Abstract:
A pseudo-esterase activity-based fluorescent probe for specific detection of albumin, which has a carboxylic ester bond that can be selectively cleaved by human serum albumin (HSA), therefore forming a hydrolysate, which has a fluorescence emission spectrum significantly different from that of the fluorescent probe. According to the fluorescence intensity of the fluorescent probe and hydrolysate, we can detect the content of HSA in a biological sample.

Description:
PRIORITY CLAIM 
     This is a U.S. national stage of application No. PCT/CN2014/00334 filed on Mar. 27, 2014, which claims priority from China Patent Application No. 201310338267.7 filed Aug. 6, 2013, the entire content of which is incorporated herein as reference. 
     FIELD OF THE INVENTION 
     The present invention belongs to the medical technology, including a pseudo-esterase activity-based fluorescent probe for specific detection of albumin and its applications. 
     BACKGROUND OF THE INVENTION 
     Human serum albumin (HSA), the most abundant, versatile and complex protein in human blood, account for 50-60% of the total protein in plasma. See  Biochem Pharmacol.  2005 Nov. 25; 70(11): 1673-84. In human body, HSA makes a large contribution to plasma colloid osmotic pressure, mediates coagulation, helps to maintain normal microvascular permeability, and serves as a carrier protein for fatty acids, cholochrome, amino acid, steroid hormone, metal ions, and drug molecules. In human serum, the normal albumin range is 34-54 g/L. See Nlm.nih.gov. Retrieved 2010 May 12. But many diseases, including liver complaint (cirrhosis especially), nephrotic syndrome, tumors, protein-losing enteropathy, empyrosis, malnutrition and so on, will cause a decline in the level of serum albumin, while a high-protein diet and chronic dehydration will raise the HSA levels. So it has been clinically used for disease diagnosis to quantitatively determine the albumin levels in human blood. It will be necessary to give patients HSA by injection, when the HSA level is much lower than the normal level. It has been confirmed by a large number of clinical practices that monitoring the HSA level will be reference for the early diagnosis of liver or kidney disease, and diseases after operation. More importantly, urinary microalbumin has been widely used as an important biomarker for patients with renal damage (like diabetes, hypertension and poststreptococcal acute glomerulonephritis), endothelial dysfunction, cardiac disease and venous thromboembolism. The normal range of the renal small albumin in human is shown in Table 1. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The diagnostic value of the microalbuminuria 
               
             
          
           
               
                   
                 Gender 
                 Lower limit 
                 Upper limit 
                 Unit 
               
               
                   
                   
               
             
          
           
               
                 24 hours urine 
                   
                 30 
                 300 
                 mg/24 h 
               
               
                 short haul urine 
                   
                 20 
                 200 
                 μg/min 
               
               
                 single-point urine 
                   
                 30 
                 300 
                 mg/L or μg/g 
               
               
                 single-point urine/ 
                 Female 
                 3.5 
                 25 or 35 
                 mg/mmol 
               
               
                 Urinary C-peptide 
                   
                 30 
                 400 
                 μg/mg 
               
               
                 Creatinine Ratio 
                 Male 
                 2.5 or 3.5 
                 25 or 35 
                 mg/mmol 
               
               
                   
                   
                 30 
                 300 
                 μg/mg 
               
               
                   
               
             
          
         
       
     
     At present there are kinds of methods for HA quantification, including electrophoresis, immunoassay methods, as well as dye-binding procedures. Wherein the electrophoresis method is time-consuming with low specificity and it is easy to overestimate the HSA concentrations. See Clinical Chemistry: Theory, Analysis, Correlation, Mosby, 5 th  edition, 2009. While immunoassay is a specific method for HSA quantification, but it is high-cost, so it is less used in clinical application. Although the application of dye-binding method is relatively wide, its specificity is unable to be guarantee, for the reason that this method is based on the conjugation of dye molecule and parts of HSA or modifying the function of individual amino acids, but other tissue proteins in body also have the ability to bind these dyes. Bromcresol green (BCG) and bromcresol purple are widely used to quantitative detect albumin in clinic on account of simplicity, cheap and relatively higher specificity. But due to its low sensitivity (see Clinical Chemistry: Theory, Analysis, Correlation, Mosby, 5th edition, 2009.), hard to ensure the quantitative accuracy, overvalued (See  J Clin Pathol.  2003, 56, 780.) or underestimated (See  Clin Chim Acta . 1986, 155, 83.) often appear, and dye-binding-based assays can be interfered by the nonspecific interference from biological samples (See  Clin Chem ., 2009, 55, 583). Therefore, it is necessary to develop an easy-operating, highly specific, accurate faithful quantitative method for HSA quantification. 
     In recent years, domestic and overseas scholars found that HSA has catalytic properties and participates in catalyzing the hydrolysis of various molecules with ester bond in structure (Biochem Pharmacol 2010; 79 (5):784-91 &amp; J Biol Chem 2008; 283 (33):22582-90), after a specific cleavage of the carboxylic ester bond, the phenylbenzoate group covalently bound to the residues of HSA accompanying with releasing the hydrolysate that contains free phenolic hydroxyl group. Guangbo Ge et al. also found that some small molecules with ester bond in structure can be catalyzed by HSA, for examples, Boc5, receptor agonist of GLP-1, can only be catalyzed by albumin in vivo, while other human esterase don&#39;t participate in the hydrolysis of this compound ( Eur. J. Pharm. Sci.  2013; 48: 360-9). Recent domestic and overseas researches suggest that we can design and develop pseudo-esterase activity-based specific fluorescent probes for activity evaluation and quantification of HSA. The present invention provides the applications of a specific fluorescent probe to HSA, it is a dibenzoyl ester derivative, which replaced the C4 hydroxyl of N-n-butyl-4-hydroxy-1,8-naphthalimide, the fluorescence emission spectrum of the pseudo-esterase activity-based hydrolysis product is significantly different from that of the substrate molecule. This hydrolysis reaction has the characteristics of high selectivity, metabolic product easy to detect. 
     SUMMARY OF THE INVENTION 
     The purpose of the present invention is to provide a specific fluorescent probe, which is a dibenzoyl ester derivative, and its applications; this probe is used to detect albumin levels in human serum, plasma, or urine by testing the fluorescence intensity of samples, there are obvious differences between the fluorescence emission wavelength of the prototype and hydrolysate, and the product is more likely to detect. 
     The present invention provides a pseudo-esterase activity-based fluorescent probe. Wherein the carboxylic ester bond of the probe could be selectively cleaved by human serum albumin (HSA) to release a fluorescent product of which the fluorescence emission spectrum is significantly different from that of the substrate molecule, according to the content of substrate and product we can deduce the content and function of HSA in the system. This specific fluorescent probe is a dibenzoyl ester derivative, which replaced the C4 hydroxyl of N-n-butyl-4-hydroxy-1,8-naphthalimide, the structural general formula are shown in Scheme (1), wherein R 1  is —H, —CH 3 , —OCH 3 , —OC 2 H 5 , and R 2  is C2-C8 alkyl, C2-C8 halogenated alkyl group or any of its derivatives. 
     
       
                 
         
             
             
         
      
     
     The present invention also provides the applications of a kind of specific fluorescent probe for quantitative determination of HSA levels in different samples, wherein the compound as shown in scheme 1 was used as a specific substrate of hydrolysis metabolism for HSA, human serum albumin levels in different samples (including recombinant albumin, single enzyme, tissue preparation liquid, all kinds of tissue cells and other biological systems) were detected by the quantitative detection of the elimination of substrate and the generation of hydrolysis products in unit time. Specific method is: 
     This specific fluorescent probe is a dibenzoyl ester derivative, which replaced the C4 hydroxyl of N-n-butyl-4-hydroxy-1,8-naphthalimide, the concentrations of the probe substrate are between 1/10 and 10 Km, Km is chosen as the optimal concentration of substrate when determination at a single point (Km is Mie kinetic constant). 
     The system is phosphate buffer, reaction temperature is between 20 to 60° C. (The optimum reaction temperature is 37° C.) and pH value of the incubation system is between 5.5 and 10.5 (The optimum pH value is 7.4) 
     The reaction time is 5-120 minutes to ensure that the hydrolysate amount corresponding to the substrate described above achieve the limit of quantification. 
     HSA activity levels in different samples are detected with the quantitative detection of the elimination of substrate and the generation of hydrolysis products in unit time as the evaluating index. 
     The present invention provides applications of a kind of specific fluorescent probe for the quantitative determination of HSA levels in different samples, wherein both the probe substrate and its hydrolysate possess fluorescent properties, the rapid and sensitive determination for substrate and product can be realized by using fluorescence detector, fluorescence detection conditions: excitation wavelength is between 300 and 500 nm, emission wavelength is between 410 and 600 nm. 
     The present invention provides applications of a kind of specific fluorescent probe for the quantitative determination of human serum albumin levels in different samples, wherein the samples described above are human serum, plasma and urine. 
     Recombinant expression of HSA incubation system were investigated, through specific inhibition experiment, single enzymes metabolic reactions, and the kinetics of enzyme reaction, and it was proved that dibenzoyl ester derivatives, which replaced the C4 hydroxyl of N-n-butyl-4-hydroxy-1,8-naphthalimide, could be selectively cleaved by HSA to release hydrolysate, that is C4 ester bond rupture of probe. (as shown in  FIG. 4 ) 
     Using the albumin-specificity probe for the detection of albumin described in the present invention, has the following outstanding advantages: 
     (1) High specificity: dibenzoyl ester derivatives, which replaced the C4 hydroxyl of N-n-but yl-4-hydroxy-1,8-naphthalimide could be selectively cleaved by HSA to release hydrolysate, that is C4 ester bond rupture of probe. 
     (2) Cheap and available: dibenzoyl ester derivatives, which replaced the C4 hydroxyl of N-n-butyl-4-hydroxy-1,8-naphthalimide and their hydrolysis products can all be obtained by chemical synthesis, and the synthesis process is simple. 
     (3) High sensitivity: derivatives of N-n-butyl-4-hydroxy-1,8-naphthalimide have good features in fluorescence emission spectrum (410-600 nm), the substrate and its hydrolysis metabolites have different fluorescence emission spectrum characteristics, which makes it easy to do a distinguish detection, at the same time, standard curve drawing can be approved by the ratio method for quantitative determination. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Structure of dibenzoyl ester derivatives, which replaced the C4 hydroxyl of N-n-butyl-4-hydroxy-1,8-naphthalimide; and A is fluorescent probe A, B is fluorescent probe B, C is fluorescent probe C, D is fluorescent probe D. 
         FIG. 2 . 1H-NMR of N-n-butyl-1,8-naphthalimide-4-phenylbenzoate. 
         FIG. 3 . 13C-NMR of N-n-butyl-1,8-naphthalimide-4-phenylbenzoate. 
         FIG. 4 . Restructuring single enzyme screening test results of N-n-butyl-1,8-naphthalimide-4-phenylbenzoate&#39;s derivatives. 
         FIG. 5 . Fluorescence emission spectra of N-n-butyl-1,8-naphthalimide-4-phenylbenzoate and its hydrolysate (λex=342/452 nm). 
         FIG. 6 . Fitting curve of the generation of dibenzoyl ester hydrolysate against HSA concentrations. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The following example will further explain the invention, but not limit the present invention. 
     EXAMPLE 1 
     Synthesis of N-n-butyl-1,8-naphthalimide-4-phenylbenzoate (Fluorescent Probe A) 
     (1) To a solution of 4-hydroxy-N-butyl-1,8-naphthalimide (0.5 mmol) and Et 3 N (0.625 mmol) in 10 mL of THF, dibenzoyl chloride (0.6 mmol, mixed with 10 mL of CH 2 Cl 2 ) was added dropwise at 0° C. 
     (2) After stirring at this temperature for 1 h, the mixture was warmed to room temperature and stirred overnight. 
     (3) The solvent was removed in vacuo, and the residual solid was purified by chromatography (silica gel, EtOAc—hexane as eluent, 1:3, v/v) to afford 113 mg of A as a white solid. 
     (4) Then validating the white solid A compound structure, its  1 H NMR and  13 C NMR are shown in  FIG. 2  and  FIG. 3 . 
     EXAMPLE 2 
     Synthesis of N-n-butyl-1,8-naphthalimide-4-(4-methylphenylbenzoate) (Probe B) 
     (1) To a solution of 4-hydroxy-N-butyl-1,8-naphthalimide (0.5 mmol) and Et 3 N (0.625 mmol) in 10 mL of THF, 4-methyl-dibenzoyl chloride (0.6 mmol, mixed with 10 mL of CH 2 Cl 2 ) was added dropwise at 0° C. 
     (2) After stirring at this temperature for 1 h, the mixture was warmed to room temperature and stirred overnight. 
     (3) The solvent was removed in vacuo, and the residual solid was purified by chromatography (silica gel, EtOAc—hexane as eluent, 1:3, v/v) to afford 241 mg of B as a white solid. 
     EXAMPLE 3 
     Synthesis of N-3-carboxy propyl-1,8-naphthalimide-4-phenylbenzoate (Probe C) 
     (1) To a solution of 4-hydroxy-N-3-carboxy propyl-1,8-naphthalimide (0.5 mmol) and Et 3 N (0.625 mmol) in 10 mL of THF, dibenzoyl chloride (0.6 mmol, mixed with 10 mL of CH 2 Cl 2 ) was added dropwise at 0° C. 
     (2) After stirring at this temperature for 1 h, the mixture was warmed to room temperature and stirred overnight. 
     (3) The solvent was removed in vacuo, and the residual solid was purified by chromatography (silica gel, EtOAc—hexane as eluent, 1:3, v/v) to afford 95 mg of C as a white solid. 
     EXAMPLE 4 
     Synthesis of N-n-butyl-1,8-naphthalimide-4-(4-methoxyphenylbenzoate) (Probe D) 
     (1) To a solution of 4-hydroxy-N-butyl-1,8-naphthalimide (0.5 mmol) and Et 3 N (0.625 mmol) in 10 mL of THF, 4-methoxy-dibenzoyl chloride (0.6 mmol, mixed with 10 mL of CH 2 Cl 2 ) was added dropwise at 0° C. 
     (2) After stirring at this temperature for 1 h, the mixture was warmed to room temperature and stirred overnight. 
     (3) The solvent was removed in vacuo, and the residual solid was purified by chromatography (silica gel, EtOAc—hexane as eluent, 1:3, v/v) to afford 143 mg of D as a white solid. 
     EXAMPLE 5 
     Hydrolysis Selectivity of Fluorescent Probe A 
     (1) 196 μL PBS (pH=6.0, 10 mM) containing CES1b, CES1c. CES2 (5 μg/mL), acetylcholin esterase (0.1 U/L), butyrylcholine esterase (20 U/L), plasma (1%), HSA (0.5 mg/mL). BSA (0.5 mg/mL) was preincubated with stirring for 10 min at 37° C. 
     (2) 4 μL fluorescent probe A (0.5 mM) was added to the above solution to make the final concentration of the fluorescent probe to 10 μM in the test sample, then incubated with stirring at 37° C. 
     (3) 30 min later, 200 μL ice-cold CH 3 CN was added to the sample and stirred, then the reaction was stopped. 
     (4) Detecting the fluorescence intensity of probe A (λex=342 nm, λem=416 nm) and hydrolysate A1 (λex=452 nm, λem=564 nm) at corresponding wave length and calculating the fluorescence intensity ratio of A1 to A. (As can be seen in  FIG. 4  and  FIG. 5 ). 
     EXAMPLE 6 
     Quantitative Working Curve of HSA 
     (1) 5 mg/mL of human serum albumin (HSA) standard solution was diluted by PBS to obtain a series of working solution with different concentrations (0, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg/L) (Table 2), then samples were incubated at 37° C. for 10 min. 
     (2) Adding 20 μL fluorescent probe B (0.5 mM) to each sample (980 μL) to make the final concentration of the fluorescent probe to 0.5 mM, then samples were incubated turmoil at 37° C. for 30 min, 1 mL ice-cold CH 3 CN was added and stirred for 15 second, then the reaction was stopped. 
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Preparation of a series of working solution of HSA 
               
             
          
           
               
                   
                   
                   
                 Final 
               
               
                   
                   
                 The volume of the 
                 concentration of 
               
               
                 NO 
                 Volume of HSA 
                 diluent 
                 HSA 
               
               
                   
               
             
          
           
               
                 0 
                 200 
                 μL 
                 780 μL 
                 1000 
                 mg/L 
               
               
                 1 
                 180 
                 μL 
                 800 μL 
                 900 
                 mg/L 
               
               
                 2 
                 160 
                 μL 
                 820 μL 
                 800 
                 mg/L 
               
               
                 3 
                 140 
                 μL 
                 840 μL 
                 700 
                 mg/L 
               
               
                 4 
                 120 
                 μL 
                 860 μL 
                 600 
                 mg/L 
               
               
                 5 
                 100 
                 μL 
                 880 μL 
                 500 
                 mg/L 
               
               
                 6 
                 80 
                 μL 
                 900 μL 
                 400 
                 mg/L 
               
               
                 7 
                 60 
                 μL 
                 920 μL 
                 300 
                 mg/L 
               
               
                 8 
                 40 
                 μL 
                 940 μL 
                 200 
                 mg/L 
               
               
                 9 
                 20 
                 μL 
                 960 μL 
                 100 
                 mg/L 
               
             
          
           
               
                 10 
                 Absorbing 100 μL 
                 880 μL 
                 50 
                 mg/L 
               
               
                   
                 from 0 tube 
               
               
                 11 
                 Absorbing 40 μL 
                 940 μL 
                 20 
                 mg/L 
               
               
                   
                 from 0 tube 
               
               
                 12 
                 Absorbing 20 μL 
                 960 μL 
                 10 
                 mg/L 
               
               
                   
                 from 0 tube 
               
             
          
           
               
                 13 
                 0 
                 μL 
                 980 μL 
                 0 
                 mg/L 
               
               
                   
               
             
          
         
       
     
     (3) Detecting the fluorescence intensity of probe B (λ ex =342 nm, λ em =416 nm) and hydrolysate B1 (λ ex =452 nm, λ em =564 nm) at corresponding wave length, the working curves (fluorescence intensity ratio of B1 to B versus the concentration of HSA) were fitted, curve equation is Y=0.005578×X+0.04533 (R 2 =0.9992) (As can be seen in  FIG. 6 ). 
     EXAMPLE 7 
     Quantitative Analysis of Albumin in Human Plasma Sample 
     (1) 1 μL of human plasma sample was diluted to 200 times by PBS (pH 7.4, 10 mM), then samples were incubated turmoil at 45° C. for 10 min. 
     (2) 4 μL fluorescent probe B (0.5 mM) was added to the above solution to make the final concentration of the fluorescent probe to 10 μM, and then samples were incubated at 45° C. 
     (3) After 30 min, 200 μL of ice-cold CH 3 CN was added to the sample and stirred, and then the reaction was stopped. 
     (4) Detecting the fluorescence intensity of probe B (λ ex =342 nm, λ em =416 nm) and hydrolysate B1 (λ ex =452 nm, λ em =564 nm) at corresponding wavelength and calculating the fluorescence intensity ratio of B1 to B, the fluorescence ratio was substituted into working curve from example 2, concentration of HSA in plasma albumin was calculated as 46.2 mg/L. 
     EXAMPLE 8 
     Quantitative Analysis of Albumin in Urine Sample 
     (1) 490 μL of human urine sample was diluted by 490 μL PBS (pH 7.4, 10 mM), and then samples were incubated at 45° C. for 10 min. 
     (2) 20 μL fluorescent probe C (0.5 mM) was added to the above solution to make the final concentration of the fluorescent probe to 10 μM, and then samples were incubated turmoil at 37° C. for 30 min, 1 mL ice-cold CH 3 CN was added to the sample and stirred for 15 second, then the reaction was stopped. 
     (3) Detecting the fluorescence intensity of probe C (λ ex =342 nm, λ em =416 nm) and hydrolysate C1 (λ ex =452 nm, λ em =564 nm) at corresponding wavelength and the fluorescence intensity ratio of C1 to C was calculated. 
     (4) Finding the corresponding albumin level in the working curve, concentration of HSA in the sample was calculated as 48 mg/L. 
     EXAMPLE 9 
     Quantitative Analysis of HSA in Recombinant Expression System 
     (1) 10 mg recombinant HSA was weighted and dissolved in PBS (pH=7.4), configuring to 1 mg/mL albumin solution at 37° C. 
     (2) 20 μL fluorescent probe D (0.5 mM) was added to the above solution to make the final concentration of the fluorescent probe to 10 μM, and then samples were incubated turmoil at 37° C. for 30 min, 1 mL ice-cold CH 3 CN was added to the sample and stirred for 15 second, then the reaction stopped. 
     (3) Detecting the fluorescence intensity of probe D (λ ex =342 nm, λ em =416 nm) and hydrolysate D1 (λ ex =452 nm, λ em =564 nm) at corresponding wavelength and the fluorescence intensity ratio of D1 to D was calculated. 
     (4) Based on the working curve, the concentration of HSA in this sample (1 mg/mL recombinant HSA solution) was calculated as 14.9 μM.