TWI454455B - Salicylic acid derivates with fluorophores and method of making and using the same - Google Patents

Description

O-hydroxybenzoic acid derivative having a fluorescent group, and method of making and using same

The present invention relates to a substance which can emit fluorescence by a biochemical reaction, and more particularly to a substance having a chemical structure having an o-hydroxybenzoic acid moiety and a fluorescent group.

Salicylate hydroxylase (SHL) belongs to a flavoprotein that catalyzes salicylic acid in the presence of an aerobic environment and NAD(P)H to produce catechol.

At present, SHL and NAD(P) ⁺ dependent dehydrogenase have been glued together to the Clark electrode to form an electrochemical sensor that can be operated by various enzymes. Molecular sensing.

However, since the aforementioned electrochemical type biosensor is difficult to implement and the fabricated electrode is difficult to store, it is necessary to propose a different solution.

Inspired by the shortcomings of the prior art, the inventor exhausted his mind and researched it, and based on his years of experience in the industry, he developed a hidden type of firefly that allows users to perform biochemical analysis by fluorescence. A latent fluorophore or pro-fluorophore, a method for producing the same, a method for performing biochemical analysis thereof, and a biochemical analysis kit containing the concealed fluorescent group.

One of the objects of the present invention is to provide a hidden fluorophore derived from o-hydroxybenzoic acid and having a fluorescent group and a moiety as shown in Formula I in the structure:

Wherein the fluorescent group is directly linked to the moiety of formula I or indirectly to a moiety of formula I via a linking structure.

In a preferred embodiment, various chemical synthesis means can be used to directly attach the fluorescent group to the moiety of Formula I or indirectly through a linking structure to provide the various concealed fluorophores exemplified below:

;as well as

Wherein R ₁ is selected from the group consisting of H and C ₁ -C ₆ alkyl.

Furthermore, the fluorophores which can be used are preferably selected from the group consisting of: Wherein R ₂ is selected from the group consisting of H and CN, and R ₃ is selected from the group consisting of and

Wherein each R ₄ is independently selected from the group consisting of C ₁ -C ₆ alkyl, benzyl, CN and halogen.

It is still another object of the present invention to provide a method of preparing a hidden fluorophore comprising: providing a protected o-hydroxybenzoic acid derivative; and attaching a molecule having a fluorescent group to the protected o-hydroxybenzene a formic acid derivative; and a protecting group for removing the o-hydroxybenzoic acid moiety to obtain the hidden fluorescent group.

In one embodiment, the protected o-hydroxybenzoic acid derivative has the structure of Formula V:

Wherein L is a leaving group.

Another object of the present invention is to provide a biochemical analysis method capable of measuring the amount of o-hydroxybenzoic acid, comprising: providing a hidden type of fluorescein containing salicylic acid hydroxylase and catalyzing by salicylic acid hydroxylase a photophore and a reagent for NADH or NADPH; a test substance is added to the reagent under aerobic conditions; and the fluorescence of the reagent is measured to determine the amount of o-hydroxybenzoic acid present in the reagent.

In addition, the present invention also provides a method for performing biochemical analysis by using a fluorescent method and a plurality of enzymes, comprising the steps of: providing a salicylic acid hydroxylase, which is a salicylic acid hydroxylase a reagent for catalyzing a hidden fluorophore, NAD(P) ^+, and NAD(P) ⁺ dependent dehydrogenase; adding a test substance to the reagent under aerobic conditions; and measuring the fluorescing of the reagent Light to determine the amount of a biochemical molecule present in the reagent.

In one embodiment, the NAD(P) ⁺ dependent dehydrogenase is selected from the group consisting of 3-hydroxybutyrate dehydrogenase, cholesterol dehydrogenase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase.

Another object of the present invention is to provide a biochemical analysis kit comprising: salicylate hydroxylase, NAD(P) ⁺ , NAD(P) ⁺ dependent dehydrogenase, and a hidden fluorophore. In addition, the kit for biochemical analysis may or may not contain a third enzyme for catalyzing the production of the NAD(P) ⁺ dependent dehydrogenase.

To fully clarify the objects, features, and advantages of the present invention, those of ordinary skill in the art of the present invention can understand the invention and practice the invention. A detailed description of the present invention will be given.

In one embodiment of the invention, there is provided a compound derived from o-hydroxybenzoic acid having a fluorescent group and a moiety of formula I in the structure:

In the foregoing structure, the fluorescent group may be directly bonded to the moiety shown in Formula I, so that the structure may be as shown in Formula II:

For example, if coumarin is used as the main structure of the fluorescent group, to prepare the structure shown in Formula II, the chemical synthesis can be carried out by the procedure shown below:

In this procedure, Compound A is a protected o-hydroxybenzoic acid derivative, and its preparation can be found in Kang, SW; Gothard, CM; Maitra, S.; Wahab, A.; Nowick, JS J. Am. .Soc. , 2007 , 129 , 1486-1487, the contents of which are incorporated herein by reference in its entirety. Undoubtedly, a protected o-hydroxybenzoic acid derivative different from Compound A can also be prepared by other means of protection known to those of ordinary skill in the art without undue experimentation. Further, in the structure of the compound A, Br is used as a leaving group for the substitution reaction, and different kinds of leaving groups such as other kinds of halogens, OTs or OMs can be used, and are not limited thereto.

In this procedure, Compound B is a coumarin derivative which emits fluorescence, and its preparation can be found in Huang, ST; Ting, KN; Wang, KL Anal. Chimica Acta , 2008 , 620 , 120-126, The contents are hereby incorporated by reference in its entirety. Undoubtedly, those skilled in the art can also react with Compound A to form an analog of Compound C using other coumarin derivatives similar to Compound B without undue experimentation. For example, in compound B, CN can be replaced by H, and or And not limited thereto; if such compounds are to be synthesized, see also Wolfbeis, OS; Koller, E.; Hochmuth, P. Bull . Chem . Soc . Jp . 1985 , 58 , 731, the contents of which are all For reference herein.

As indicated above, the aforementioned procedure is performed in a two-step manner. In the first step, the hydroxyl moiety of Compound B is benzylated by Compound A under basic conditions to give the structure of monoanisole (not shown). In this case, Na ₂ CO _{3 is} used as a base to neutralize the acid produced in the reaction, but other bases which do not affect the reaction, such as KOH, may also be used. In the second step, the protecting group in the anisole produced in the first step is removed. In this case, TFA is used for deprotection, but other strong acids can also be used.

By the foregoing procedure, the compound B which originally emits fluorescence can be converted into a compound C which does not emit fluorescence, and can be used as a hidden fluorescent group, which can generate a compound capable of releasing fluorescence after the action of the enzyme, so Can be used for biochemical testing.

In addition, the fluorescent group can also be indirectly connected to the moiety shown in Formula I through a linking structure to form a hidden fluorophore as shown below:

Wherein R ₁ is selected from the group consisting of H and C ₁ -C ₆ alkyl; or

To synthesize the hidden fluorophore of Formula III, in addition to the use of protected o-hydroxybenzoic acid derivatives and fluorophores, phosgene is still required. If coumarin is used as the main structure of the fluorescent group as an example, to prepare the structure shown in Formula III, refer to the following, and also in a two-step manner (ie, form anisole first, then remove the protection). ) for chemical synthesis:

Wherein R ₂ is selected from the group consisting of H and CN, and R ₃ is selected from and

For the synthesis of the hidden fluorophores of the formula IV, see the method disclosed in Huang, ST; Peng, YX; Wang, KL Biosensor & Bioelectronic 2008 , 23 , 1793-1798, and the contents thereof are all incorporated herein. Reference.

In the present invention, the fluorescent group in the hidden fluorophore molecule is not particularly limited. For example, in certain embodiments, the fluorophore can be coumarin or a derivative thereof. In other embodiments, the luminescent group can be other than coumarin or a derivative thereof.

In a preferred embodiment, the fluorophore is selected from the group consisting of: Wherein R ₂ is selected from the group consisting of H and CN, and R ₃ is selected from and

Wherein each R ₄ is independently selected from the group consisting of C ₁ -C ₆ alkyl, benzyl, CN, and halogen.

In a preferred embodiment, the fluorescent group emits fluorescence having a wavelength of 500 nm or more after the action of SHL. Since many biochemical molecules also have fluorescent properties, and most of their fluorescence wavelengths fall in the blue region below 500 nm, the use of fluorescent groups that can release wavelengths above 500 nm will avoid other biochemical analysis. Interference with biochemical molecules. In a preferred embodiment, the fluorescent light emitted by the fluorescent groups in the hidden fluorophore molecules has a peak emission wavelength of about 550-650 nm.

In addition to this, in order to synthesize a hidden fluorophore, an ortho-hydroxybenzoic acid derivative and a fluorescent group of a starting material may be provided using an equivalent ratio other than 1:1. For example, if a rhodamine fluorophore is used to prepare a hidden fluorophore, it is necessary to provide twice the equivalent of the hydroxybenzoic acid derivative of the fluorophore, as shown below:

In other words, the hidden fluorophore structure provided by the present invention may have only one o-hydroxybenzoic acid moiety, and may also contain two or more o-hydroxybenzoic acid moieties.

Various biochemical analyses can be performed by various hidden fluorophores provided by embodiments of the present invention. For example, a kit may be provided, which comprises a SHL solution, any of the aforementioned hidden fluorophore solutions, and a solution containing NADH or NADPH, and then a sample to be tested and the aforementioned solution in an aerobic environment. Mixing, at this time, if o-hydroxybenzoic acid (which is a metabolite of aspirin) is present in the sample to be tested, the amount of fluorescence will be reduced, so that the adjacent hydroxyl group in the analyte can be judged by the change in the amount of fluorescence The amount of benzoic acid present.

In addition, biochemical analysis of multiple enzymes can also be performed by various hidden fluorophores provided by the embodiments of the present invention. For example, a set may be provided comprising a SHL solution, any of the aforementioned concealed fluorophore solutions, a NAD(P) ⁺ solution, and a NAD(P) ⁺ dependent dehydrogenase solution, followed by an aerobic environment. A sample to be tested is mixed with the solution. At this time, if the NAD(P) ⁺ dependent dehydrogenase is present in the sample to be tested, the amount of fluorescence increases, so that the amount of fluorescence can be changed by the amount of fluorescence. The amount of o-hydroxybenzoic acid present in the test object is judged.

In addition, a set of SHL solution, any of the aforementioned hidden fluorophore solution, NAD (P) ⁺ solution, NAD (P) ⁺ dependent dehydrogenase solution, and a third enzyme solution may be provided. The third enzyme can catalyze a biochemical molecule to be measured to produce the NAD(P) ⁺ dependent dehydrogenase; then, a sample to be tested is mixed with the solution in an aerobic environment. If the biochemical molecule is present in the sample to be tested, the amount of fluorescence increases, so that the amount of specific biochemical molecules in the analyte can be judged by the change in the amount of fluorescence. In a preferred embodiment, the NAD(P) ⁺ dependent dehydrogenase is selected from the group consisting of 3-hydroxybutyrate dehydrogenase, cholesterol dehydrogenase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase.

Accordingly, an embodiment of the present invention provides a single enzyme (ie, SHL) biochemical analysis method for measuring the amount of o-hydroxybenzoic acid by fluorescence. An embodiment of the present invention provides a biochemical analysis method for measuring the presence of a specific biochemical molecule by a dual-enzyme (ie, SHL and NAD(P) ⁺ dependent dehydrogenase) by fluorescence. An embodiment of the present invention provides a method for biochemical analysis of three enzymes (ie, SHL, NAD(P) ⁺ dependent dehydrogenase, and a third enzyme) for measuring the presence of a specific biochemical molecule by fluorescence. In a preferred embodiment, the NAD(P) ⁺ dependent dehydrogenase is glucose-6-phosphate dehydrogenase and the third enzyme is hexokinase.

Furthermore, other embodiments of the present invention provide a multi-enzyme (ie, SHL, NAD(P) ⁺ dependent dehydrogenase, and one or more other enzymes) biochemical analysis methods, and wherein NAD(P) ⁺ dependent dehydrogenase and Other enzymes can be selected according to the biochemical molecular characteristics to be analyzed by those having ordinary knowledge in the art without undue experimentation.

Instance

Material and instrument description

¹ H and¹³ C NMR was measured on a Bruker AMX-500 spectrometer; chemical shifts were expressed in ppm relative to tetramethyl decane (δ units); fluorescence measurements were made using a fluorescent grade quartz colorimetric tube and Horiba Jobin Yvon Fluoromax -4 Fluorescence spectrometer. Other chemicals were purchased from Acros, Aldrich, TCI or Sigma Chemical and were not specifically purified prior to use. The o-hydroxybenzoic acid derivative of the starting material and the fluorescent group are according to Cham, B. E.; John, D.; Bochner, F.; Imhoff, D. M.; Rowland, M.Clin .Chem . 1979 ,25 , 1420, Fuman, F.; Firberg, L.J .Pediatr . 1976 ,70 , 287. c) Grahm, G.; Rowland, J.J .Pharm .Sci . 1972 ,61 , 1219. d) Trinder, P.Biochem .J . 1954 ,57 , 301 and Wolfbeis, O. S.; Koller, E.; Hochmuth, P.Bull .Chem .Soc .Jp . 1985 ,58 , 731 et al., the entire disclosure of which is incorporated herein by reference. SHL is available from GDS Technology Inc. (USA). D-3-hydroxybutyrate dehydrogenase (III) was purchased from TOYOBO (product number HBD-301). Cholesterol dehydrogenase was purchased from Genzyme. All measurements were performed in 50 mM Tris-HCl (TRIZMA Base, Tris, pH 8). The synthesized hidden fluorophores were prepared in DMSO and added to Tris-HCl buffer before use, wherein the concentration of DMSO did not exceed 0.1% (v/v).

Main reaction process description

The main reaction flow of an embodiment of the present invention is as follows:

Analysis of Fluorescence Characteristics of Concealed Fluorescent Clusters

The fluorescence characteristics of Compound C were analyzed under an aerobic environment in the presence of SHL and NADH. Fluorescence emission was maintained near baseline when Compound C (20 μM) was used alone in the presence of SHL, alone in the presence of NADH or in Tris-HCl buffer at pH 8. However, if Compound C was placed in 1 unit of SHL and 1 mM of NADH solution, it was found that the fluorescence characteristic of Compound B increased 57-fold at 90 °C at 90 °C, as shown in Figure 1 (dashed line is the baseline) ). After adding 1 unit of SHL and 1 mM of NADH to Tris-HCl buffer containing Compound C, fluorescence can be detected within a few seconds, but if only Compound C and SHL are present, even after 10 minutes, The light value is still maintained near the baseline.

Effect of o-hydroxybenzoic acid on fluorescence intensity

If o-hydroxybenzoic acid is added to a solution containing SHL, Compound C and NADH under aerobic conditions, the fluorescence conversion of Compound C will be inhibited, and the fluorescence intensity will decrease as the concentration of o-hydroxybenzoic acid increases. As shown in Figure 2 (dashed line is the fluorescence intensity before the addition). If the concentration of o-hydroxybenzoic acid was plotted by fluorescence intensity, it was found that the linear relationship was in the range of 200 to 700 μM. Since o-hydroxybenzoic acid is a metabolite of aspirin and its toxicity is higher than 2.2 mM in blood, and may be fatal when it is higher than 4.3 mM, the detection of o-hydroxybenzoic acid is clinically important. Sex. As can be seen from this example, Compound C can be used to detect the concentration of o-hydroxybenzoic acid.

Double enzyme analysis using a hidden fluorophore

Double enzyme analysis was performed using 3-hydroxybutyrate dehydrogenase and cholesterol dehydrogenase in combination with SHL, in which the former can oxidize 3-hydroxybutyric acid (related to diabetes) and NAD ⁺ to produce acetamidine acetate and NADH. The latter oxidizes cholesterol (associated with cardiovascular disease) and uses NADH as an electron acceptor. As shown in Figures 3 and 4, the fluorescence of Compound C (10 μM) was maintained near the baseline prior to the addition of the substrate. When added to the substrate, there is a significant increase in fluorescence intensity. If the fluorescence intensity and the substrate concentration are plotted, it is known that there is a linear relationship between the concentration of 3-hydroxybutyric acid between 60 nM and 280 nM and the concentration of cholesterol between 100 nM and 800 nM, so compound C can be used in nM. Detect 3-hydroxybutyrate or cholesterol under range.

Double enzyme analysis using a hidden fluorophore

Double enzyme analysis was performed using glucose dehydrogenase in combination with SHL, and the results are shown in Fig. 5. It can be seen from Fig. 5 that the fluorescence intensity of the compound C and the concentration of glucose have a linear relationship within a certain range.

Three enzyme assays using hidden fluorophores

In the presence of glucose-6-phosphate dehydrogenase, SHL and hexokinase, in combination with NAD ⁺ , glucose and compound C, the ATP three enzyme assay can be performed as shown below. Figure 6 shows.

Preparation of protected hidden fluorophores

Compound A (0.43 mmole), Compound B (0.31 mmole), KI ( 0.042 mmole) and K ₂ CO ₃ (0.37 mmole) in dry DMF (2 mL) and the solution was stirred at room temperature under argon for overnight. The resulting mixture was filtered with water (50 mL). In CH ₂ Cl ₂ the organic layer was extracted (3 x 50 mL), and is dried over MgSO ₄ and concentrated in vacuo, followed by further purification _{(CH 2 Cl 2 / toluene =} 1/4) to flash chromatography to obtain Protected concealed fluorophore (42%, 70 mg) as an orange solid, and the analytical data is as follows: mp 187-190 ° C. ¹ H NMR (DMSO-d ₆ , 500 MH _Z, ) δ = 1.71 (s , 6H); 5.42 (s, 2H); 7.22 (s, 1H); 7.32 (m, 3H); 7.56 (t, J = 7.5 Hz, 1H); 7.62 (t, J = 7.5 Hz, 1H); (d, J = 8.0 Hz, 1H); 7.96 (d, J = 8.0 Hz, 1H); 8.13 (d, J = 8.1 Hz, 1H); 8.21 (d, J = 8.1 Hz, 1H). MS (EI C ₂₈ H ₁₈ N ₂ O ₆ S calc. 510.5 m/z = 510.0. FT-IR: v/cm ^-1 = 3075, 2941, 2868, 2371, 1742, 1724, 1615, 1587, 1536, 1502, 1440 , 1378, 1288, 1268, 1191, 1144, 1042, 953, 902, 867, 842, 772, 693, 672 cm ^-1

Deprotection of protected hidden fluorescent clusters

The protected concealed fluorophore (0.38 mmole) and trifluoroacetic acid (TFA)-H ₂ O (1.75 mL, 9/1, v/v) were added to a 50 mL round bottom flask equipped with a magnetic stir bar and The solution was stirred for 12 hours. Water (50 mL) was added to the mixture, and the resulting mixture was stirred for 30 min. The precipitate was filtered and washed with cold water to give pure compound C (0.08 mg, 0.17 mmol, 94%) as an orange solid, and the analytical data is as follows: mp 176-178 ° C. ¹ H NMR (DMSO-d ₆ ,500 MH _Z ) δ = 5.37 (s, 2H); 7.02 (d, J = 8.1 Hz, 1H); 7.06 (s, 1H); 7.30 (d, J = 8.1 Hz, 1H); 7.33 (s, 1H); 7.56 ( t, J = 7.5 Hz, 1H); 7.62 (t, J = 7.5 Hz, 1H); 7.81 (d, J = 8.1 Hz, 1H); 7.93 (d, J = 8.1 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H); 8.23 (d, J = 8.1 Hz, 1H) MS (ESI) C ₂₅ H ₁₄ N ₂ O ₆ S m/z = 469 (M-1); MS (HESI): calc. 470.0573, m/z = 469.470. FT-IR: v /cm ^-1 = 3447,2924,2852,2224,1724,1672,1619,1536,1507,1452,1433,1379,1312,1288,1255,1208, 1187,1142,1041,1015,948 cm ^-1

Analysis of concentration of double enzymes

Fluorescence measurements were taken at excitation wavelength λ _ex =500 nm and emission wavelength λ _em =595 nm. At 25 ° C, the total volume is 1 mL, containing compound C (40 μM), SHL (0.2 units), 3-hydroxybutyrate dehydrogenase (2 units) or cholesterol dehydrogenase (1 unit), NAD (10 μM) and 3-hydroxybutyrate (0 to 0.5 μM) or cholesterol (0 to 0.5 μM) in Tris-HCl buffer for 30 minutes. After subtracting the intensity of the fluorescence intensity of each solution from the baseline (the concentration of the substance to be tested is 0), the concentration of the substance to be tested is plotted.

The present invention has been disclosed in its preferred embodiments, and it should be understood by those of ordinary skill in the art that the present invention is not intended to limit the scope of the invention. It should be noted that variations and permutations equivalent to those of the embodiments are considered to be within the scope of the invention. Therefore, the scope of the invention is defined by the scope of the following claims.

1 is a view showing the fluorescence characteristics of a hidden fluorophore according to an embodiment of the present invention; FIG. 2 is a view showing the effect of o-hydroxybenzoic acid on a hidden fluorophore according to an embodiment of the present invention; and FIG. 3 is a view showing the effect of 3-hydroxyl The effect of butyric acid on the hidden fluorophore of one embodiment of the present invention; the fourth figure illustrates the effect of cholesterol on the hidden fluorophore of one embodiment of the present invention; and the fifth figure illustrates the hiding of glucose for an embodiment of the present invention. Effect of type fluorophores; Figure 6 illustrates the effect of ATP on the hidden fluorophores of an embodiment of the invention.

Claims (23)

An o-hydroxybenzoic acid derivative having a fluorescent group and a moiety as shown in Formula I in the structure: Wherein the fluorescent group is indirectly connected to the moiety of formula I through a linking structure. An ortho-hydroxybenzoic acid derivative as described in claim 1, wherein the structure is as shown in any one of the following formulae: ;as well as Wherein R ₁ is selected from the group consisting of H and C ₁ -C ₆ alkyl. An ortho-hydroxybenzoic acid derivative as described in claim 2, wherein the fluorescent group is selected from the group consisting of: Wherein R ₂ is selected from the group consisting of H and CN, and R ₃ is selected from the group consisting of and And wherein each R ₄ is independently selected from the group consisting of C ₁ -C ₆ alkyl, benzyl, CN, and halogen. The o-hydroxybenzoic acid derivative according to claim 1, wherein the fluorescent group is selected from the group consisting of coumarin and a derivative thereof. An ortho-hydroxybenzoic acid derivative as described in claim 1, which releases a fluorophore after the action of salicylic acid hydroxylase. The o-hydroxybenzoic acid derivative according to claim 5, wherein the fluorescent light emitted by the fluorescent group has a wavelength of 500 nm or more. The o-hydroxybenzoic acid derivative according to claim 6, wherein the fluorescent light emitted by the fluorescent group has a peak emission wavelength of 550 to 650 nm. A method of preparing a hidden fluorophore, comprising: (1) providing a protected o-hydroxybenzoic acid derivative; (2) linking a molecule having a fluorescent group to the protected o-hydroxybenzoic acid derivative And (3) removing the protecting group of the o-hydroxybenzoic acid moiety of the product of step (2) to obtain the hidden fluorescent group, wherein the structure of the hidden fluorescent group is as follows: As shown: ;as well as Wherein R ₁ is selected from the group consisting of H and C ₁ -C ₆ alkyl. The method of claim 8, wherein the protected o-hydroxybenzoic acid derivative has the structure shown in Formula V in the step (1): Wherein L is a leaving group. The method of claim 9, wherein the L is selected from the group consisting of halogen, OTs, and OMs. The method of claim 8, wherein the fluorescent group is selected from the group consisting of: Wherein R ₂ is selected from the group consisting of H and CN, and R ₃ is selected from the group consisting of and ;as well as Wherein each R ₄ is independently selected from the group consisting of C ₁ -C ₆ alkyl, benzyl, CN, and halogen. The method of claim 8, wherein in step (3), a strong acid is used for deprotection. The method of claim 12, wherein the strong acid is trifluoroacetic acid. A biochemical analysis method comprising: (1) providing a reagent comprising: salicylic acid hydroxylase; and derivatizing o-hydroxybenzoic acid as described in claim 3 And NADH or NADPH; (2) adding a test substance to the reagent under aerobic conditions; and (3) measuring the fluorescence of the reagent to determine the amount of o-hydroxybenzoic acid present in the reagent . A biochemical analysis method comprising: (1) providing a reagent comprising: salicylic acid hydroxylase; an o-hydroxybenzoic acid derivative as described in claim 1; NAD ⁺ or NADP ⁺ ; and NAD ⁺ or a NADP ⁺ dependent dehydrogenase; (2) adding a test substance to the reagent under aerobic conditions; and (3) measuring the fluorescence of the reagent to determine the presence of a biochemical molecule in the reagent . The method of claim 15, wherein the biochemical molecule is a substrate of the NAD+ or NADP+ dependent dehydrogenase. The method of claim 15, wherein the reagent further comprises a third enzyme for catalyzing the biochemical molecule to produce a substrate for the NAD ⁺ or NADP ⁺ dependent dehydrogenase. The method of claim 17, wherein the third enzyme is hexokinase. The method of claim 15, wherein the NAD ⁺ or NADP ⁺ -dependent dehydrogenase is selected from the group consisting of 3-hydroxybutyrate dehydrogenase, cholesterol dehydrogenase, glucose dehydrogenase, and glucose-6- Phosphophosphate dehydrogenase. The method of claim 15, wherein the structure of the o-hydroxybenzoic acid derivative is as shown in any one of the following formulae: ;as well as Wherein R ₁ is selected from the group consisting of H and C ₁ -C ₆ alkyl. The method of claim 20, wherein the fluorescent group is selected from the group consisting of: Wherein R ₂ is selected from the group consisting of H and CN, and R ₃ is selected from the group consisting of and ;as well as Wherein each R ₄ is independently selected from the group consisting of C ₁ -C ₆ alkyl, benzyl, CN, and halogen. A biochemical analysis kit comprising: salicylic acid hydroxylase; NAD ⁺ or NADP ⁺ dependent dehydrogenase; NAD ⁺ or NADP ⁺ ; and an o-hydroxybenzoic acid derivative as described in claim 3 . The biochemical analysis kit according to claim 22, further comprising a third enzyme for catalyzing the production of the NAD+ or NADP+ dependent dehydrogenase.