WO2009067031A1

WO2009067031A1 - Method for detection and/or assay of lovastatin esterase with use of fluorogenic/chromogenic reagent, lovastatin esterase isolated and/or purified by this method, assembly for detection and/or assay and use of fluorogenic/chromogenic reagent for detection and/or assay of lovastatin esterase

Info

Publication number: WO2009067031A1
Application number: PCT/PL2008/000085
Authority: WO
Inventors: Ryszard Ostaszewski; Dominik Koszelewski; Waldemar Kurek; Olga Piotrowska
Original assignee: Instytut Chemii Organicznej Polska Akademia Nauk
Current assignee: Instytut Chemii Organicznej Polska Akademia Nauk
Priority date: 2007-11-19
Filing date: 2008-11-18
Publication date: 2009-05-28
Anticipated expiration: 2010-05-19
Also published as: PL383818A1; PL212182B1

Abstract

The invention relates to a method for detection and/or assay of the lovastatin esterase enzymatic activity, with use of the fiuorogenic/chromogenic reagent, comprising use of the fiuorogenic/chromogenic reagent of formula II wherein R represents hydrogen or C1-C6-alkyl, R' represents one or two substituents of the ring A, R' represents one or two or three substituents of the ring B. According to a method, the reagent is added to a material of enzymatic activity and the hydrolysis reaction under enzyme action is carried out, followed by detection and/or assay of the liberated coumarin compound of formula III. Also the invention relates to lovastatin esterase isolated and/or purified using this method, an assembly for detection and/or assay of the lovastatin esterase enzymatic activity, an use of the fiuorogenic/chromogenic reagent of formula II for detection and/or assay of the lovastatin esterase enzymatic activity.

Description

Method for detection and/or assay of lovastatin esterase with use of fluorogenic/chromogenic reagent, lovastatin esterase isolated and/or purified by this method, assembly for detection and/or assay and use of fluorogenic/chromogenic reagent for detection and/or assay of lovastatin esterase

The invention relates to a method for detection and/or assay of the lovastatin esterase enzymatic activity, with use of the fluorogenic/chromogenic reagent, lovastatin esterase isolated and/or purified using this method, an assembly for detection and/or assay of the lovastatin esterase enzymatic activity and use of the fluorogenic/chromogenic reagent for detection and/or assay of the lovastatin esterase enzymatic activity. The lovastatin esterase enzyme, independently of a source of origin, is an enzyme catalyzing the hydrolysis reaction of an ester bond in 2-methylbutyric acid side chain of lovastatin and its derivatives. The enzyme may be obtained inter alia from Clonostachys compactiuscula culture, said fungus being deposited under No. ATCC 38009 or ATCC 74178. The enzyme features by high selectivity with respect to a structure of lovastatin side chain and does not catalyze^" the hydrolysis of another statin being structurally similar, i.e. simvastatin and its derivatives. Such capability is employed in the process for manufacturing of purified simvastatin. The use of lovastatin esterase in the process of simvastatin synthesis is disclosed in U.S. patent No. 5,223,415. In the course of a synthetic process, from the potassium salt of lovastatin (Ia: M=K) the mixture of ammonium salts of lovastatin (Ia: M=NH₄) and simvastatin (Ib: M=NH₄), is obtained which is subjected to the selective hydrolysis reaction in a presence of material of biological origin containing lovastatin esterase, or to the hydrolysis reaction using a purified enzyme, according to Scheme I. In order to achieve a high productivity and selectivity of the process, the isolation of the enzyme from a biological material and purification is required. An assay of the lovastatin esterase enzymatic activity using a chromogenic reagent, such as butyric acid o- nitrophenyl ester is disclosed in U.S. patent No. 5,223,415. However, susceptibility of said ester for the hydrolysis reaction on treatment by other enzymes present in a biological material makes the assay accuracy problematic. Also the measurement is deteriorated due to the low extinction level of o-nitrophenol produced by said ester hydrolysis. Scheme I

Lovastatin esterase enzyme

According to the method disclosed by Schimmel et al, (Appl. Environ. Microbiology, 1997, 63, pp. 1307-1311), the enzyme activity is assayed using lovastatin ammonium salt (Ia: M=NH₄), by measurement of the conversion level of lovastatin (Ia: M=NH₄) to triol (Ic: M=NH₄) determined by means of an isocratic high performance liquid chromatography in reverse phases. Such assay technique and the type of substrate used render this method expensive. Moreover, the method cannot be applied for analyzing of small biological samples and for detection of an enzyme on a gel in a electrophoresis method.

The object of this invention is to present a method for detection and/or assay of lovastatin esterase enzymatic activity, with use of a fluorogenic/chromogenic reagent, lovastatin esterase isolated and/or purified using this method, an assembly for detection and/or assay of the lovastatin esterase enzymatic activity and use of the fluorogenic/chromogenic reagent for detection and/or assay of the lovastatin esterase enzymatic activity.

A method for detection and/or assay of the lovastatin esterase enzymatic activity, with use of the fluorogenic/chromogenic reagent, according to the invention is characterized in that, it comprises use of the fluorogenic/chromogenic reagent of formula II

wherein R represents hydrogen or Ci-C₆-alkyl,

R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, Ci-C₆-alkyl, Ci-C₆-alkenyl, Cj-C₆-alkoxy, C]-C₆-alkylcarboxy, d-C_ό-alkylcarbonyl, OH, CN, NO₂,

CONR R where each of R and R independently represents hydrogen, C₁ -C₆alkyl or R⁷ and R⁸ taken together represent C₃-C₆alkylene substituent, NR⁹R¹⁰ where each of R⁹ and R¹⁰ independently represents hydrogen, Ci-C₆alkyl or R⁹ and R¹⁰ taken together represent C₃-C₆alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with the benzene ring,

R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C₆-alkyl, Q-C_ό-alkenyl, d-C₆-alkoxy, Ci-C₆-alkylcarboxy, d-C₆-alkylcarboxy-Ci-C₆-alkyl, NO₂, NR¹¹R¹² where each of R¹¹ and R¹² independently represents hydrogen, Ci-C₆alkyl or R¹¹ and R¹² taken together represent C₃-C₆alkylene substituent, by adding said reagent to a material of enzymatic activity and carrying out the hydrolysis reaction under enzyme action, followed by detection and/or assay of the liberated coumarin compound of formula III

where R' and R" posses the meaning indicated above, in order to detect and/or assay the lovastatin esterase enzymatic activity. Preferably, a method comprises use of the fluorogenic/chromogenic reagent of formula II, wherein

R represents hydrogen or d-C₆-alkyl,

R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, d-C₆-alkyl, Ci-Cβ-alkenyl, Ci-C₆-alkoxy, Ci-C₆- alkylcarboxy, Ci-Q-alkylcarbonyl, aminocarbonyl, OH, CN, NO₂, NH₂, - A -

R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, C!-C₆-alkyl, Q-C_ό-alkenyl, CrC_ό-alkoxy, C i -C₆-alky lcarboxy, C i -C_ό-alkylcarbonyl .

More preferably, a method comprises use of the fluorogenic/chromogenic reagent of formula II, wherein

R represents hydrogen or Ci-Q-alkyl,

R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, Q-C_ό-alkyl, Cj-C_ό-alkenyl, Q-C_δ-alkoxy, R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Cj-C_ό-alkyl, Q-Co-alkenyl, CrC₆-alkoxy.

Even more preferably, a method comprises use of the fluorogenic/chromogenic reagent of formula II, wherein R represents methyl or C₃-C₆-alkyl, said reagent selected from a group comprising a racemic mixture, a mixture enriched in R enantiomer, a mixture enriched in S enantiomer, an optically pure S enantiomer and an optically pure R enantiomer.

Specifically, a method comprises use of the fluorogenic/chromogenic reagent selected from the group comprising:

- butyric acid 2-oxo-2H-chromen-7-yl ester,

- 2-methylbutyric acid 2-oxo-2H-chromen-7-yl ester, - butyric acid 4-methyl-2-oxo-2H-chromen-7-yl ester,

- 2-methylbutyric acid 4-methyl-2-oxo-2H-chromen-7-yl ester.

Very specifically, a method comprises use of the fluorogenic/chromogenic reagent selected from the group comprising:

- 2-methylbutyric acid 2-oxo-2H-chromen-7-yl ester, - 2-methylbutyric acid 4-methyl-2-oxo-2H-chromen-7-yl ester in a form of racemic mixture, a mixture enriched in R enantiomer, a mixture enriched in S enantiomer, an optically pure S enantiomer and/or an optically pure R enantiomer.

In a preferred method of the invention, the hydrolysis reaction is conducted in a buffered solution. Preferably, the hydrolysis reaction is conducted under a control of pΗ change and an adjustment of pΗ by use of a base. More preferably, the hydrolysis reaction is conducted at pΗ of 6-11.5, and even more preferably, the hydrolysis reaction is conducted at pΗ of 7.5-11. Most preferably, the hydrolysis reaction is conducted at pΗ of 8.5-10.5. Also preferably, the hydrolysis reaction is conducted at the temperature of 20°C-50°C. More preferably, the hydrolysis reaction is conducted at the temperature of 26°C-40°C.

In the preferred method of the invention, the liberated coumarin compound of formula III is detected and/or assayed by means of a spectrophotometric method. Optionally, the liberated coumarin compound of formula III is detected and/or assayed by means of a fluorescent method. More preferably, the coumarin compound is qualitatively assayed and then the content/activity of lovastatin esterase is determined.

Lovastatin esterase, according to the invention is characterized in that, said esterase is isolated and/or purified using the method indicated above. An assembly for detection and/or assay of lovastatin esterase enzymatic activity, according to the invention is characterized in that, it comprises the fluorogenic/chromogenic reagent of the formula II

wherein R represents hydrogen or d-C₆-alkyl

R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, d-C₆-alkyl, Ci-C₆-alkenyl, d-C₆-alkoxy, d-C₆-alkylcarboxy, d-C₆-alkylcarbonyl, OH, CN, NO₂, CONR⁷R⁸ where each of R⁷ and R⁸ independently represents hydrogen, Ci-C₆alkyl, or R⁷ and R⁸ taken together represent d-Cβalkylene substituent, NR⁹R¹⁰ where each of R⁹ and R¹⁰ independently represents hydrogen, d-C_όalkyl or R⁹ and R¹⁰ taken together represent C₃-C₆alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with benzene ring,

R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, d-C₆-alkyl, d-C₆-alkenyl, d-C₆-alkoxy, Ci-C₆-alkylcarboxy, d-C₆-alkylcarboxy-C,-C₆-alkyl, NO₂, NR¹¹R¹² where R¹¹ and R¹² independently represents hydrogen, C!-C₆alkyl or R¹¹ and R¹² taken together represent C₃-C₆alkylene substituent, and a compatible solvent.

According to the invention, the fluorogenic/chromogenic reagent of the formula II

wherein

R represents hydrogen or Cj-C₆-alkyl

R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, Q-C_ό-alkyl, Cj-C_ό-alkenyl, Q-C_ό-alkoxy, Ci-C_ό-alkylcarboxy, d-Ce-alkylcarbonyl, OH, CN, NO₂,

CONR⁷R⁸ where each of R⁷ and R⁸ independently represents hydrogen, Ci-C₆alkyl, or R and R taken together represent C₃-C₆alkylene substituent, NR⁹R¹⁰ where each of R⁹ and R¹⁰ independently represents hydrogen, Ci-Qalkyl, or R⁹ and R¹⁰ taken together represent C₃-C₆alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with benzene ring,

R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Cj-Cβ-alkyl, d-C₆-alkenyl, CpCβ-alkoxy, Ci-C_δ-alkylcarboxy, d-Qj-alkylcarboxy-d-Ce-alkyl, NO₂, NR¹¹R¹² where each of R¹¹ and R¹² independently represents hydrogen, Cj-C₆alkyl, or R¹¹ and R¹² taken together represent C₃-C₆alkylene substituent, is used for detection and/or assay of the lovastatin esterase enzymatic activity. Preferably, the fluorogenic/chromogenic reagent is used for detection of lovastatin esterase on a gel in a electrophoresis method. Optionally, the fluorogenic/chromogenic reagent is used for detection and/or assay of lovastatin esterase in an eluate in a chromatography method. More preferably, the fluorogenic/chromogenic reagent is used for detection and/or assay of lovastatin esterase in the method for manufacturing and/or purifying of simvastatin. In order to effective employ LE enzyme in biotechnological process, the activity of the enzyme has to be assayed and the purification has to be carried out to isolate the suitable fraction. The method for detection and/or assay of the activity according to the invention is distinguished by high sensitivity, reproducibility and simplicity of experimen- tation. Due to the factj that a reaction product is a substance exhibiting strong absorption in the visible or UV region, and/or exhibiting strong UV fluorescence, the hydrolytic action of an enzyme is detected by means of optical detection methods (such as photometry, spectrophotometry, fluorescence, luminescence) assuring a high quality of assay.

Especially, by the usage of fluorogenic/chromogenic reagent the assay of very low quantities of enzyme is possible, when in the course of reaction catalyzed by the enzyme assayed the product exhibiting strong fluorescence is formed. Under circumstances that the fluorogenic/chromogemc reagent itself does not exhibit a fluorescent activity, use of said reagent for qualitative and quantitative assay of even very low amounts of an enzyme is possible. Since changes of a fluorescence in the respect of time unit are proportional to the enzyme concentration, determination of an enzyme amount even in very diluted samples is feasible. Using a method for detection and/or assay according to the invention, the process for isolation and purification of an enzyme is simplified to a great extend.

Throughout the description of this invention and the patent claims, the term fluorogenic/chromogenic reagent denotes a reagent of enzymatic reaction, where the fluorogenic/chromogenic reagent on treatment of the lovastatin esterase enzyme is cleaved to form fluorescent and/or chromatic compound, which may be detected by optical methods, especially by measurement of fluorescence and/or absorption in visible and UV region of light.

Throughout the description of this invention and the patent claims, the term "triol" denotes the compound of the formula Ic,

wherein M represents a hydrogen atom, and also wherein M represents a metal cation or ammonium cation, i.e., the compound in the free-acid form or in the salt form, respectively, if not specified otherwise. Since the compound Ic in the free-acid form (M=H) is easily lactonised, the term "triol" may also comprise the lactone-diol form of the formula Id, if not specified otherwise.

The names ,,lovastatin" and simvastatin" refer to the compounds of the formulae Ie and If, respectively.

Ie If

Throughout the description of this invention and the patent claims, the names ,,lovastatin" and simvastatin" comprise also the carboxylic acid forms of these compounds, having the formulae Ia and Ib (M=H), respectively.

Ia Ib, and also salts of the compounds of the formulae Ia and Ib. if not specified otherwise. The "lovastatin salt" represents the compound of the formula Ia, wherein M represents a metal cation or an ammonium cation, and the "simvastatin salt" represents the compound of the formula Ib, wherein M represents a metal cation or an ammonium cation.

Throughout the description of this invention and the patent claims, the "lovastatin esterase" denotes a cellular or non-cellular material of the natural or recombinant origin having an enzymatic activity that consists in catalysing the hydrolysis of lovastatin and salts thereof, to form the above-defined triol of formula Ic (M=H) or salts thereof or derivative, where, under analogous conditions, the simvastatin and its salts do not undergo enzymatic hydrolysis or undergo enzymatic hydrolysis at a rate lower of at least one order of magnitude.

The fluorogenic/chromogenic reagent according to the invention is the compound of formula II

wherein

R represents hydrogen or Cj-C₆-alkyl,

R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, C!-C₆-alkyl, Q-Co-alkenyl, CrQ-alkoxy, Cj-Q-alkylcarboxy, Ci-Cβ-alkylcarbonyl, OH, CN, NO₂,

CONR⁷R⁸ where each of R⁷ and R⁸ independently represents hydrogen, Q-Cβalkyl or R and R taken together represent C₃-C₆alkylene substituent, NR⁹R¹⁰ where each of R⁹ and R¹⁰ independently represents hydrogen, C₁-QaIlCyI or

R⁹ and R¹⁰ taken together represent C₃-C₆alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with the benzene ring, R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C₆-alkyl, C]-C₆-alkenyl, Cj-C₆-alkoxy, Cι-C₆-alkylcarboxy, d-Q-alkylcarboxy-Ci-Ce-alkyl, NO₂, NR¹¹R¹² where each of R¹¹ and R¹² independently represents hydrogen, Ci-C₆alkyl or R¹¹ and R¹² taken together represent C₃-C₆alkylene substituent. Preferably, R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, Ci-Cβ-alkyl, d-C_ό-alkenyl, Cj-C_ό-alkoxy, Ci-Cβ-alkylcarboxy, Cj-Cβ-alkylcarbonyl, aminocarbonyl, OH, CN, NO₂, NH₂, and R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C_ό-alkyl, Ci-C₆-alkenyl, Q-C_ό-alkoxy, Q-Q-alkylcarboxy, Cj-Co-alkylcarbonyl.

More preferably, R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, Ci-C₆-alkil, Ci-C₆-alkenyl, Ci-C₆- alkoxy, and R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C₆-alkyl, Ci-C₆-alkenyl, Q-Q-alkoxy. Under circumstances that a substituent R in the formula II represents methyl or C₃-C₆-alkyl, the carbon atom of the group C₂H₅-*CH(R)-C(O)-O- marked with an asterisk constitutes a chiral atom, hence the fluorogenic/chromogenic reagent may exist in a form of racemic mixture comprising R and S enantiomers. According to the invention, in the method for detection and/or assay of LE enzyme, the fluorogenic/chromogenic reagent used is selected from a group comprising a racemic mixture, a reagent enriched in R enantiomer, a reagent enriched in S enantiomer, an optically pure S enantiomer and an optically pure R enantiomer.

The fluorogenic/chromogenic reagent of formula II, according to the invention, is used for detection and/or assay of lovastatin esterase activity in crude and purified biological preparations obtained from native and/or genetically modified organisms. In the course of an enzyme catalyzed reaction, illustrated on Scheme II,

Scheme II

II ^" III the coumarin compounds of the formula III are produced (where R' and R" possess the meaning indicated above), which strongly absorb the radiation in the visible and/or UV region, and/or strongly fluoresce allowing the detection and/or assay of a liberated coumarin compound by means of optical methods. Therefore rapid qualitative and quantitative assay of the lovastatin esterase enzyme in test samples is possible. Especially, the detection and/or assay of corresponding coumarin compounds of formula III is conducted by means of spectrophotometric and/or spectrofluorimetric methods.

The assay may be performed for different biological preparations containing the enzyme of such activity, being of various origin, in aqueous solutions, in the course of an enzyme purification process, an immobilization process, in the course of analyze of test samples derived from the enzyme purification process following separation by means of medium pressure liquid chromatography and high performance liquid chromatography, inokulum culturing, a fermentation on a laboratory and industrial scale. Moreover by applying the compound of formula II as the reagent in a method for detection and/or assay of enzymatic activity, detecting of the lovastatin esterase enzyme on gels following a sample separation by an electrophoresis method is possible. Such identification comprises lighting of a gel surface e.g. by UV light of a wavelength 366 run, to track a fluorescence band on a surface at location of a presence of an enzyme catalyzed reaction product, i.e. the coumarin compound of formula III.

The technical solutions according to the invention are explained in more detail by the examples and drawings, where Fig. 1 presents the change of fluorescence intensity (in relative measures) of the reagent governed by a kinetic catalytic effect of the enzyme versus time, and Fig. 2 presents the change of enzyme specific activity versus pH, determined using said reagent. Within the scope of examples below, the term LE enzyme represents the lovastatin esterase enzyme.

EXAMPLES

The fluorogenic/chromogenic reagents of the formula II are obtained in conventional manner, according to Scheme III, by reacting a substituted coumarin compound and an aliphatic carboxylic acid derivative of formula C₂HsCH(R)COOH, where R represents hydrogen or CrC₆-alkyl. According to the preferred embodiment, the derivative of 2-methylbutyric acid is used (R represents methyl). Preferably, an aliphatic carboxylic acid derivative is an activated acid form, such as acid halide, active ester, acid anhydride or mixed anhydride prepared in situ or separately prepared. The reaction usually is carried out in aprotic solvents, e.g. in tetrahydrofurane, methylene chloride or aromatic hydrocarbons, or in mixtures thereof, in a presence of an organic base (such as triethylamine, pyridine, lutidine) or an inorganic base (such as sodium or potassium carbonate), at temperatures from 0 to 100⁰C, for 1-24 hours.

Scheme III

acylation

Example 1. Preparation of racemic 2-methylbutyric acid 2-oxo-2H-chromen-7-yl ester

A solution of 7-hydroxycoumarin (145 mg, 1 mmol), triethylamine (140 μL, 1 mmol) and N,N'-dimethylpyridine (2 mg) in DME (10 mL) is cooled to the temperature 0°C and then a solution of 2-methylbutyric acid chloride (140 μL, 1.1 mmol) in DME (1 mL) is added. The mixture is allowed to react at ambient temperature for 30 minutes, and then is heated to reflux under reflux condenser for 5 hours. The solvent is evaporated under reduced pressure, and the residue is dissolved in chloroform (5 mL) and washed sequentially with hydrochloric acid (3%, 2 mL) and saturated potassium carbonate solution (5 mL). The title compound is obtained (165 mg, 75% yield). TLC, Rf=0.32 in the system of n-hexane/ethyl acetate (8:2 v/v).

¹H NMR (200 MHz, CDCl₃) δ: 6.4-7.7 (m, 5H), 2.7 (m, 1 H), 1.6-1.9 (m, 2H), 1.3 (d, J=7 Hz, 3H), 1.0 (t, J=7.3 Hz, 3H).

¹³C NMR (50 MHz, CDCl₃) δ: 175.0, 160.8, 155.2, 154.0, 143.4, 129.0, 118.9, 117.0, 116.6, 116.5, 110.9, 41.7, 27.3, 17.0, 12.2.

HR-MS, calculated for Ci₄H₁₅O₄ [M+H]⁺: 247.0970; found: 247.1028. Elemental analysis, calculated for Ci₄Hj₄O₄: C 68.27%, H 5.73%; found: C 68.34%, H 5.67%.

UV (c=0.093 mmol/dm³, 20 mM buffer TRIS/acetone 20/1 v/v) λ_max: 329 (ε=l 1120), 266 (ε=18260) λ_min: 302 (ε=7810), 258 (ε=18030). IR (KBr) cm^"1: 2969, 1757, 1726, 1621, 1656, 1400, 1268, 1224, 1124, 1101, 1053,

1019, 989, 906, 855.

Following the above procedure, but substituting 2-methylbutyric acid chloride by (S^ -2-methylbutyric acid chloride (prepared according to disclosure of Kharasch et ah, J. Org. Chem.; 1954; 19; 1283-1288) the (S) enantiomer of 2-methylbutyric acid 2-oxo- 2H-chromen-7-yl ester is obtained. The spectral analyses provide spectra of analogous signals and shifts, as indicated above for racemic 2-methylbutyric acid 2-oxo-2H-chromen- 7-yl ester.

Following the above procedure, but substituting 2-methylbutyric acid chloride by

acid chloride (prepared according to disclosure of Weisenborn et ah, J. Am. Chem. Soc; 1954; 76; 1792-1795) the (R) enantiomer of 2-methylbutyric acid 2-oxo-2H-chromen-7-yl ester is obtained. The spectral analyses provide spectra of analogous signals and shifts, as indicated above for racemic 2-methylbutyric acid 2-oxo- 2H-chromen-7-yl ester.

Example 2. Preparation of racemic 2-methylbutyric acid 4-methyl-2-oxo-2H- chromen-7-yl ester

A solution of 4-methyl-7-hydroxycoumarin (352 mg, 2 mmol), triethylamine (280 μL, 2 mmol) and N,N'-dimethylpyridine (5 mg) in tetrahydrofurane (2.5 mL) is cooled to the temperature 0⁰C and then a solution of 2-methylbutyric acid chloride (275 μL, 2.2 mmol) in tetrahydrofurane (1 mL) is added. After 30 minutes the cooling bath is removed and the mixture is allowed to react at ambient temperature for 12 hours. The solvent is evaporated under reduced pressure, and the residue is subjected to column chromatography on silica gel, eluting with the mixture of hexane and ethyl acetate (9:1, v/v). The title compound is obtained (338 mg, 65% yield). TLC, R_f=0.25 in the system of n-hexane/ethyl acetate (8:2 v/v).

¹H NMR (200 MHz, CDCl₃) δ: 7.6 (m, IH), 7.0 (m, 2H), 6.2 (s, IH), 2.7 (d.k, J=6.9 Hz, J=7.0 Hz, 1 H), 2.4 (s, 3H), 1.7 (m, 2H), 1.3 (d, J=7.0 Hz, 3H), 1.0 (t, J=7.3 Hz, 3H). 1³C NMR (50 MHz, CDCl₃) δ: 175.0, 160.9, 154.7, 153.9, 152.4, 125.8, 118.6,

118.6, 118.2, 114.9, 110.9, 41.6, 27.2, 19.2, 16.9, 12.0.

HR-MS, calculated for C₁₅H₁₆O₄ [M+H]: 261.1127 found: 261.1126. Elemental analysis, calculated for C₁₅H₁₆O₄: C 69.22%, H 6.20%; found: C 69.47%, H 6.46%.

UV (c=0.014 mmol/dm³, 20 mM buffer TRIS/acetone 20/1 v/v) λ_max: 324 (ε=l 1870), 268 (ε=13840), λ_min: 299 (ε=7800), 256 (ε=13100).

IR (KBr) cm^'1: 2971, 1758, 1732, 1615, 1388, 1263, 1148, 1131, 1108, 1017, 858.

Following the above procedure, but substituting 2-methylbutyric acid chloride by (S) -2-methylbutyric acid chloride (prepared according to disclosure of Kharasch et ah, J. Org. Chem.; 1954; 19; 1283-1288) the (S) enantiomer of 2-methylbutyric acid 4-methyl-2- oxo-2i/-chromen-7-yl ester is obtained. The spectral analyses provide spectra of analogous signals and shifts, as indicated above for racemic 2-methylbutyric acid 4-methyl-2-oxo- 2H-chromen-7-yl ester.

Following the above procedure, but substituting 2-methylbutyric acid chloride by (R) -2-methylbutyric acid chloride (prepared according to disclosure of Weisenborn et al., J. Am. Chem. Soc; 1954; 76; 1792-1795) the (R) enantiomer of 2-methylbutyric acid 4- methyl-2-oxo-2H-chromen-7-yl ester is obtained. The spectral analyses provide spectra of analogous signals and shifts, as indicated above for racemic 2-methylbutyric acid 4- methyl-2-oxo-2H-chromen-7-yl ester.

Example 3. Preparation of butyric acid 2-oxo-2H-chromen-7-yl ester A solution of 7-hydroxycoumarin (1 g, 6.17 mmol), triethylamine (1.12 mL, 8.02 mmol) and N,N'-dimethylpyridine (5 mg) in tetrahydrofurane (10 mL) is cooled to the temperature 0⁰C and then a solution of butyric acid chloride (710 μL, 6.8 mmol) in tetrahydrofurane (2 mL) is added. After 30 minutes the cooling bath is removed and the mixture is allowed to react at ambient temperature for 12 hours. The solvent is evaporated under reduced pressure, and the residue is subjected to column chromatography on silicagel, eluting with the mixture of n-hexane and ethyl acetate (9:1, v/v). The title compound is obtained (1.36 g, 95% yield). Following recrystallization from the mixture of hexane / ethyl acetate the product of melting point 72°C is obtained [72-73°C, according to R. B. Shaπna, et al, Indian. J. Chem. 1983, 22B, 538-541].

¹H NMR (200 MHz, CDCl₃) δ: 1.03 (t, J=7.1 Hz, 3H), 1.65-1.90(m, 2H), 2.56 (t, J=7.0, 2H), 6.35 (d, J=9.6 Hz, Hl), 6.98-7.15 (m, 2H), 7.45 (d, J=8.4 Hz, IH), 7.72 (d, J=9.6, Hz IH).

¹³C NMR (50 MHz, CDCl₃) δ: 13.8; 18.5, 36.3, 110.6, 116.1, 116.8, 118.7, 128.8,

143.1, 153.4, 154.8, 160.6, 171.6. .

IR (KBr) cm^'1: 2976, 1746, 1625, 1418, 1400, 1378, 1268, 1229, 1159, 1124, 1096, 991, 856., 843. Example 4. Preparation of butyric acid 4-methyl-2-oxo-2H-chromen-7-yl ester

A solution of 4-methyl-7-hydroxycoumarin (1.1 g, 6.24 mmol), triethylamine (1.12 mL, 8.02 mmol) and N,N'-dimethylpyridine (5 mg) in tetrahydrofurane (10 mL) is cooled to the temperature 0°C and then a solution of butyric acid chloride (710 μL, 6.8 mmol) in tetrahydrofurane (2 mL) is added. After 30 minutes the cooling bath is removed and the mixture is allowed to react at ambient temperature for 12 hours. The solvent is evaporated under reduced pressure, and the residue is subjected to column chromatography on silica gel, eluting with the mixture of n-hexane and ethyl acetate (9:1, v/v). The title compound is obtained (1.39 g, 92% yield). Following recrystallization from the mixture of n-hexane / ethyl acetate the product of melting point 94°C is obtained. 1H NMR (200 MHz, CDCl₃) δ: 1.04 (t, J=7.1 Hz, 3H), 1.67-1.88 (m, 2H), 2.43 (s,

3H), 2.57 (t, J=7.1 Hz, 2H), 6.27 (s, IH), 7.03-7.12 (m, 2H), 7.65 (d, J=8.5 Hz, IH).

¹³C NMR (50 MHz, CDCl₃) δ: 13.9, 18.6, 19.1, 36.5, 110.7, 114.7, 118.4, 125.6,

152.2, 153.5, 154.5, 160.8, 171.7.

IR(KBr) cm^"1: 2977, 1754, 1729, 1627, 1615, 1420, 1388, 1271, 1264, 1147, 1133, 1091,984,871,857. Examples 5-32

Following the procedure established in Example 3 and substituting 7-hydroxy- coumarin by correspondingly substituted derivatives of this compound, the fluorogenic/chromogenic reagents 5-32 are obtained (indicated in Table 1 below).

Example 33

Following the procedure established in Example 1 and substituting 2-methylbutyric acid by 2-ethylcaproic acid the fluorogenic/chromogenic reagent 33 is obtained (indicated in Table 1 below).

Example 34

Following the procedure established in Example 2 and substituting 2-methylbutyric acid by 2-ethylcaproic acid the fluorogenic/chromogenic reagent 34 is obtained (indicated in Table 1 below).

Table 1

Further, the embodiment examples illustrate use of the compounds of formula V for rapid detection of LE enzyme in a biological material (employing the enzymatic reaction according to Scheme IV), for spectrophotometric assay of the enzymatic activity of LE enzyme in the samples obtained and in the process of enzyme purification.

Scheme IV

Example 35. Use of the compound of formula V as the fluorogenic/chromogenic reagent for optical assay of lovastatin esterase The lovastatin esterase enzyme (LE) is produced by fungus Clonostachys compactiuscula ATCC 38009. The biological preparation of fungus is prepared according to the protocol disclosed in U.S. patent No. 5,223,415.

A frozen mycelium (50 g) is placed in a prechilled ceramic mortar, flooded with liquid nitrogen and grinded with glass beads (d=0.4 mm, 6 mg) to obtain a material of powder consistency (about 7 hours). The frozen homogenate is transferred to the centrifuge tubes and allowed to thaw. The enzyme is extracted with the glicyne buffer (50 mM, pH 9.4, 4°C) of a volume being proportional to the weight of mycelium (50 mL). Following cellular material centrifugation (20 min., 14000 rpm, 4°C) the pellet is extracted once again to obtain 130 mL of supernatant, in total. The concentration of protein is determined by a direct measurement of a solution absorbance or by Lowry method [Cwiczenia z biochemii (Exercises in biochemistry), ed. L. Klyszejko-Stefanowic, PWN, 2005]. The accurate protocol is listed below.

The protein concentration (c) is determined by the known method, comprising the measurement of absorbance [a] of a protein in a buffer, at wavelength λ=280 nm, by loading a sample of volume vo (given in milliliters) into a calibrated measure and filling-up with a buffer (glycine buffer 50 mM, pH 9.4) to 10 mL. The concentration is red out from the standard curve formerly prepared for BSA protein (calf albumin) given by the equation

(1434.4-a + 5.3228H0 fmg] C 1000-vo [mL]

Prior to the column chromatography purification, the protein extract (0.5 mL) is diluted 20-fold for assay; following purification the dilution of protein extract is not required, since the protein concentration is lowered. Each assay is repeated three-fold.

For activity assay a sample of 15 μL is drawn. The protein mass being present in a sample is determined by multiplication of the concentration and volume values.

Detection of a LE enzyme presence in a supernatant obtained by applying the fluorogenic/chromogenic reagent of formula V

A compound of formula V is added (50 μL of the solution in acetone, the concentration of 0.0 IM) to 1 mL of the supernatant obtained and the mixture is allowed to stay for 1 minute at ambient temperature. Next, the solution is placed under the UV lamp emitting the light of wavelength λ=366 nm and the result is compared to the blank sample result (containing a buffer only). Following the irradiation of the LE enzyme-containing sample, the characteristic blue coloration is observed originated from the hydrolysis product being the compound of formula IV, this is observed for the assayed solution only, but not in the case of the blank sample. The test is repeated for the supernatant diluted with distilled water, in the ratio of 1 : 100; and after 1 minute of the reaction, the solution is placed under the UV lamp emitting the light of wavelength λ=366 nm. Also for this trial the characteristic blue coloration is observed originated from the hydrolysis product - the compound of the formula V.

Example 36

To a buffer (3 mL, Tris, 20 mMol, pH=9.6) the solution of the compound from Example 1 (1 μL, 0.01 M in acetone) is added and a fluorescence spectrum is recorded by

Hitachi F-7000 apparatus using cuvette 10 mm Quartz Suprasil R of Hellma. Next, to this solution 300 mg of a mycelium in 2 mL of a buffer is added, the mixture is pulped and 200 μL of a supernatant is sampled. The total protein concentration is below 1 μg/mL. The changes of a fluorescence recorded with respect to time are shown on Fig. 1. The sample content of the LE enzyme may be assayed quantitatively under conditions of the experiment, since changes of the concentration versus time are linear and corresponds to the zero order reaction.

Example 37. Use of compounds of formula V for spectrophotometric assay of the enzymatic activity of the LE enzyme-containing material - general method

Assembly description. The measurement assembly comprises a thermostatted reactor (30°C±l) of capacity 20 mL positioned on a magnetic stirrer. The stirring rate of 300 rpm is applied. The absorbance changes are recorded by the detector Shiniadzu SPD-6A.

Performing an analysis. To a thermostatted buffer Tris (20 mM, pH 7.8), formerly subjected to filtration and sterilization procedure in autoclave, acetone (0.5 mL) is added and the mixture is degassed on a ultrasonic bath. The volume of the sample consists of 12 mL. The self-hydrolysis rate of the compounds of formula V is determined by recording the spectrophometer indications over 3 minutes period, in 15 second intervals, starting from the time point of adding a specific compound (50 μL of acetone solution, concentration of 0.01 M). Then the LE enzyme solution is added (the fraction of supernatant salted out with ammonium sulfate; the saturation range 40-80%, 0.487 mg protein). After 1 minute recording of spectrophometer indications is started, in 15 second intervals, over 3 minutes period. On the basis of results obtained, the graph of absorbance changes being the function of time is plotted. The measurement is made twice. The resulted and recalculated data are summarized in Tables 2, 3, 4, 5 (the specific activity for the reaction of zero order is calculated from linear relationship: concentration [nmol/mg]/time [min], as a gradient graph).

Example 38. Use of compounds from Examples 1 and 2 as the fluorogenic/chromogenic reagents for the chromatic and spectrophotometric assays of lovastatin esterase

The specific activities determined for the compounds from Examples 1 and 2 in the course of assay the LE enzyme activity are summarized in Table 2. The high values of specific activities for both compounds prove that these compounds constitute adequate chromogenic reagents for the enzyme. The comparative measurement performed by applying the known method (using o-nitrophenyl butyrate) reveals the significantly lower value of specific activity assayed for o-nitrophenyl butyrate.

The specific activity for the reaction of zero order is calculated from linear relationship: concentration [nmol/mg]/time [min], as a gradient graph.

Table 2. Specific activities of the LE enzyme solutions for the enzyme preparation at pH 7.8 assayed for reagents from Examples 1 and 2, in comparison with o-nitrophenyl butyrate

Example 39

The measurements according to Example 38 are repeated in Tris buffer (20 mM) of pH 9.6, i.e. at such condition parameters under which (according to literature data) the LE enzyme exhibits maximum activity. The results obtained are summarized in Table 3. Even at such pH value, the specific activity values for the compounds of formula V are significantly higher than the value obtained in the comparative measurement performed according to the known method (using o-nitrophenyl butyrate).

Table 3. Specific activities of the LE enzyme solutions for the enzyme preparation at pH 9.6 assayed for fluorogenic/chromogenic reagents from Examples 1, 2, 3 and 4, in comparison with o-nitrophenyl butyrate

Example 40. Use of compounds from Examples 1 and 2 for qualitative and quantitative assay of the LE enzyme activity in the course of enzyme purification process - general method The protein fraction obtained in Example 44 by. precipitation on salting with ammonium sulfate (the saturation range 40-80%), dissolved in the glicyne buffer (5 mL, 12.8 mg/mL), is loaded on the hydrophobic interaction column (HIC) packed with Phenyl- Sepharose (1.5 x 18 cm). The proteins are eluted with the same glycine buffer of pH 9.4 (flow: 1.5 mL/min, pressure 69 kPa). Next to elution of proteins non-absorbed on the column (detection by means of spectrophotometer UV, 254 nm), the former elution system is substituted by water as a eluent. The resulted chromatogram is shown on Fig. 2. The presence of the LE enzyme in subsequent fractions is detected by adding to the fractions of the acetone solution of fluorogenic/chromogenic reagent (0.01 M). The activity is exhibited by the fraction eluted just after substitution of the eluting system by water only (retention time 20 minutes). The activity is assayed by means of the method described in Example 37.

Also, the specific activity of the protein purified is assayed by means of a spectrophotometric method described in Example 44 below. The results are summarized in Table 4.

Table 4. The specific activities of active fraction solutions containing LE enzyme assayed for the fluorogenic/chromogenic reagents from Examples 1 and 2, compared with the state of art (o-nitrophenyl butyrate)

The results obtained prove that the compounds from Example 1 and 2 constitute very convenient reagents for the LE enzyme assays.

Example 41

The changes of LE enzyme activity with respect to pH are determined with use of 2-methylbutyric acid 2-oxo-2H-chromen-7-yl ester from Example 1, according to the protocol described in Example 37. The activity values are determined in the course of sequential adjustments of pH of a buffer used, from the value 7.8 to 9.9, and are presented in the form of bar diagram on Fig. 2. The data obtained reveal the fact that the enzyme activity is the highest one at the pH value of 9.6.

Example 42

The influence of a buffer type on the enzymatic activity of LE enzyme is determined according to the procedure described in Example 37, using 2-methylbutyric acid 2-oxo-2H-chromen-7-yl ester from Example 1.

The series of measurements are made by conducting the hydrolysis reaction in different buffers. The experiments performed do not revealed any dependence between the buffer type and the enzymatic activity. The results obtained (summarized in Table 5) differ to the less extend than the calculated error limits (5%). In spite of the buffer used, the enzymatic reaction runs at similar rate.

Table 5. The influence of the buffer type on the enzymatic activity of LE enzyme solution

Example 43. Use of the compounds from Examples 1 and 2 for LE enzymatic activity assay in the course of enzyme purification process by means of the polyacrylamide gel electrophoresis

The protein fraction obtained by precipitation on salting with ammonium sulfate

(the saturation range 40-80%) is dissolved in glycine buffer (5 mL, 12.8 mg/mL) and a portion is sampled (50 μL). The fraction obtained is subjected to the analysis by means of

SDS/PAGE method at the reductive conditions (according to the protocol disclosed in Appl. Environ. Microbiolog. 63, 1997, 1307-1311).

For gel dyeing, the acetone solution of the compound from Example 1 or 2 is used (50 μL acetone solution of concentration 0.01 M) which solution is applied onto a gel. Following 5 minutes period, the gel surface is irradiated by UV lamp (λ=366 run) to render visible the band of blue fluorescence corresponding to lovastatin esterase. Example 44

A frozen mycelia of Clonostachys compactiuscula (70 g) is placed in a prechilled ceramic mortar, flooded with liquid nitrogen and grinded with glass beads (d=0.4 mm, 30 mg) to obtain a material of powder consistency (about 13 hours). The homogenate of cellular material is extracted with phosphorane buffer (20 mM, pH 7.8, 4°C, Tween 80 (0.1%), NaN₃ (0.01%), 50 mL) with careful mixing of the extract with a glass spatula in order to avoid formation of a foam. Following the cellular material centrifugation (20 minutes, 11000 rpm, 4°C), the supernatant 1 is obtained. The extraction of a pellet is repeated and the sequentially obtained supernatants are subjected to the determination of a protein concentration and specific activities, using the compound from Example 3 as the fluorogenic/chromogenic reagent. The results obtained are summarized in Table 5.

The protein concentration is determined by the direct measurement of solution absorbance or by Lowry method [Cwiczenia z biochemii (Exercises in biochemistry), ed. L. Klyszejko-Stefanowic, PWN, 2005). The accurate protocol is summarized below.

The protein concentration is determined on the basis of an absorbance measurement at the wavelength λ=280 nm. The concentration is red out from standard curve formerly prepared for BSA protein BSA (calf albumin) (y=1434.4 x + 5.3). Prior to the column chromatography purification, the protein extract (0.5 mL) is diluted 20-fold for assay; following purification the dilution of protein extract is not required, since the protein concentration is lowered. Each assay is repeated three-fold.

Table 6

The supernatants Nos. 1 and 2 (exhibiting the highest activity) are pooled and cooled to the temperature 4°C. Then the LE enzyme is purified by salting out. To the supernatant obtained ammonium sulfate is added in portions (58 g) up to the level of 40% solution saturation, the solution is stirred for 1 hour and allowed to stay for 14 hours. The precipitated fraction A is centrifuged off (20 minutes, 14000 rpm, 4°C), dissolved in phosphorane buffer (15 mL, pH=7.8) and the specific activity is assayed (the result of <30, indicates a low level of specific activity). To the supernatant having the volume of 275 mL ammonium sulfate is added (98.3 g) up to saturation of 85%. After 24 hours the precipitated fraction B is centrifuged off (20 minutes, 11000 rpm, 4°C), dissolved in phosphorane buffer (15.65 mL, 20 mM, pH 7.8, 4°C) to obtain a solution exhibiting a very high specific activity. To the contrary, the supernatant obtained at this stage (260 mL) exhibits a very low specific activity (<30).

The fraction B is purified by the method of hydrophobic interaction chromatography (HIC) using column packed with Phenyl-Sepharose and eluting with phosphorane buffer (20 mM, pH 6.5, EDTA 5mM, NaCl 0.5 M; flow: 1.6 mL/min, pressure 10 psi, detector sensivity 0.1). Following washing out of the proteins non- adsorbed on the column, (0-15 minute period, UV 254 run), the eluent system is changed to water. The presence of the LE enzyme in the specific fractions is detected by an addition of the fluorogenic/chromogenic reagent solution (0.1 mL, 0.05 mol). The fraction collected is the fraction exhibiting the activity in the reaction with the fluorogenic/chromogenic reagent, and is featured by the retention time of 20 minutes and by the volume of 50 mL (fraction C). The analysis of the fraction C activity leads to the conclusion, that the fraction C contains purified LE enzyme, in 8% yield (defined as a ratio of total activity of a given fraction to the total activity of the homogenate, and expressed in percent), of the purity level 25 (defined as a ratio of specific activity of a given fraction to specific activity of homogenate).

The mixture of ammonium salts of simvastatin (18 mg) and lovastatin (2 mg) is dissolved in 25 mL of glycine buffer (50 mM, pH=9.4). To 1 mL of that solution fraction C is added (being a purified solution of the LE enzyme comprising 0.666 mg of protein) and the mixture is stirred at the temperature of 20°C. The progress of the reaction is controlled by the HPLC method. After 24 hours it has been found that the reaction mixture do not contain lovastatin, but simvastatin only and a corresponding amount of triol.

The HPLC analyses were performed on the liquid chromatograph apparatus Pro Star by Varian, equipped with ProStar 330 Photodiode Array Detector, Prostar 410 AutoSampler, ProStar 410 Solvent Delivery Module. The separation of compounds was performed on the column RP LiChro Cart 55-4, packed with octadecylsilyl silica gel for chromatography R (RP- 18), 3 μm. The elution of molecules was carried out using the solvent of the following content: acetonitrile (350 mL), orthophosphoric acid (0.375 mL), water (150 mL), at the flow speed 1.5 mL/min, at wavelength 238 nm. The retention times of the consecutive components of the reaction mixture were as follows: 0.51 minutes - triol, 2.21 minutes - lovastatin, 3.22 minutes - simvastatin.

Claims

1. A method for detection and/or assay of the lovastatin esterase enzymatic activity, with use of a fluorogenic/chromogenic reagent, characterized in that, it comprises use of the fluorogenic/chromogenic reagent of formula II

wherein

R represents hydrogen or Ci-C₆-alkyl,

R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, Ci-Cβ-alkyl, Ci-C₆-alkenyl, Ci-C₆-alkoxy, C₁-C₆- alkylcarboxy, d-Q-alkylcarbonyl, OH, CN, NO₂,

CONR⁷R⁸ where each of R⁷ and R⁸ independently represents hydrogen, d-Cβalkyl or R⁷ and R⁸ taken together represent C₃-C₆alkylene substituent, NR⁹R¹⁰ where each of R⁹ and R¹⁰ independently represents hydrogen, C_!-C₆alkyl or R⁹ and R¹⁰ taken together represent C₃-C₆alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with the benzene ring,

R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C₆-alkyl, CrC₆-alkenyl, Q-C_ό-alkoxy,

Ci-C₆-alkylcarboxy, d-Q-alkylcarboxy-Ci-Ce-alkyl, NO₂, NR¹¹R¹² where each of R¹¹ and

R¹² independently represents hydrogen, d-C₆alkyl or R¹¹ and R¹² taken together represent

C₃-C₆alkylene substituent, by adding said reagent to the material of enzymatic activity and carrying out the hydrolysis reaction under enzyme action, followed by the detection and/or assay of the liberated coumarin compound of formula III

where R' and R" posses the meaning indicated above, in order to detect and/or assay the lovastatin esterase enzymatic activity.

2. A method for detection and/or assay of enzymatic activity according to claim 1, characterized in that, it comprises use of the fluorogenic/chromogenic reagent of formula II, wherein R represents hydrogen or d-C₆-alkyl,

R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, Q-Co-alkyl, Ci-C₆-alkenyl, d-C₆-alkoxy, C₁-C₆- alkylcarboxy, Cj-C_ό-alkylcarbonyl, aminocarbonyl, OH, CN, NO₂, NH₂, R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-Ce-alkyl, d-Q-alkenyl, Q-C_δ-alkoxy, C i -C_ό-alkylcarboxy, C i -C_ό-alkylcarbonyl.

3. A method for detection and/or assay of enzymatic activity according to claim 2, characterized in that, it comprises use of the fluorogenic/chromogenic reagent of formula II, wherein R represents hydrogen or C i -C₆-alkyl, R' represents one or two substituents of the ring A independently selected from the group comprising hydrogen, halogens, Ci-C₆-alkyl, Ci-C₆-alkenyl, C]-C₆-alkoxy, R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, Ci-C₆-alkyl, CrC₆-alkenyl, Ci-C₆-alkoxy.

4. A method for detection and/or assay of enzymatic activity according to claim 1, 2, or 3, characterized in that, it comprises use of the fluorogenic/chromogenic reagent of formula II, wherein R represents methyl or C₃-C₆-alkyl, said reagent selected from a group comprising a racemic mixture, a mixture enriched in R enantiomer, a mixture enriched in S enantiomer, an optically pure S enantiomer and an optically pure R enantiomer.

5. A method for detection and/or assay of enzymatic activity according to claim 3, characterized in that, it comprises use of the fluorogenic/chromogenic reagent selected from the group comprising:

- butyric acid 2-oxo-2H-chromen-7-yl ester,

- 2-methylbutyric acid 4-methyl-2-oxo-2H-chromen-7-yl ester.

6. A method for detection and/or assay of enzymatic activity according to claim 5, characterized in that, it comprises use of the fluorogenic/chromogenic reagent selected from the group comprising

- 2-methylbutyric acid 2-oxo-2H-chromen-7-yl ester, - 2-methylbutyric acid 4-methyl-2-oxo-2H-chromen-7-yl ester in a form of a racemic mixture, a mixture enriched in R enantiomer, a mixture enriched in S enantiomer, an optically pure S enantiomer and/or an optically pure R enantiomer.

7. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 4, characterized in that, the hydrolysis reaction is conducted hi a buffered solution.

8. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 5, characterized in that, the hydrolysis reaction is conducted under a control of pΗ change and an adjustment of pΗ by use of a base.

9. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 5, characterized in that, the hydrolysis reaction is conducted at pΗ of 6-11.5.

10. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 5, characterized in that, the hydrolysis reaction is conducted at pΗ of 7.5-11.

11. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 5, characterized in that, the hydrolysis reaction is conducted at pΗ of 8.5-10.5.

12. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 5, characterized in that, the hydrolysis reaction is conducted at the temperature 20°C-

50°C.

13. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 5, characterized in that, the hydrolysis reaction is conducted at the temperature 26⁰C- 40°C.

14. A method for detection and/or assay of enzymatic activity, according to claim 1, 2, 3, or 5, characterized in that, the liberated coumarin compound of the formula III is detected and/or assayed by means of a spectrophotometric method.

15. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 5, characterized in that, the liberated coumarin compound of the formula III is detected and/or assayed by means of a fluorescent method.

16. A method for detection and/or assay of enzymatic activity according to claim 1, 2, 3, or 5, or 12, or 13, characterized in that, the coumarin compound is qualitatively assayed and then the content/activity of lovastatin esterase is determined.

17. Lovastatin esterase isolated and/or purified using the method according to claim 1.

18. An assembly for detection and/or assay of the lovastatin esterase enzymatic activity, characterized in that, it comprises the fluorogenic/chromogenic reagent of formula II

wherein R represents hydrogen or C i -C₆-alkyl

R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, Ci-Cβ-alkyl, Cj-C_ό-alkenyl, CpCβ-alkoxy, Q-C_δ-alkylcarboxy, Ci-Ce-alkylcarbonyl, OH, CN, NO₂, CONR R where each of R and R independently represents hydrogen,

or R⁷ and R⁸ taken together represent C₃-C₆alkylene substituent, NR⁹R¹⁰ where each of R⁹ and R¹⁰ independently represents hydrogen, C_!-C₆alkyl or R⁹ and R¹⁰ taken together represent C₃-C₆alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with benzene ring,

R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, d-C₆-alkyl, Ci-C₆-alkenyl, Ci-C₆-alkoxy, Ci-C₆-alkylcarboxy, d-Ce-alkylcarboxy-Ci-Ceralkyl, NO₂, NR¹¹R¹² where R¹¹ and R¹² independently represents hydrogen, Ci-C₆alkyl or R¹¹ and R¹² taken together represent C₃-C₆alkylene substituent, and a compatible solvent.

19. Use of the fluorogenic/chromogenic reagent of the formula II

wherein

R represents hydrogen or d-C₆-alkyl R' represents one or two substituents of the ring A independently selected from the group comprising: hydrogen, halogens, Ci-Cβ-alkyl, C]-C₆-alkenyl, CrC_ό-alkoxy, Q-Ce-alkylcarboxy, Ci-Cβ-alkylcarbonyl, OH, CN, NO₂,

7 S 7 8

CONR R where each of R and R independently represents hydrogen, C₁-C₆HIlCyI,

7 8 or R and R taken together represent C₃-C₆alkylene substituent,

NR⁹R¹⁰ where each of R⁹ and R¹⁰ independently represents hydrogen, d-C₆alkyl, or R⁹ and R¹⁰ taken together represent C₃-C₆alkylene substituent, heteroaryl substituent having nitrogen atom as a ring atom and optionally an additional heteroatom selected from the group comprising O and S atoms, said heteroaryl substituent optionally fused with benzene ring,

R" represents one or two or three substituents of the ring B independently selected from the group comprising hydrogen, halogens, CrC₆-alkyl, Ci-C₆-alkenyl, CrQ-alkoxy, Ci-Cβ-alkylcarboxy, Ci-C₆-aJkylcaΛoxy-Ci-C₆-alkyl, NO₂, NR¹¹R¹² where each of R¹¹ and R¹² independently represents hydrogen, CrC₆alkyl, or R¹¹ and R¹² taken together represent C₃-C₆alkylene substituent, for detection and/or assay of the lovastatin esterase enzymatic activity.

20. Use according to claim 17, characterized in that, the fluorogenic/chromogenic reagent is used for detection of lovastatin esterase on a gel in an electrophoresis method.

21. Use according to claim 17, characterized in that, the fluorogenic/chromogenic reagent is used for detection and/or assay of lovastatin esterase in an eluate in a chromatography method.

22. Use according to claim 17, characterized in that, the fluorogenic/chromogenic reagent is used for detection and/or assay of lovastatin esterase in the method for manufacture and/or purification of simvastatin.