer solution, such as 1×10 -11 mol ~ 1 × 10 -22 1 x 10 mol alkaline phosphatase in a composition -11 mol ~ 1 × 10 -22 mol of alkaline phosphatase (e.g., 1 × 10 -14 mol, 1 × 10 -15 mol, 1 × 10 -16 mol, 1 × 10 -17 mol, 1 × 10 -18 mol, 1 × 10 -19 mol, 1 × 10 -20 mol, 1 × 10 -21 mol, 1 × 10 -22 mol, 1 × 10 -23 mol; 1×10 -13 mol ~ 1 × 10 -22 mol, 1 × 10 -11 mol ~ 1 × 10 -20 mol, 1 × 10 -13 mol ~ 1 × 10 -18 mol, 1 × 10 -15 mol ~ 1 × 10 20 or 1×10 -14 mol ~ 1 × 10 -17 mol of alkaline phosphatase can be detected.

[0032] In some embodiments, the luminescence intensity of the compounds of the present disclosure is described in units of relative light units (RLU). The term "RLU" refers to the relative light unit relative to the chemiluminescence signal (S) in the presence of alkaline phosphatase (ALP). In some embodiments, RLU can be corrected for background chemiluminescence (B) in the absence of ALP, for example, S minus B.

[0033] Specifically, this disclosure describes compositions comprising a combination of dioxetanes and selected surfactants that provide unexpected radiative results in aqueous environments. The dioxetane concentration can be selected by the artisan. Exemplary dioxetane concentrations that can be used include 0.5 mg / L to 5000 mg / L (e.g., 1 mg / L to 1000 mg / L, 1 mg / L to 500 mg / L, 25 mg / L to 2000 mg / L, 50 mg / L to 500 mg / L, 50 mg / L to 250 mg / L, 100 mg / L to 150 mg / L, 50 mg / L to 1,000 mg / L, 200 mg / L to 1200 mg / L, 50 mg / L to 500 mg / L, 50 mg / L to 150 mg / L, 100 mg / L to 200 mg / L, 100 mg / L to 500 mg / L, and 100 mg / L to 200 mg / L). In some embodiments, dioxetane concentrations that can be used are at least 0.5 mg / L, at least 1 mg / L, at least 25 mg / L, at least 50 mg / L, at least 100 mg / L, at least 200 mg / L, at least 500 mg / L, at least 1000 mg / L, at least 3000 mg / L, or at least 4000 mg / L. In some embodiments, dioxetane concentrations that can be used are at most 5000 mg / L, at most 4000 mg / L, at most 3000 mg / L, at most 1000 mg / L, at most 500 mg / L, at most 200 mg / L, 100 mg / L, at most 25 mg / L, or at most 1 mg / L.

[0034] The surfactant concentration can also be selected by the artisan. Exemplary surfactant concentrations that can be used include concentrations from 5 mg / L to 25,000 mg / L (e.g., 1,000 mg / L to 10,000 mg / L, 500 mg / L to 5,000 mg / L, 50 mg / L to 10,000 mg / L, 100 mg / L to 5,000 mg / mL, 100 mg / L to 500 mg / L, 250 mg / L to 1,000 mg / L, 250 mg / L to 300 mg / L, 300 mg / L to 5 ... (e.g., 50 mg / L to 500 mg / L, 50 mg / L to 500 mg / L, 50 mg / L to 5,000 mg / L, 500 mg / L to 1,000 mg / L, 100 mg / L to 500 mg / L, 100 mg / L to 1,000 mg / L, 100 mg / L to 300 mg / L, 150 mg / L to 250 mg / L, and 200 mg / L to 300 mg / L). In some embodiments, surfactant concentrations that can be used can be at least 5 mg / L, at least 50 mg / L, at least 100 mg / L, at least 500 mg / L, at least 1,000 mg / L, at least 3,000 mg / L, at least 4,000 mg / L, at least 5,000 mg / L, at least 10,000 mg / L, at least 15,000 mg / L, or at least 20,000 mg / L. In some embodiments, surfactant concentrations that may be used may be at most 25,000 mg / L, at most 20,000 mg / L, at most 15,000 mg / L, at most 10,000 mg / L, at most 5000 mg / L, at most 4000 mg / L, at most 3000 mg / L, at most 1000 mg / L, at most 500 mg / L, at most 100 mg / L, or at most 50 mg / L.

[0035] The concentration ratio of the dioxetane compound to the surfactant can be in the range of 5:1 to 1:10 (e.g., 1:1 to 1:5, 1:2 to 1:8; 1:1 to 1:3, 1:2 to 1:5, and 1:3 to 1:9). For example, the concentration ratio of the dioxetane compound to the surfactant can be 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0036] As shown in Figure 1,

[0037] [ka] Dioxetane compounds having the following structure can be synthesized in a relatively short time when a fluorescein surfactant is used (e.g., LP530):

[0038] [ka] VPPD exhibits a greater RLU than dioxetane compounds having the formula (I). As used herein, the term "LP530" refers to the Lumigen, Inc. product "Lumi-Phos 530," which includes a dioxetane compound, referred to herein as "PPD," and a fluorescein surfactant. However, as shown in Figures 2A and 3, when VPPD is used in combination with a polymeric phosphonium surfactant / enhancer (EXL), a surprisingly large difference in brightness (as measured in relative light units (RLU)) occurs when compared to either PPD or VMPD. For example, the brightness of VPPD, when combined with a polymeric phosphonium surfactant / enhancer, is more than 20-fold greater than the brightness of VPPD combined with a fluorescein surfactant (approximately 9500 RLU in the EXL surfactant compared to approximately 380 in the fluorescein surfactant). See Figure 3.

[0039] As an added benefit, polymeric phosphonium surfactants / enhancements provide approximately a 50-fold (and as much as a 100-fold) increase in signal-to-noise ratio. For example, a VPPD enhancer in a polymeric phosphonium enhancer can be used to detect AP10 (1.2 x 10) using 10 µL of AP10 added to 100 µL of VPPD and enhancer solution. -21 However, to generate essentially the same signal as LP530, 100 times more ALP (10 μL of AP8; AP8 is 1.2 × 10 -19See Figure 6. Figure 7 is a plot of RLU as a function of time for VPPD EXL and LP530 background, where "background" refers to the signal obtained for a VPPD composition containing EXL or LP530, respectively, without any ALP. The signal is generated from the thermal decomposition of the dioxetane when incubated at 37°C. The background signal for a composition containing VPPD and EXL is approximately one-third of the signal obtained for LP530. The lower signal (background) without ALP, as seen in Figure 7, also means that the limit of detection, i.e., the lowest practical amount of analyte conjugated with ALP that can be reliably detected and distinguished from the background, is lower.

[0040] Moreover, as shown in Figures 2A and 3, the brightness of VPPD in combination with a polymeric phosphonium surfactant / activator is higher than other dioxetanes in combination with a polymeric phosphonium surfactant / activator, including, for example, PPD in combination with a polymeric phosphonium surfactant / activator or VMPD in combination with a polymeric phosphonium surfactant / activator, where VMPD has the following structure:

[0041] [ka] For example, VMPD in combination with a polymeric phosphonium surfactant / enhancer has a higher brightness than LP530. In fact, as shown in these exemplary figures, VMPD in combination with a polymeric phosphonium surfactant / enhancer exhibits a much brighter signal than LP530. Furthermore, VMPD in a polymeric phosphonium surfactant reaches a signal maximum more quickly (in less than one minute) than VPPD in the same surfactant.

[0042] These results suggest that the combination of a particular dioxetane compound and a phosphonium surfactant / activator provides unexpectedly improved emission results in an aqueous environment.

[0043] The present disclosure provides compounds of formula I and salts thereof

[0044] [ka] (In the formula, R 1 and R 2 Each of these is independently C3 to C 10 alkyl or R 1 and R 2 together with the carbon to which they are attached, C5 to C 10 providing a cycloalkyl ring, R 3 is C1~C 10 Alkyl, C6-C 10 is aryl or heteroaryl, R 4 is C2~C 10 is alkenyl, R 5 is H or C1~C 10 is alkyl, X is a phosphate (e.g., a phosphate group of the formula -OP(O)(ONa)(ONa)); and at least one surfactant.

[0045] For example, the present disclosure provides a compound of formula II or a salt thereof:

[0046] [ka] (In the formula, R 10 and R 11 each independently represents H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 aryl, and R 3 is C1~C 10 Alkyl, C6-C 10 aryl or heteroaryl, and R 4 is C2~C 10 alkenyl, and R 5is H or C1~C 10 In some embodiments, R is an alkyl group, and at least one surfactant. 10 and R 11 are independently H or halogen.

[0047] The present disclosure provides a compound of formula III or a salt thereof

[0048] [ka] (In the formula, R 10 and R 11 each independently represents H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 aryl, and R 3 is C1~C 10 Alkyl, C6-C 10 aryl or heteroaryl, and R 4 is C2~C 10 alkenyl, and R 5 is H or C1~C 10 In some embodiments, R 10 and R 11 are independently H or halogen.

[0049] The present disclosure further provides a compound of formula IV or a salt thereof:

[0050] [ka] (In the formula, R 10 and R 11 each independently represents H, halogen, C1-C 10 Alkyl, C2-C 10 Alkenyl, C6-C 10 aryl, and R 3 is C1~C 10 Alkyl, C6-C 10 aryl or heteroaryl, and R4 is C2~C 10 alkenyl, and R 5 is H or C1~C 10 In some embodiments, R 10 and R 11 are independently H or halogen.

[0051] The compounds of formula I-IV are represented by the formula

[0052] [ka] or a salt thereof.

[0053] Compositions containing compounds of Formulas I-IV can be aqueous or non-aqueous. The compositions can be mixtures of both aqueous and non-aqueous solvents and can contain other additives (e.g., magnesium ions, e.g., magnesium ions derived from magnesium salts, e.g., magnesium chloride). When compositions containing compounds of Formulas I-IV contain magnesium ions, they can contain 50 mg / L to 500 mg / L (e.g., 50 mg / L to 250 mg / L, 100 mg / L to 190 mg / L, 150 mg / L to 200 mg / L, or 150 mg / L to 190 mg / L) of magnesium(II) chloride (MgCl2), although other magnesium(II) salts can be used. In one example, the magnesium ion concentration can be 0 mM to 10 mM, 0 mM to 3 mM, 0.5 mM to 2 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, or 2 mM to 5 mM.

[0054] In various embodiments, such surfactants used in combination with compositions containing compounds of Formulas I-IV include, but are not limited to, phosphonium surfactants, which are also interchangeably referred to herein as "phosphonium enhancers." Phosphonium surfactants include polymeric phosphonium surfactants and small molecule phosphonium surfactants. Examples of polymeric phosphonium surfactants include polyvinyl-type polymers with pendant quaternary phosphonium groups, which are disclosed in U.S. Pat. No. 5,393,469. For example, U.S. Pat. No. 5,393,469 describes a polymer having the general formula:

[0055] [ka] The polymeric phosphonium surfactants are described as follows: (wherein each A is selected from a lower alkyl, aryl, or aralkyl group containing 1 to 20 carbon atoms; the F1 group is a fluorescent group; m is an integer between 1 and 14; and n and p are integers between about 10 and 1000). The A groups on a particular phosphorus atom can all be the same group, or two different groups, or all three can be different. The sets of A groups on adjacent phosphorus atoms can be the same set or different sets, where the sets are as described above. The relative positions of the substituents on the aromatic rings can be ortho, meta, para, or a mixture of the three in any proportion. The attached fluorescent group can be any fluorescent agent that can be chemically bonded to the polymer and has a lower energy for its singlet electronic excited state compared to the excited state of the dioxetane (e.g., compounds of Formulas I-IV). The fluorescent group can enhance the chemiluminescence efficiency of the dioxetane by acting as an energy acceptor, which becomes excited and releases the excitation energy in the form of light. Examples of fluorescent agents useful in practicing the present invention include, but are not limited to, any fluorescent dye; aromatic compounds including polycyclic aromatic compounds, biphenyls, terphenyls, stilbenes, heteroaromatic and polycyclic heteroaromatic compounds such as acridines, coumarins, phthalocyanines, furans, oxazoles, oxadiazoles, benzothiazoles, quinolines, xanthenes, fluorescein and fluorescein derivatives such as amidofluorescein, eosin and eosin derivatives, rhodamines, and resorufins.

[0056] Exemplary polymeric phosphonium enhancers include polyvinylbenzyltributylphosphonium chloride copolymers having polyvinylbenzyltrioctylphosphonium chloride and polyvinylbenzyltributylphosphonium chloride, as well as dicationic compounds having two quaternary ammonium or phosphonium groups, such as those disclosed in U.S. Patent No. 5,451,347, which discloses dicationic compounds having two quaternary ammonium or phosphonium groups of the formula 1-27:

[0057] [ka] The present invention describes compounds having a phosphonium group of the formula:

[0058] Examples of polymeric phosphonium enhancers include repeating units (A), repeating units (B), or both.

[0059] [ka] (In the formula, "Bu3" refers to "tributyl" and "Oct3" refers to "trioctyl") The molar ratio of repeating unit (A) to repeating unit (B) can be in the range of 1:100 to 100:1 (e.g., 100:1 to 1:1, including, for example, 80:1, 70:1; 60:1, 50:1; 40:1, 30:1, 20:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 70:30, 75:25, 60:40, or 50:50). The weight average molecular weight (Mw) can be, for example, in the range of 100,000 g / mol to 400,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary embodiments, Mw can be 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, 360,000, 380,000, or 400,000 g / mol. In some embodiments, Mw is 220,000 g / mol. In some embodiments, Mw is 240,000 g / mol. In some embodiments, Mw is 260,000 g / mol. In some embodiments, Mw is 280,000 g / mol. In some embodiments, Mw is 300,000 g / mol. The copolymers can be random copolymers, block copolymers or random-block copolymers.

[0060] Examples of polymeric phosphonium enhancers include those of the general formula

[0061] [ka] (In the formula, m and n each independently represent an integer of 0 to 1000 (e.g., an integer of 1 to 500, 250 to 1000, 300 to 900, 50 to 500, or 250 to 750). The m:n ratio may be in the range of 100:1 to 1:1, 4:1, for example, 70:30, 75:25, 60:40, or 50:50, and m+n may be in the range of 100 to 1000 (for example, m+n may be 100 to 600, 200 to 500, or 300 to 600), and the weight average molecular weight (Mw) may be in the range of, for example, 100,000 g / mol to 400,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary embodiments, Mw can be 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, 360,000, 380,000, or 400,000 g / mol. In some embodiments, Mw is 220,000 g / mol. In some embodiments, Mw is 240,000 g / mol. In some embodiments, Mw is 260,000 g / mol. In some embodiments, Mw is 280,000 g / mol. In some embodiments, Mw is 300,000 g / mol. The copolymers can be random copolymers, block copolymers or random-block copolymers.

[0062] Exemplary polymeric phosphonium enhancers / surfactants include those having the structure

[0063] [ka] (In the formula, z is an integer ranging from 2 to 1000 (e.g., 2 to 500, 100 to 600, 450 to 800, 300 to 600, 2 to 250, 50 to 250, 100 to 300, 150 to 400, 175 to 400, 50 to 300, 100 to 500, 150 to 400, or 10 to 200) The weight average molecular weight (Mw) can be in the range of, for example, 500 g / mol to 500,000 g / mol, 500 g / mol to 100,000 g / mol, 50,000 g / mol to 100,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary embodiments, Mw can be 50,000, 60,000, 70,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, or 350,000 g / mol. In some embodiments, Mw is 70,000, 80,000, 100,000, or 220,000 g / mol. In some embodiments, Mw is 240,000 g / mol. In some embodiments, Mw is 260,000 g / mol. In some embodiments, Mw is 280,000 g / mol. In some embodiments, the Mw is 300,000 g / mol. Examples of such polymeric phosphonium enhancers / surfactants include those having the structure

[0064] [ka] (In the formula, z is an integer of 2 to 1000 (e.g., 2 to 500, 100 to 600, 450 to 800, 300 to 600, 2 to 250, 50 to 250, 100 to 300, 150 to 400, 175 to 400, 50 to 300, 100 to 500, 150 to 400, or 10 to 200) The weight average molecular weight (Mw) can be in the range of, for example, 500 g / mol to 500,000 g / mol, 500 g / mol to 100,000 g / mol, 50,000 g / mol to 100,000 g / mol, 150,000 g / mol to 350,000 g / mol, or 200,000 g / mol to 300,000 g / mol. For example, in exemplary embodiments, Mw can be 50,000, 60,000, 70,000, 100,000, 120,000, 140,000, 160,000, 180,000, 200,000, 220,000, 240,000, 260,000, 280,000, 300,000, 320,000, 340,000, or 350,000 g / mol. In some embodiments, Mw is 70,000, 80,000, 100,000, or 220,000 g / mol. In some embodiments, Mw is 240,000 g / mol. In some embodiments, Mw is 260,000 g / mol. In some embodiments, Mw is 280,000 g / mol. In some embodiments, the Mw is 300,000 g / mol.

[0065] Exemplary small molecule phosphonium enhancers / surfactants include those of the formula

[0066] [ka] (In the formula, R 12 ~R 14 are each independently C1 to C 10 is alkyl, R 15 is arylalkyl, X - is the counterion (e.g., chloride) Examples of small molecule surfactants include R 15 is a C2-C8 alkenyl group or a group of the formula

[0067] [ka] (wherein k is 0, 1, or 2; R 16~R 18 are each independently C1 to C 16 R may be an arylalkyl substituted with a group of 16 ~R 18 are each independently C4 to C 10 The C2-C8 alkenyl group can be represented by the formula

[0068] [ka] (In the formula, R 19 is C1-C8 alkyl, C2-C8 alkenyl, or C2-C8 alkynyl) It can be a group of the formula:

[0069] Examples of small molecule phosphonium enhancers / surfactants include:

[0070] [ka] Examples include:

[0071] Other surfactants contemplated herein include poly(vinylbenzyltributylphosphonium chloride) (TBE) (e.g., TBE having a molecular weight of 175,000 g / mol), polyvinylpyridinium salts (e.g., polyvinylpyridinium salts having a molecular weight of 10,000 g / mol to 100,000 g / mol), alkylammonium salts (e.g., (C8 to C 20 alkyl)2N(C1-C4 alkyl)2Cl, for example (C8H 17 )3N(CH3)Cl, (C 12 H 25 )2N(CH3)2Cl and (C 18 H 37 )2N(CH3)2), alkyl-glycol ammonium salts (e.g., (C 12 ~C 20 alkyl)2N(C1-C4 alkyl-O)2Cl, for example (C 12 H 25 )2N((CH2CH2O)5)2Cl and (C 18 H 37)N((CHCHO)Cl), as well as arylalkyl-alkylammonium salts (e.g., (C 18 H 37 )2N(CH3)(CH2Ph)Cl).

[0072] Combinations of any of the aforementioned surfactants / enhancers (e.g., combinations of two or more of small molecule phosphonium, polymeric phosphonium, and fluorescein surfactants / enhancers) are also contemplated herein.For example, the composition can include a small molecule phosphonium and a polymeric phosphonium.In some examples, the composition can include a small molecule phosphonium and a fluorescein surfactant / enhancer.In some examples, the composition can include a small molecule phosphonium, a polymeric phosphonium, and a fluorescein surfactant / enhancer.For example, the composition can include the following combinations: Formula (a)

[0073] [ka] Polymeric phosphonium and

[0074] [ka] Small molecule phosphonium Formula (b)

[0075] [ka] Polymeric phosphonium and

[0076] [ka] Small molecule phosphonium (c) formula

[0077] [ka] Small molecule phosphonium and

[0078] [ka] Fluorescein surfactant / enhancing agent Formula (d)

[0079] [ka] Small molecule phosphonium and

[0080] [ka] Fluorescein surfactant / enhancing agent Formula (e)

[0081] [ka] Polymeric phosphonium of formula

[0082] [ka] Small molecule phosphonium and

[0083] [ka] fluorescein surfactant / enhancer, or (f) formula

[0084] [ka] Polymeric phosphonium of formula

[0085] [ka] Small molecule phosphonium and

[0086] [ka] Fluorescein surfactant / enhancing agent.

[0087] In various embodiments, the composition contains a buffer solution. The buffer solution can be, but is not necessarily, an alkaline or amine-based buffer solution. As an example, an amine-based buffer solution is 221 buffer (2-amino-2-methyl-1-propanol (AMP)-based buffer) available from Sigma-Aldrich (St. Louis, MO). Therefore, the buffer solution contemplated herein includes amine-based buffer solutions, including 2-amino-2-methyl-1-propanol (AMP), 2-amino-2-methyl-1,3-propanediol (AMPD), tris(2-amino-2-(hydroxymethyl)propane-1,3-diol), TAPS (3-{[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}propane-1-sulfonic acid), bicine (N,N-bis(2-hydroxyethyl)glycine), and tricine (N-[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]glycine) and combinations thereof, such as buffer solutions containing AMPD and AMP.Similarly, the composition can have a basic pH, but does not necessarily need to have one. In some exemplary embodiments, the composition can have a pH of 4-12, 5-12, 6-12, 8-11, 7-12, 8-12, 9-12, 10-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, or 4-5, with a buffer concentration of, for example, 0.1-1 M, 0.1-0.5 M, 0.2-0.5 M, 0.2-0.8 M, 0.1-0.25 M, 0.2-0.3 M, or 0.15 M-0.3 M. The pH of the composition can be selected depending on whether luminescence is intended to be immediately induced upon analyte-induced removal of the X group of the compounds described herein by ALP, as would be typical for alkaline pH values, or the pH of the composition can be acidic so as to luminesce upon treatment with base.

[0088] In various embodiments, the composition has / exhibits an RLU of greater than 2,000 RLU, greater than 4,000 RLU, greater than 6,000 RLU, greater than 8,000 RLU, greater than 9,000 RLU; up to 50,000 RLU, up to 30,000 RLU, up to 25,000 RLU, up to 20,000 RLU, up to 15,000 RLU, up to 10,000 RLU, or up to 5,000 RLU; between 2,000 RLU and 10,000 RLU, between 4,000 RLU and 10,000 RLU, between 2,000 RLU and 5,000 RLU, between 2,500 RLU and 4,000 RLU, or between 8,000 RLU and 10,000 RLU, RLU can be measured by any suitable means, including, for example, using a Tuner TD-20 luminometer to measure light intensity at 37° C. In some embodiments, 10 μL of ALP solution (e.g., AP8) can be mixed with 100 μL of formulated reagent. Additionally, or alternatively, the composition can be incubated (e.g., at 37° C.) for 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, 30 seconds to 4 minutes, 30 seconds to 2 minutes, 30 seconds to 1 minute, 1 minute to 3 minutes, or 1 minute to 2 minutes. 1 / 2 Shows.

[0089] In various embodiments, the composition has / exhibits, in the absence of an enzyme (e.g., ALP), a peak RLU of at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, or at most 10. In various embodiments, the composition has / exhibits, in the absence of an enzyme (e.g., ALP), a peak RLU of at least 0.5, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7. In various embodiments, the composition has / exhibits, in the absence of an enzyme (e.g., ALP), a peak RLU in the range of 1-20; 1-15; 1-14; 1-13; 1-12; 2-20; 2-15; 2-14; 2-13; 2-12; 3-20; 3-15; 3-14; 3-13; or 3-12.

[0090] The compositions including the dioxetanes and surfactants described herein may contain a magnesium salt in an amount of 50 mg / L to 500 mg / L (e.g., 50 mg / L to 250 mg / L, 100 mg / L to 190 mg / L, 150 mg / L to 200 mg / L, or 150 mg / L to 190 mg / L) of magnesium(II) chloride (MgCl), although other magnesium(II) salts can be used.

[0091] The present disclosure also provides methods for detecting an analyte (e.g., ALP, or ALP conjugated to an analyte of interest) in a sample, comprising contacting the sample with one or more of the compounds or salts thereof described herein, or compositions comprising same, and then monitoring the sample for luminescence. In various embodiments, the method involves measuring the intensity of the resulting luminescence and correlating the intensity with at least one of the presence and concentration of the analyte (e.g., ALP, or ALP conjugated to an analyte of interest). In various embodiments, the method comprises measuring at most 1×10 -18 , 1×10 -19 , 1×10 -20 or 1×10 -21 In various embodiments, the method detects ALP concentrations of at least 1×10 mol / μL. -22 mol / µL or at least 1 x 10 -23 In various embodiments, the method detects ALP concentrations of 1×10 mol / μL. -23 mol / μL to 1×10 -18 mol / μL (e.g., 1 × 10 -23 mol / μL to 1×10 -19 mol / μL; 1×10 -22 mol / μL to 1×10 -18 mol / µL; or 1 x 10 -23 mol / μL to 1×10 -20 In various embodiments, the method detects ALP concentrations in the range of at least 1×10 -22 mol ALP or at least 1 x 10 -21 In various embodiments, the method detects up to 1 x 10 mol of ALP.-17 , up to 1×10 -18 , up to 1×10 -19 or up to 1×10 -20 In various embodiments, the method detects 1×10 mol of ALP. -22 mol ALP ~ 1 x 10 -17 mol of ALP (e.g., 1 × 10 -22 mol ALP ~ 1 x 10 -18 mol ALP: 1 x 10 -22 mol ALP ~ 1 x 10 -19 mol ALP; or 1 x 10 -21 mol ALP ~ 1 x 10 -17 Detects ALP in the range of mol ALP.

[0092] In some embodiments, the method further involves increasing the pH in the sample. For example, the pH can be adjusted to 7 or higher, 8 or higher, 9 or higher, 10 or higher, or 11 or higher.

[0093] In various embodiments, the analyte is detected in 10 minutes or less, 6 minutes or less, 4 minutes or less, 2 minutes or less, 1 minute or less, 55 seconds or less, 45 seconds or less, or 30 seconds or less.

[0094] The present disclosure further provides a kit for determining at least one of the presence and concentration of analyte.In some embodiments, the kit comprises any one or more compounds described herein, its olefin precursor, its salt, or the composition comprising it.The kit can contain instructions according to the method described herein.

[0095] The compounds and compositions described herein can be triggered directly by the addition of an analyte to produce a signal that identifies at least one of the presence and concentration of the analyte and probe, or can be triggered in a two-step process in which one step involves contacting with the analyte and another step involves increasing the pH.

[0096] The compounds of the present disclosure can be configured as probes for detecting ALP or ALP conjugated to an analyte of interest.

[0097] It is desirable for the compound to emit light for the shortest possible time when triggered by the analyte to produce the strongest possible signal. If the chemiluminescence is emitted gradually over a period of time, the light intensity (photons / second) will decrease, potentially compromising the sensitivity of detection.

[0098] The rate of increase in luminescence or rise time is the time to maximum emission (t max ) or radioactive half-life (T 1 / 2 ) can be explained according to either

[0099] The compounds described herein can be used as enzyme substrates for alkaline phosphatase (ALP) enzymes, and the like. Without being limited by theory, one example mechanism involves the ALP enzyme hydrolyzing the phosphate group, X, to provide a phenol that is rapidly deprotonated due to the alkaline environment of the solution (e.g., a pH 9.7 buffer). Formation of the oxyanion triggers decomposition of the 1,2-dioxetane into two compounds, 2-adamantanone and an excited-state phenyl ester. The excited phenyl ester then rapidly decays to the ground state by emitting light.

[0100] The resulting light intensity is a linear function of the amount of enzyme. The compounds described herein can therefore be used to detect labeled enzymes used in assays. For example, the steps in the chemical process by which a dioxetane provides light can be represented by the following equations: (i) X + S → X + S' (ii) S' → P * and (iii) P * →P+ light. Step (i) represents the catalytic turnover of the substrate, where X is an enzyme or other component that converts the substrate (S) to its activated form (S'), and step (ii) represents the conversion of a transiently excited species (P) of the activated substrate. * ), and step (iii) represents the decay of the excited species to the ground state (P) and emission of light. The light intensity is determined by the product of the catalytic turnover of the substrate in step (i) and the resulting photogenerated compound P in step (ii). * Step (ii) is usually first order with a rate constant k and its half-life: T 1 / 2 = (In2) / k. Step (iii) is extremely short compared to the other steps and generally does not have a significant effect on the reaction kinetics.

[0101] The chemiluminescence intensity / time profile contains an initial rising period of emission intensity, followed by a period of steady-state intensity. It takes time to reach a steady-state concentration of S', so S' → P * The slow first-order reaction of S' → P corresponds to the extended rise time. *A faster reaction corresponds to a shorter initial rise period and therefore provides a rapid rise. For enzymatic chemiluminescent reactions, the intensity typically remains steady at a high level. The absence of a constant intensity indicates either depletion of the substrate or subsequent inactivation of the enzyme. Although detection of enzyme-generated chemiluminescence provides flexibility in the measurement process because the light intensity at any time can be related to the amount of enzyme, enzyme-generated processes can have drawbacks due to, for example, the size and "sticky" nature of the enzyme label. However, the most sensitive measurement is achieved by detecting the maximum intensity (I) during the period of steady-state intensity. max ) or thereabouts.

[0102] Compounds of the present disclosure can generally be prepared according to the synthetic procedures depicted, for example, in Scheme 1.

[0103] [ka] wherein G is any suitable oxygen protecting group.

[0104] Briefly, 2-adamantanone and 3-substituted benzoate esters can be coupled together by subjecting them to McMurry reaction conditions involving oxophilic titanium and a reducing agent. For example, protecting group G can be removed or replaced, or protecting group R can be removed or replaced. 4 The resulting olefin can be further modified by further functionalizing the R position. The olefin is then subjected to photooxidation conditions to provide the 1,2-dioxetane product. In various embodiments, R 4 and X are as described in any of the various embodiments of the present application. In some embodiments, R 10 and R 11 is H. In some embodiments, R 3 is substituted or unsubstituted alkyl.

[0105] The term "intensity" (e.g., light intensity or luminescence intensity), as used herein, refers to the rate of emission in photons / second. Intensity can be measured through the use of a luminometer. A luminometer is a light detector in a housing that blocks ambient light. Any suitable luminometer can be used, including photomultiplier tubes and / or photodiodes.

[0106] The term "speed of luminescence" refers to the rate of luminescence increase, i.e., the change in light intensity over time.

[0107] The term "sensitivity," as used herein, refers to the lowest level at which a signal for the analyte or product being measured can be reproducibly detected.

[0108] The term "alkyl," as used herein, refers to a substituted or unsubstituted, straight-chain, branched-chain, or cyclic, saturated monovalent or divalent group having 1 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 1 to 10 carbon atoms, 1 to 8 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 1 to 6 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 1 to 3 carbon atoms. 20 Examples of branched chain monovalent (C1-C3) alkyl groups include those having 1 to 8 carbon atoms, such as methyl (i.e., CH3), ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. 20 Examples of alkyl groups include isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, and isopentyl. 20Examples of alkyl groups include those having 1 to 6 carbon atoms, such as -CH-, -CHCH-, -CHCHCH-, -CHCHCHCH-, -CHCHCHCHCH-, and -CHCHCHCHCHCH-. Examples of branched divalent alkyl groups include -CH(CH)CH- and -CHCH(CH)CH-. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, bicyclo[1.1.1]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, and adamantyl. Cycloalkyl groups further include substituted and unsubstituted polycyclic cycloalkyl groups, such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, as well as fused rings, such as, but not limited to, decalinyl, and the like. For example, cycloalkyl includes adamantyl substituted with one, two, three, four, or more substituents, for example, at the tertiary bridgehead positions of the methylene bridge. In some embodiments, alkyl includes a combination of substituted and unsubstituted alkyl. As an example, alkyl, and also (C1)alkyl, includes methyl and substituted methyl. As a specific example, (C1)alkyl includes benzyl. As a further example, alkyl can include methyl and substituted (C2-C8)alkyl. Alkyl can also include substituted methyl and unsubstituted (C2-C8)alkyl. In some embodiments, alkyl can be methyl and C2-C8 straight-chain alkyl. In some embodiments, alkyl can be methyl and C2-C8 branched-chain alkyl. The term methyl is understood to be unsubstituted -CH3. The term methylene is understood to be unsubstituted -CH2-. For comparison, the term (C1)alkyl is understood to be substituted or unsubstituted -CH3 or substituted or unsubstituted -CH2-.Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, such as cycloalkyl, heterocyclyl, aryl, amino, haloalkyl, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.As a further example, representative substituted alkyl groups can be substituted with one or more fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido.In some embodiments, representative substituted alkyl groups can be substituted from the set of groups including amino, hydroxy, cyano, carboxy, nitro, thio, and alkoxy, but not including halogen groups.Therefore, in some embodiments, alkyl can be substituted with non-halogen groups. For example, representative substituted alkyl groups can be substituted with fluoro groups, bromo groups, halogens other than bromo, or halogens other than fluoro.In some embodiments, representative substituted alkyl groups can be substituted with one, two, three or more fluoro groups, or they can be substituted with one, two, three or more non-fluoro groups.For example, alkyl can be trifluoromethyl, difluoromethyl or fluoromethyl, or alkyl can be substituted alkyl other than trifluoromethyl, difluoromethyl or fluoromethyl.Alkyl can be haloalkyl, or alkyl can be substituted alkyl other than haloalkyl.

[0109] The term "alkenyl," as used herein, refers to a substituted or unsubstituted, linear, branched, or cyclic, saturated monovalent or divalent group having at least one carbon-carbon double bond and 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. The double bond can be in the trans or cis orientation. The double bond can be terminal or internal. Alkenyl groups can be attached through an alkenyl group containing a double bond, such as vinyl, propen-1-yl, and buten-1-yl moiety, or the alkenyl group can be attached through an alkenyl group not containing a double bond, such as penten-4-yl moiety. When specified, it should be understood that the parent moiety is attached to the alkenyl group at the vinyl position of the double bond, rather than at a non-vinyl position. For example, if an aromatic ring is substituted with a π-conjugated alkenyl group, it should be understood to be substituted at the vinyl position, rather than at a non-vinyl position. As a further example, an aromatic ring substituted with a π-conjugated propenyl group would be understood to be a propen-1-yl or propen-2-yl group, rather than a propen-3-yl group. Monovalent (C2-C 20 Examples of branched-chain monovalent (C2-C3)-alkenyl groups include those having 1 to 8 carbon atoms, such as vinyl, propenyl, propen-1-yl, propen-2-yl, butenyl, buten-1-yl, buten-2-yl, sec-buten-1-yl, sec-buten-3-yl, pentenyl, hexenyl, heptenyl, and octenyl groups. 20 Examples of alkenyl groups include isopropenyl, isobutenyl, sec-butenyl, t-butenyl, neopentenyl, and isopentenyl. 20Examples of alkenyl groups include those having 2 to 6 carbon atoms, such as -CHCH-, -CHCHCH2-, -CHCHCH2CH2-, and -CHCHCH2CH2CH2-. Examples of branched divalent alkyl groups include -C(CH3)CH- and -CHC(CH3)CH2-. Examples of cyclic alkenyl groups include cyclopentenyl, cyclohexenyl, and cyclooctenyl. For example, alkenyl can be vinyl and substituted vinyl. For example, alkenyl can be vinyl and substituted (C3-C8) alkenyl. Alkenyl can also include substituted vinyl and unsubstituted (C3-C8) alkenyl. Representative substituted alkenyl groups can be substituted one or more times with any of the groups enumerated herein, such as monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio, alkoxy, and halogen groups. As a further example, representative substituted alkenyl groups can be substituted with one or more of fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. In some embodiments, representative substituted alkenyl groups can be substituted from the set of groups including monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio, and alkoxy, but not including halogen groups. Thus, in some embodiments, alkenyl can be substituted with a non-halogen group. In some embodiments, representative substituted alkenyl groups can be substituted with a fluoro group, a bromo group, a halogen other than bromo, or a halogen other than fluoro.For example, alkenyl can be 1-fluorovinyl, 2-fluorovinyl, 1,2-difluorovinyl, 1,2,2-trifluorovinyl, 2,2-difluorovinyl, trifluoropropen-2-yl, 3,3,3-trifluoropropenyl, 1-fluoropropenyl, 1-chlorovinyl, 2-chlorovinyl, 1,2-dichlorovinyl, 1,2,2-trichlorovinyl, or 2,2-dichlorovinyl. In some embodiments, representative substituted alkenyl groups can be substituted with one, two, three, or more fluoro groups, or they can be substituted with one, two, three, or more non-fluoro groups.

[0110] The term "alkynyl," as used herein, refers to substituted or unsubstituted straight- and branched-chain alkyl groups, provided that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have 2 to 50 carbon atoms, 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples include, but are not limited to, ethynyl, propynyl, propyn-1-yl, propyn-2-yl, butynyl, butyn-1-yl, butyn-2-yl, butyn-3-yl, butyn-4-yl, pentynyl, pentyn-1-yl, hexynyl, among others, and examples include, but are not limited to, -C≡CH, -C≡C(CH), C≡C(CHCH), -CHC≡CH, -CHC≡C(CH) and -CHC≡C(CHCH).

[0111] The term "aryl," as used herein, refers to a substituted or unsubstituted monovalent group having 6 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 20 carbon atoms, 6 to 10 carbon atoms, or 6 to 8 carbon atoms derived by removing a hydrogen atom from an arene, a cyclic aromatic hydrocarbon. (C6-C 20Examples of aryl groups include phenyl, naphthalenyl, azulenyl, biphenylyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, and anthracenyl groups. Examples include substituted phenyl, substituted naphthalenyl, substituted azulenyl, substituted biphenylyl, substituted indacenyl, substituted fluorenyl, substituted phenanthrenyl, substituted triphenylenyl, substituted pyrenyl, substituted naphthacenyl, substituted chrysenyl, and substituted anthracenyl groups. Examples also include unsubstituted phenyl, unsubstituted naphthalenyl, unsubstituted azulenyl, unsubstituted biphenylyl, unsubstituted indacenyl, unsubstituted fluorenyl, unsubstituted phenanthrenyl, unsubstituted triphenylenyl, unsubstituted pyrenyl, unsubstituted naphthacenyl, unsubstituted chrysenyl, and unsubstituted anthracenyl groups. Aryl includes phenyl groups and also non-phenylaryl groups. From these examples, the term (C-C 20 ) Aryl is a fused and non-fused polycyclic (C6-C 20 ) Monocyclic and polycyclic (C6-C) containing aryl groups 20 ) aryl groups.

[0112] The term "heterocyclyl," as used herein, refers to substituted aromatic, unsubstituted aromatic, substituted nonaromatic, and unsubstituted nonaromatic rings containing three or more atoms in the ring, one or more of which are heteroatoms, such as, but not limited to, N, O, and S. The term "heteroaryl" refers to an all-aromatic heterocyclyl and is therefore a subset of the term heterocyclyl. The term "heterocycloalkenyl" refers to a heterocyclyl group containing an olefin in the nonaromatic ring such that the olefin is the point of attachment to the parent moiety. A heterocyclyl group can therefore be a heterocycloalkyl, heterocycloalkenyl, or heteroaryl, or any combination thereof if polycyclic. In some embodiments, heterocyclyl groups contain 3 to 20 ring members, while other such groups contain 3 to 15 ring members. In some embodiments, heterocyclyl groups include heterocyclyl groups containing 3 to 8 carbon atoms (C3-C8), 3 to 6 carbon atoms (C3-C6), or 6 to 8 carbon atoms (C6-C8). A heterocyclyl group designated as a C2 heterocyclyl can be a 5-membered ring having 2 carbon atoms and 3 heteroatoms, a 6-membered ring having 2 carbon atoms and 4 heteroatoms, etc. Similarly, a C4 heterocyclyl can be a 5-membered ring having 1 heteroatom, a 6-membered ring having 2 heteroatoms, etc. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also contain one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase heterocyclyl group includes fused ring species, including those containing fused aromatic and non-aromatic groups. Representative heterocyclyl groups include, but are not limited to, piperidinyl, pyrrolidinyl, piperazinyl, and morpholinyl. For example, heterocyclyl groups include, but are not limited to,

[0113] [ka] (X in the formula 1 is H, (C1~C 20 ) Alkyl, (C6-C 20) represents an aryl or amine protecting group (e.g., a t-butyloxycarbonyl group), and the heterocyclyl group can be substituted or unsubstituted). Representative heteroaryl groups include furanyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, imidazolyl, triazolyl, tetrazolyl, benzoxazolinyl, and benzimidazolinyl groups. In some embodiments, the heteroaryl is a 5-membered heteroaryl. In some embodiments, the heteroaryl is other than pyridine, pyrimidine, pyridazine, pyrazine, or a fused derivative thereof. A π-excess heteroaryl is an electron-rich heteroaryl that can function as an electron-donating group. Examples of π-excess heteroaryls are furan, thiophene, indole, pyrrole, benzofuran, and benzothiophene.

[0114] The term "alkoxy," as used herein, refers to an alkyl group, including cycloalkyl groups, as defined herein, linked to an oxygen atom. Examples of straight-chain alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched-chain alkoxy groups include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy groups include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can contain 1 to 12 to 20 or 12 to 40 carbon atoms bonded to the oxygen atom, can further contain double or triple bonds, and can also contain heteroatoms. Thus, alkoxy includes both an oxygen atom linked to an alkenyl group and an oxygen atom linked to an alkynyl group. For example, an allyloxy group is an alkoxy group within the meaning of this specification. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group within the context in which two adjacent atoms of the structure are substituted therewith.

[0115] The term "aryloxy," as used herein, refers to an aryl group, as defined herein, linked to an oxygen atom. The point of attachment to the parent moiety is the oxygen atom.

[0116] The term "arylcarbonyl," as used herein, refers to a carbonyl (CO) group attached to an aryl group, as defined herein. The point of substitution to the parent moiety is the carbonyl group.

[0117] The term "heteroarylcarbonyl," as used herein, refers to a carbonyl (CO) group attached to a heteroaryl group, as defined herein. The point of substitution onto the parent moiety is the carbonyl group.

[0118] The term "arylalkyl" as used herein refers to an alkyl group, as defined herein, in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to an aryl group, as defined herein.Representative aralkyl groups include benzyl, biphenylmethyl and phenylethyl groups, and fused (cycloalkylaryl) alkyl groups, such as 4-ethyl-indanyl.An aralkenyl group is an alkenyl group, as defined herein, in which a hydrogen or carbon bond of the alkenyl group is replaced with a bond to an aryl group, as defined herein.The substitution point to the parent moiety is the alkyl group.

[0119] The terms "halo," "halogen," or "halide" group, as used herein by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0120] The term "amino" as used herein includes -NH2, -NHR, -NR2, -NR3 + wherein each R is independently selected and cannot be protonated -NR + "Amino" refers to a substituent in the form of an amine, where each of the amines is a protonated form, except for the protonated form of each of the amines. Thus, any compound substituted with an amino group can be considered an amine. An "amino group" within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An "alkylamino" group includes monoalkylamino, dialkylamino, and trialkylamino groups.

[0121] The term "acyl," as used herein, refers to a group containing a carbonyl moiety, where the group is bonded via the carbonyl carbon atom, which is also bonded to another carbon atom and can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl group, etc.

[0122] The term "formyl," as used herein, refers to a group containing an aldehyde moiety. The point of substitution onto the parent moiety is the carbonyl group.

[0123] The term "alkoxycarbonyl," as used herein, refers to a group containing a carbonyl moiety, wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to an oxygen atom, which is further bonded to an alkyl group. Alkoxycarbonyl also includes groups in which the carbonyl carbon atom is also bonded to an oxygen atom, which is further bonded to an alkenyl group. Alkoxycarbonyl also includes groups in which the carbonyl carbon atom is also bonded to an oxygen atom, which is further bonded to an alkynyl group. In a further case, where the term is included within the definition of alkoxycarbonyl as defined herein and is also included within the term "aryloxycarbonyl," the carbonyl carbon atom is bonded to an oxygen atom, instead of an alkyl group, which is bonded to an aryl group.

[0124] The term "alkylamide" as used herein refers to a group containing a carbonyl moiety, wherein the group is bonded via a carbonyl carbon atom.The carbonyl carbon atom is also bonded to a nitrogen group that is bonded to one or more alkyl groups.In another case of alkylamide as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom that is bonded to one or more aryl groups instead of or in addition to one or more alkyl groups.In another case of alkylamide as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom that is bonded to one or more alkenyl groups instead of or in addition to one or more alkyl and / or aryl groups.In another case of alkylamide as defined herein, the carbonyl carbon atom is bonded to a nitrogen atom that is bonded to one or more alkynyl groups instead of or in addition to one or more alkyl, alkenyl and / or aryl groups.

[0125] The term "carboxy" as used herein refers to a group containing a carbonyl moiety, where the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to a hydroxyl group or an oxygen anion to produce a carboxylic acid or carboxylate. Carboxy also includes both the protonated form and the salt form of carboxylic acid. For example, carboxy can be understood as COOH or COH.

[0126] The term "alkylthio," as used herein, refers to a sulfur atom linked to an alkyl, alkenyl, or alkynyl group, as defined herein. The point of substitution onto the parent moiety is the sulfur atom.

[0127] The term "arylthio," as used herein, refers to a sulfur atom linked to an aryl group, as defined herein. The point of substitution to the parent moiety is the sulfur atom.

[0128] The term "alkylsulfonyl," as used herein, refers to a sulfonyl group linked to an alkyl, alkenyl, or alkynyl group, as defined herein. The point of substitution onto the parent moiety is the sulfonyl group.

[0129] The term "alkylsulfinyl," as used herein, refers to a sulfinyl group linked to an alkyl, alkenyl, or alkynyl group, as defined herein. The point of substitution to the parent moiety is the sulfinyl group.

[0130] The term "dialkylaminosulfonyl" as used herein refers to a sulfonyl group connected to a nitrogen atom that is further connected to two alkyl groups, as defined herein, and which can optionally be bonded together to form a ring with the nitrogen atom. This term also includes groups in which the nitrogen atom is further connected to one or two alkenyl groups instead of an alkyl group. The substitution point on the parent moiety is the sulfonyl group.

[0131] The term "dialkylamino" as used herein refers to an amino group linked to two alkyl groups, as defined herein, which can optionally be bonded together to form a ring with the nitrogen. This term also includes groups in which the nitrogen is further linked to one or two alkenyl groups instead of an alkyl group. The substitution point to the parent moiety is the nitrogen atom.

[0132] The term "dialkylamide" as used herein refers to an amide group linked to two alkyl groups, as defined herein, which can optionally be joined together to form a ring with the nitrogen. This term also includes groups in which the nitrogen is further linked to one or two alkenyl groups instead of an alkyl group. The substitution point to the parent moiety is the amide group.

[0133] Each of the various substituents described herein can be substituted or unsubstituted. The term "substituted," as used herein (e.g., in the context of "optionally substituted arylalkyl"), includes the following groups: deuterium (D), halogen (e.g., F, Cl, Br, and I), R, OR, OC(O)N(R), CN, NO, NO, ONO, azido, CF, OCF, methylenedioxy, ethylenedioxy, (C-C 20 )Heteroaryl, N(R)2, Si(R)3, SR, SOR, SO2R, SO2N(R)2, SO3R, P(O)(OR)2, OP(O)(OR)2, C(O)R, C(O)C(O )R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, C(O)N(R)OH, OC(O)N(R)2, C(S)N(R)2, (CH2) 0~2 N(R)C(O)R, (CH2) 0~2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N( R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R or C(=NOR)R (where R is hydrogen, (C1~C 20 ) alkyl or (C6-C 20) aryl). Substitution also includes groups substituted with one or more groups, including, but not limited to, the following groups: fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino, and dialkylamido. Where two or more adjacent substituents are present, the substituents can be linked to form a carbocyclic or heterocyclic ring. Such adjacent groups can have a vicinal or germinal relationship, or they can be adjacent to the ring, for example, in an ortho configuration. Each instance of substitution is understood to be independent. For example, a substituted aryl can be substituted with bromo, and a substituted heterocycle on the same compound can be substituted with alkyl. It is expected that a substituent can be substituted with one or more non-fluoro groups. As another example, a substituent can be substituted with one or more non-cyano groups. As another example, a substituent can be substituted with one or more groups other than haloalkyl. As yet another example, a substituent can be substituted with one or more groups other than tert-butyl. As a still further example, a substituent can be substituted with one or more groups other than trifluoromethyl. As an even further example, a substituent can be substituted with one or more groups other than nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophenyl, or nitrophenyl, or a combination of such descriptions.Furthermore, substitution is also understood to include fluoro, cyano, haloalkyl, tert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophenyl, and nitrophenyl groups. In various embodiments, substituents may be substituted with groups other than carbonyl-containing groups, nitro, cyano, sulfinyl, sulfonyl, or halogen-containing groups. In various embodiments, substituents may be substituted with groups other than electron-withdrawing groups. Some substituents in certain embodiments may be independently substituted with one or more electron-donating groups.

[0134] As used herein, the term alcohol protecting group refers to a substituent on an oxy group that renders the alcohol oxygen inert to various conditions under which it would typically react, but is easily removed when subjected to certain conditions. Alcohol protecting groups, as described herein, typically improve the stability of the dioxetane moiety and promote decomposition of the dioxetane upon their removal. Thus, alcohol protecting groups include phosphates, such as PO3Na2, PO3Cl2, and PO3H2, glycosyl groups, dinitrobenzenesulfonylaminobenzyl groups, and other groups that can be enzymatically hydrolyzed to provide the unprotected alcohol. Some alcohol protecting groups are described in Theodora W. Greene, Peter GM Wuts (1999). Protecting Groups in Organic Synthesis (3rd ed.). J. Wiley. Alcohol protecting groups include acetyl, benzoyl, benzyl, methoxyethoxymethyl, dimethyltrityl, methoxylmethyl, methylthiomethyl, pivaloyl, tetrahydropyranyl, tetrahydrofuranyl, trityl, trialkylsilyl, trialkylsiloxymethyl, dialkylarylsilyl, glycosyl, pyranyl, galactosyl and ethoxyethyl groups.Alcohol protecting groups also include groups in which the alcohol is substituted with a fragmentable linker that is further substituted with a protecting group, and when the protecting group is deprotonated, the linker is fragmented and removed from the alcohol.The following compounds are further examples of alcohols substituted with alcohol protecting groups:

[0135] [ka]

[0136] In various embodiments, the protecting group G is an enzyme-cleavable group, the removal of which by an analyte of interest, e.g., in the presence of an enzyme capable of cleaving the enzyme-cleavable group, provides an unstable phenolate-dioxetane species that subsequently decomposes and emits light. For example, G can be a peptide moiety of two or more amino acid residues that is cleavable by a specific enzyme.

[0137] In some instances, the compounds described herein (e.g., compounds of Formulas I-IV) may contain chiral centers. All diastereomers of the compounds described herein are contemplated herein, as are racemates.

[0138] As used herein, the term "salt" refers to a derivative of the disclosed compound in which the parent compound is modified by forming its acid or base salt. Examples of salts include alkali salts and alkaline earth salts of the ionized forms of the disclosed compounds. For example, lithium salts, sodium salts, potassium salts, calcium salts, or magnesium salts. The disclosed compounds can also be salts containing cationic metals and anionic organic compounds, such as compounds with oxyanions and sodium cations. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic groups, such as amines; and alkali or organic salts of acidic groups, such as carboxylic acids. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid; and organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like.

[0139] Salts can be synthesized from parent compounds containing basic or acidic moieties by conventional chemical methods. In some cases, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount (or more) of an appropriate base or acid in water or an organic solvent, or a mixture of the two, and generally non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985.

[0140] Unless otherwise specified, all numbers used in the specification and claims expressing amounts of components, molecular weights, and the like are understood to be modified in all instances by the term "about." When used herein in connection with a measured quantity, the term "about" refers to the variation in the measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring instrument used. Thus, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0141] Values ​​expressed in range format should be interpreted flexibly to include not only the numerical values ​​explicitly recited as limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. For example, a range of "0.1% to 5%" or "0.1% to 5%" should be interpreted not only to include 0.1% to 5%, but also to include individual values ​​(e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the stated range. In addition, the terms "in a range" or "within" (and similar expressions) include the endpoints of the stated range. As used herein, for example, a "up to" number (e.g., "up to 50") includes that number (e.g., 50).

[0142] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless stated otherwise. Furthermore, the term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0143] Additionally, any phraseology or terminology employed herein and not otherwise defined shall be understood to be for purposes of description only and not of limitation. Any use of section headings is intended to aid in the reading and comprehension of the document and shall not be construed as limiting. Furthermore, the information associated with a section heading may occur within or outside that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of a conflict of usage between this document and those documents so incorporated by reference, the usage in the incorporated references shall be considered supplementary to the usage in this document, and in the event of any irreconcilable conflict, the usage in this document shall prevail.

[0144] In the methods described herein, steps may be performed in any order without departing from the principles of the invention, unless a temporal or operational order is explicitly stated. Furthermore, certain steps may be performed simultaneously unless the express language of a claim recites them otherwise. For example, a claimed step of performing X and a claimed step of performing Y may be performed simultaneously in a single operation, and the resulting process falls within the language of the claimed process.

[0145] Terms such as "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms are understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements.

[0146] By "consisting of," it is meant to include and be limited to everything that follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that no other elements may be present. By "consisting essentially of," it is meant to include any elements listed after the phrase, and to be limited to other elements that do not interfere with or contribute to the activity or action specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or essential, but that other elements are optional and may or may not be present depending on whether they materially affect the activity or action of the listed elements.

[0147] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0148] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Therefore, the present description is not intended to be, and should not be construed as, limited to the examples shown, but the full breadth of protection afforded by the appended claims and their equivalents should be accorded. Additionally, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Thus, the above description or exemplary embodiments are provided for the purpose of illustrating the principles of the present disclosure, and may include modifications thereto and permutations thereof, without limitation thereto. [Example]

[0149] The present invention may be better understood by reference to the following examples, which are provided by reference: The present invention is not limited to the examples set forth herein.

[0150] General method The various compounds of the present disclosure can be synthesized according to a variety of methods, including, but not limited to, the synthetic procedures described in PCT Publication WO 1996 / 015122, U.S. Pat. No. 4,962,192, or U.S. Pat. No. 5,004,565.

[0151] Chemiluminescence (emission) intensity can be measured using a Turner Designs (Sunnyvale, CA) Model TD-20e luminometer, a BMG Labtech luminescence plate reader, or a charge-coupled device (CCD) camera luminometer, or any other suitable light intensity measuring device. In the examples listed below, solutions containing different concentrations of alkaline phosphatase (e.g., AP4, 1.2 x 10 -15 with ALP concentrations of 1.2 × 10 mol / µL; AP6, 1.2 × 10 -17 with ALP concentrations of 1.2 × 10 mol / μL; AP8, 1.2 × 10 -19 mol / µL; and AP9, 1.2 x 10 -20 A 100 μL initial solution was combined with 10 μL of ALP solution and data were collected over 240 seconds.

[0152] Nuclear magnetic resonance (NMR) spectra were obtained using a 400 MHz spectrometer in solutions of D2O and CDCl3.

[0153] The amine-based buffer "221" or "Sigma-221" is available from Sigma-Aldrich (St. Louis, MO). However, other commercially available buffers can be used in the compositions described herein.

[0154] [Example 1] structure

[0155] [ka] The dioxetane compound having the formula (Lumigen® PPD) was obtained from Lumigen, Inc. The synthesis of PPD is described in WO 2021 / 086977 (Example 12). 1 H NMR (400 MHz, D2O) δ ppm 7.40-7.15 (m, 4H), 3.24 (s, 3H), 2.89 (s, 1 H), 2.28 (s, 1 H), 1.90-1.50 (m, 10H), 1.28 (d, J = 13.2Hz, 1H), 0.99 (d, J = 10Hz, 1H).

[0156] [Example 2] structure

[0157] [ka] VPPD having the formula: was obtained using the method described in WO 2021 / 086977 (Example 16).

[0158] 1H NMR (400 MHz, D2O) δ ppm 7.67 (d, J = 8.4Hz,1 H), 7.66 (br, 1 H), 7.26 (br, 1 H), 7.16-6.06 (m, 1 H), 5.88 (d, J = 16Hz, 1 H), 5.37 (d, J = 12.4Hz, 1H), 3.27 (s, 3H), 2.88 (s, 1H), 2.30 (s, 1H), 1.84 -1.56 (m, 10H), 1.28 (d, J = 9.2Hz, 1H), 1.05 (d, J = 12.8Hz, 1H).

[0159] [Example 3] structure

[0160] [ka] VMPD having the formula: was obtained using the method described in WO 2021 / 086977 (Example 18). 1 H NMR (400 MHz, D2O) δ ppm (D2O ppm): 7.51-7.41 (m, 2H), 7.29-7.20 (m, 1 H), 5.66 (d, J = 17.2Hz, 1 H), 5.19 (d, J = 11.2Hz, 1 H), 3.07 (s, 3H), 2.72 (s, 1 H), 2.36 (s, 3H), 1.99 (s, 1 H), 1.95-1.38 (m, 10H), 1.19-1.15 (m, 1 H), 0.92-0.88 (m, 1 H).

[0161] [Example 4] Initial solutions of the dioxetane compound and surfactant of Examples 1-3 were prepared in an appropriate buffer (e.g., 221 buffer). For each example, the dioxetane concentration used ranged from 50 mg / L to 500 mg / L. Figure 2B, discussed further below, shows various concentrations of the dioxetane VPPD (ranging from 50 mg / L to 500 mg / L) while the concentration of the surfactant EXL was held constant. The surfactant concentrations used ranged from 100 mg / L to 500 mg / L. Figure 2C, discussed further below, shows various concentrations of the surfactant EXL (ranging from 100 mg / L to 500 mg / L) while the concentration of the dioxetane VPPD was held constant. The ratio of the dioxetane compound to the surfactant concentration was a simple ratio ranging from 5:1 to 1:10. In one example, the dioxetane (e.g., VPPD) concentration was 100 mg / L to 150 mg / L, the surfactant (e.g., EXL) concentration was 200 mg / L to 300 mg / L, and the ratio of dioxetane (e.g., VPPD) to surfactant (e.g., EXL) concentrations ranged from 1:1 to 1:3. The compositions tested contained 50 mg / L to 500 mg / L of magnesium(II) chloride (MgCl), although other magnesium(II) salts can be used.

[0162] Next, 100 μL of the initial solution was combined with 10 μL of a solution of alkaline phosphatase (AP8) at 37° C. The intensity of chemiluminescence was measured over time as the compounds were combined with the alkaline phosphatase solution. Graphs showing the intensity of light emission (relative light units (RLU) over time) specifically for 100 μL of the initial solution combined with 10 μL of AP8 solution are provided in FIGS. 1 and 2A, with FIG. 1 showing the RLU data as a function of time for LP530. The fluorescein surfactant in LP530 is combined with cetyltrimethylammonium bromide (CTAB) to form a compound of the formula

[0163] [ka] (wherein p is an integer of 2 to 10, for example, 5 to 9 or 5 to 7).

[0164] In another experiment, EXL surfactants were used. A graph showing the intensity of light emission (relative light units (RLU) over time) is provided in Figure 2A. EXL surfactants containing repeat unit (A), repeat unit (B), or both.

[0165] [ka] (In the formula, "Bu3" refers to "tributyl" and "Oct3" refers to "trioctyl"). The molar ratio of repeat unit (A) to repeat unit (B) used was 4:1. Figures 2B and 2C are plots of RLU over time for various VPPD concentrations (50 mg / L to 500 mg / L) and various EXL surfactant concentrations (100 mg / L to 500 mg / L). In both experiments, VPPD and surfactant solutions were mixed with 6 μL of AP8 and then incubated at 37 °C. Figures 2B and 2C show that various concentrations of the dioxetane VPPD (while keeping the surfactant EXL concentration constant) or various concentrations of the surfactant EXL (while keeping the dioxetane VPPD concentration constant) provide improved luminescence (as measured in RLU units) compared to EXL and PPD. Note that 6 μL of AP8 was used in Figures 2B and 2C, and 10 μL of AP8 was used in Figure 2A. With 10 μL of AP8 (not shown), the observed RLUs were higher and less variable than with 6 μL of AP8, and were even more improved for EXL and PPD.

[0166] Figure 3 shows a comparison of the emission observed for VPPD with EXL surfactant compared to VPPD, PPD and LP530 normalized to the intensity of VPPD in EXL surfactant.

[0167] One observation from Figures 1, 2A-2C, and 3 is that VPPD exhibits higher RLUs in a relatively short period of time (e.g., approximately 4 minutes) compared to PPD and VMPD, regardless of the surfactant system (LP530 vs. EXL surfactant). However, Figure 3 highlights the large relative difference in RLU intensity between VPPD in EXL compared to LP530. For example, the intensity of VPPD is over 20-fold higher in EXL relative to LP530 (approximately 9,500 RLU in EXL surfactant vs. approximately 380 in LP530). One benefit of the compositions described herein is that alkaline phosphatase detection limits can be improved. Using the compositions described herein, alkaline phosphatase detection limits can be improved by detecting AP10 (1.2 x 10 -21 mol / μL ALP), as well as AP11 and AP12 (1.2 × 10 -22 mol / μL and 1.2 × 10 -23 ALP in the presence of α-glucan can also be detected.

[0168] [Example 5] Dioxetane compositions containing the dioxetane concentrations, surfactant / enhancement concentrations, oxetane:surfactant ratios, and magnesium amounts described in Example 4 were prepared using the following surfactants / enhancements (1)-(4).

[0169] [Table 1]

[0170] The oxetane and enhancer combinations used to generate the data in Figure 4 are listed in Table 1 below.

[0171] [Table 2]

[0172] Figure 4 shows a comparison of the luminescence observed for VPPD with surfactants / enhancers (1)-(4) when mixed with AP8 compared to LP530. Specifically, 100 μL of a solution containing VPPD and surfactant / enhancer was combined with 10 μL of AP8 and then incubated at 37 °C.

[0173] One observation from Figure 4 is that VPPD among the phosphonium surfactants / enhancers, regardless of whether they are polymeric or small molecule phosphonium surfactants / enhancers, exhibits significantly higher RLUs over a relatively short period of time (e.g., approximately 4 minutes) compared to LP530. Another observation is that the small molecule phosphonium surfactant (2) and polymeric phosphonium surfactant / enhancer (4) have roughly the same RLU at 4 minutes. And there appears to be a difference in RLU between the isomeric polymeric surfactants / enhancers (3) and (4) at 4 minutes (and beyond), with (3) being brighter than (4).

Claims

1. Compounds of Formula I and Salts Thereof 【Chemical 1】 (In the formula, R １ and R ２ Each of the is independently C ３ ~C １０ alkyl or R １ and R ２ together with the carbon to which they are attached form C ５ ~C １０ providing a cycloalkyl ring, R ３ is C １ ~C １０ Alkyl, C ６ ~C １０ is aryl or heteroaryl, R ４ is C ２ ~C １０ is alkenyl, R ５ is H or C １ ~C １０ is alkyl, X is phosphate), and a composition comprising at least one phosphonium surfactant.

2. The composition of claim 1 comprising one surfactant.

3. The composition of claim 1 comprising two or more surfactants.

4. 4. The composition of claim 1, wherein the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

5. The phosphonium surfactant may comprise repeating units (A), repeating units (B), or both. 【Chemistry 2】 (In the formula, “Bu ３ " refers to "tributyl" and "Oct ３ 10. The composition of claim 1 or 4, comprising a polymeric phosphonium surfactant comprising:

6. 6. The composition according to claim 5, wherein the molar ratio of the repeating unit (A) to the repeating unit (B) is 1:100 to 100:

1.

7. The polymeric phosphonium surfactant has the structure 【Chemistry 3】 (wherein z is an integer of 2 to 1000 or 100 to 600) 10. The composition of claim 1 or 4, comprising:

8. The polymeric phosphonium surfactant has the formula 【Chemistry 4】 (In the formula, R １２ ~R １４ are each independently C １ ~C １０ is alkyl, R １５ is arylalkyl, X － is the counterion) 5. The composition of claim 1 or 4, comprising a small molecule phosphonium surfactant of formula:

9. R １５ But C ２ ~C ８ Alkenyl group or formula 【Chemistry 5】 (wherein k is 0, 1 or 2; R １６ ~R １８ are each independently C １ ~C １６ alkyl) The composition of claim 8, wherein the arylalkyl is substituted with a group of the formula:

10. R １６ ~R １８ are each independently C ４ ~C １０ The composition of claim 9 wherein the alkyl is alkyl.

11. Said C ２ ~C ８ The alkenyl group is of the formula 【Chemistry 6】 (In the formula, R １９ is C １ ~C ８ Alkyl, C ２ ~C ８ alkenyl or C ２ ~C ８ alkynyl) The composition of claim 9, wherein the group is

12. The composition of claim 1 further comprising a fluorescein surfactant.

13. The fluorescein surfactant is represented by the formula (V): 【Chemistry 7】 (In the formula, R ６ is C ６ ~C ２０ alkyl group) and optionally comprising an ammonium salt such as cetyltrimethylammonium bromide, and optionally wherein the fluorescein surfactant is 5-dodecanoylaminofluorescein, 5-hexadecanoylaminofluorescein or 5-stearylaminofluorescein.

14. 2. The composition of claim 1, wherein the phosphate group is of the formula -O-P(O)(ONa)(ONa).

15. The compound of formula I is a compound of formula II or a salt thereof 【Chemistry 8】 (In the formula, R １０ and R １１ each independently represents H, halogen, C １ ~C １０ Alkyl, C ２ ~C １０ Alkenyl, C ６ ~C １０ aryl) The composition of claim 1 ,

16. R １０ and R １１ 16. The composition of claim 15, wherein each is independently H or halogen.

17. The compound of formula I is a compound of formula III or a salt thereof 【Chemistry 9】 17. The composition of any one of claims 1 to 16, wherein

18. The compound of formula I is a compound of formula IV or a salt thereof 【Chemistry 10】 17. The composition of any one of claims 1 to 16, wherein

19. The compound of formula I is of the formula 【Chemistry 11】 17. The composition of any one of claims 1 to 16, wherein the compound is:

20. The compound of formula I is of the formula 【Chemistry 12】 17. The composition of any one of claims 1 to 16, wherein the compound is:

21. 21. The composition of any of claims 1 to 20, further comprising an amine buffer.

22. 22. The composition of any one of claims 1 to 21, in the form of an aqueous composition.

23. alkaline phosphatase, wherein the alkaline phosphatase is present in the composition in an amount of at most 1 x 10 －１８ mol / μL, 1×10 －１９ mol / μL, 1×10 －２０ mol / μL, or 1×10 －２１ 23. The composition of any one of claims 1 to 22, having a concentration of mol / μL.

24. The alkaline phosphatase is present in the composition at a concentration of at least 1.2 x 10 －２２ mol / μL or at least 1.2×10 －２３ 24. The composition of claim 23, having a concentration of mol / μL.

25. At least 1 x 10 －２２ or at least 1 x 10 －２１ 25. The composition of claim 23 or 24, comprising mol of ALP.

26. At most 1 x 10 －１７ mol of ALP, at most 1 × 10 －１８ mol of ALP, at most 1 × 10 －１９ mol of ALP or at most 1 × 10 －２０ 26. The composition of claim 23, 24 or 25, comprising mol of ALP.

27. 27. A method for detecting alkaline phosphatase in a sample, comprising contacting the sample with a composition described in any one of claims 1 to 26, thereby producing a mixture, and monitoring the mixture for luminescence.

28. The alkaline phosphatase in the mixture is at most 1 x 10 －１８ mol / μL, at most 1 × 10 －１９ mol / μL, at most 1 × 10 －２０ mol / μL, or at most 1 × 10 －２１ 28. The method of claim 27, wherein the concentration is mol / μL.

29. The alkaline phosphatase in the mixture is at least 1×10 －２２ mol / μL or at least 1×10 －２３ 29. The method of claim 27 or 28, wherein the concentration is mol / μL.

30. The mixture is at most 1×10 －１７ mol of ALP, at most 1 × 10 －１８ mol of ALP, at most 1 × 10 －１９ mol of ALP or at most 1 × 10 －２０ 30. The method of any one of claims 27, 28 or 29, comprising mol of ALP.

31. The mixture is at least 1×10 －２１ mol of ALP or at least 1 x 10 －２２ 31. The method of any one of claims 27, 28, 29 or 30, comprising mol of ALP.

32. 32. The method of any of claims 27, 28, 29, 30 or 31, further comprising measuring the intensity of the resulting luminescence and correlating said intensity with the presence or concentration of said alkaline phosphatase.

33. The luminescence has a peak RLU of greater than 8,000 and a T of 3 minutes or less. １／２ 33. The method of any of claims 27 to 32, comprising at least one of:

34. 34. The method of any of claims 27 to 33, wherein the sample is an aqueous sample having a pH of from 4 to 12.

35. 28. The method of claim 27, wherein the sample is an aqueous sample having a pH of 8 to 12.

36. contacting the sample with a composition comprising the phosphonium surfactant, wherein the sample is combined with PPD, and cetyltrimethylammonium bromide (CTAB) to form a phosphonium surfactant of the formula 【Chemistry 13】 (wherein p is an integer from 2 to 10, for example, from 5 to 9 or from 5 to 7).

36. The method of any of claims 27-35, wherein the method has an increased signal to noise ratio when compared to contacting with a composition comprising a fluorescein surfactant having the formula:

37. 37. The method of claim 36, wherein the increased signal to noise ratio is at least 50-fold.

38. 37. The method of claim 36, wherein the composition comprising the phosphonium surfactant has a luminescence of less than 15 RLU in the absence of the sample.

39. 37. The method of claim 36, wherein the composition comprising the phosphonium surfactant in the absence of the sample has a luminescence of greater than 1 and less than 15 RLU.

40. A kit for determining the presence of alkaline phosphatase, comprising a compound of formula I and its salts. 【Chemistry 14】 (In the formula, R １ and R ２ Each of the is independently C ３ ~C １０ alkyl or R １ and R ２ together with the carbon to which they are attached form C ５ ~C １０ providing a cycloalkyl ring, R ３ is C １ ~C １０ Alkyl, C ６ ~C １０ is aryl or heteroaryl, R ４ is C ２ ~C １０ is alkenyl, R ５ is H or C １ ~C １０ alkyl), and A kit comprising at least one phosphonium surfactant, optionally wherein said composition comprises one surfactant.

41. 41. The kit of claim 40, wherein the phosphonium surfactant is selected from the group consisting of small molecule phosphonium surfactants and polymeric phosphonium surfactants.

42. The polymeric phosphonium surfactant comprises repeating units (A), repeating units (B), or both 【Chemistry 15】 (In the formula, “Bu ３ " refers to "tributyl" and "Oct ３ " refers to "trioctyl") 42. The kit of claim 40 or 41, comprising:

43. The kit according to claim 42, wherein the molar ratio of the repeating unit (A) to the repeating unit (B) is 1:100 to 100:

1.

44. The polymeric phosphonium surfactant has the structure 【Chemistry 16】 (wherein z is an integer of 2 to 1000 or 100 to 600) 42. The kit of claim 40 or 41, comprising:

45. The small molecule phosphonium surfactant has the formula 【Chemistry 17】 (In the formula, R １２ ~R １４ are each independently C １ ~C １０ is alkyl, R １５ is an optionally substituted arylalkyl; X － is the counterion) 42. The kit of claim 40 or 41,

46. R １５ But C ２ ~C ８ Alkenyl group or formula 【Chemistry 18】 (wherein k is 0, 1 or 2; R １６ ~R １８ are each independently C １ ~C １６ alkyl) 46. ​​The kit of claim 45, wherein the arylalkyl is substituted with a group:

47. R １６ ~R １８ are each independently C ４ ~C １０ 47. The kit of claim 46, wherein the alkyl is alkyl.

48. Said C ２ ~C ８ The alkenyl group is of the formula 【Chemistry 19】 (In the formula, R １９ is C １ ~C ８ Alkyl, C ２ ~C ８ alkenyl or C ２ ~C ８ 47. The kit of claim 46, wherein the group is alkynyl.

49. The method further comprises the step of: 【Chemistry 20】 (In the formula, R ６ is C ６ ~C ２０ alkyl group) and optionally comprising an ammonium salt such as cetyltrimethylammonium bromide, and optionally wherein the fluorescein surfactant is 5-dodecanoylaminofluorescein, 5-hexadecanoylaminofluorescein or 5-stearylaminofluorescein.

50. 50. The kit of claim 49, wherein the surfactant is 5-dodecanoylaminofluorescein, 5-hexadecanoylaminofluorescein, or 5-stearylaminofluorescein.

51. 51. The kit of any of claims 40 to 50, wherein the compound of formula I and its salts and the at least one surfactant are in separate containers.

52. 51. The kit of any of claims 40 to 50, wherein the compound of formula I and its salts and the at least one surfactant are in a single container.

53. 53. The composition, method or kit of any preceding claim, wherein the concentration of the dioxetane is from 100 mg / L to 150 mg / L.

54. 54. The composition, method or kit of any preceding claim, wherein the concentration of the surfactant is from 200 mg / L to 300 mg / L.

55. 55. The composition, method or kit of any preceding claim, wherein the ratio of dioxetane to surfactant is from 1:1 to 1:

3.

56. 56. The composition, method, or kit of any of claims 1 to 55, wherein the composition comprises 50 mg / L to 500 mg / L of a magnesium(II) salt.

57. The composition contains 50 mg / L to 500 mg / L of MgCl ２ 57. The composition, method or kit of any of claims 1 to 56, comprising:

58. 1. A method for detecting alkaline phosphatase in a sample, comprising: a. providing a mixture of a dioxetane compound and a phosphonium surfactant; b. contacting the sample with the mixture of the dioxetane compound and the phosphonium surfactant to produce a mixture comprising the sample; and c. Monitoring the luminescence of the mixture containing the sample, wherein a peak RLU of greater than 8,000 or a T of 3 minutes or less is obtained. １／２ monitoring the emission, including detecting A method comprising:

59. 59. The method of claim 58, wherein the emission of the combination of the dioxetane compound and the phosphonium surfactant in the absence of the sample has a peak RLU of less than 15.

60. 59. The method of claim 58, wherein the emission of the combination of the dioxetane compound and the phosphonium surfactant in the absence of the sample has a peak RLU of greater than 1 and less than 15.

61. The alkaline phosphatase in the mixture containing the sample is at most 1 x 10 －１８ mol / μL, 1×10 －１９ mol / μL, 1×10 －２０ mol / μL or 1×10 －２１ 61. The method of any of claims 58 to 60, having a concentration of mol / μL.

62. The alkaline phosphatase in the mixture containing the sample is at least 1×10 －２２ mol / μL or 1×10 －２３ 62. The method of any one of claims 58 to 61, wherein the concentration is mol / μL.

63. The mixture containing the sample has at most 1×10 －１７ mol, 1 x 10 －１８ mol, 1 x 10 －１９ mol or 1 x 10 －２０ 63. The method of any of claims 58 to 62, comprising mol of ALP.

64. The mixture is at least 1×10 －２１ mol or 1 x 10 －２２ 64. The method of any one of claims 58 to 63, comprising mol of ALP.

65. 1. A method for detecting alkaline phosphatase in a sample, comprising: a. contacting the sample with a first mixture of a dioxetane compound and a phosphonium surfactant to produce a second mixture comprising the sample; and b. Monitoring the luminescence of the second mixture, wherein a peak RLU of greater than 8,000 or a T of 3 minutes or less is reached. １／２ monitoring the emission, including detecting A method comprising:

66. 66. The method of claim 65, wherein the emission of the combination of the dioxetane compound and the phosphonium surfactant in the absence of the sample has a peak RLU of less than 15.

67. 66. The method of claim 65, wherein the emission of the combination of the dioxetane compound and the phosphonium surfactant in the absence of the sample has a peak RLU of greater than 1 and less than 15.

68. The alkaline phosphatase in the second mixture is at most 1 x 10 －１８ mol / μL, 1×10 －１９ mol / μL, 1×10 －２０ mol / μL or 1×10 －２１ 68. The method of any one of claims 65 to 67, having a concentration of mol / μL.

69. The alkaline phosphatase in the second mixture is at least 1 x 10 －２２ mol / μL or 1×10 －２３ 69. The method of any of claims 65 to 68, wherein the concentration is mol / μL.

70. The second mixture is at most 1×10 －１７ mol, 1 x 10 －１８ mol, 1 x 10 －１９ mol or 1 x 10 －２０ 70. The method of any one of claims 65 to 69, comprising mol of ALP.

71. The second mixture is at least 1×10 －２１ mol or 1 x 10 －２２ 71. The method of any one of claims 65 to 70, comprising mol of ALP.

72. the second mixture comprising: at least 10 μL, at least 50 μL, at least 100 μL; and / or 71. The method of any of claims 65 to 70, comprising up to 100 μL, up to 110 μL, up to 150 μL, up to 200 μL, or up to 500 μL.

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